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Surgery_Schwartz. along with evidence that neocortical brain cells were a source of HMGB1 following TBI.14 More recently, in an animal model of hemorrhagic shock, HMGB1 release from intestinal epithelium was linked to acute lung injury.15 Finally, increased plasma levels of HMGB1 have been shown to cor-relate with immune suppression and increased infection risk in patients undergoing major surgical procedures.16 The identifica-tion of the receptor for advanced glycation end products as the receptor for HMGB1 in this setting has identified new therapeu-tic strategy to ameliorate ALI following TBI.17A Role for Mitochondrial DAMPs in the Injury-Mediated Inflammatory Response. Mitochondrial proteins and/or DNA can act as DAMPs by triggering an inflammatory response to cellular necrosis and stress. Specifically, mitochondrial DNA (mtDNA) released from damaged or dysfunctional mitochon-dria leads both to inflammasome activation and activation of the stimulator of interferon gene pathway (STING).18Cell-free


Surgery_Schwartz

Surgery_Schwartz. mitochondrial DNA (mtDNA) released from damaged or dysfunctional mitochon-dria leads both to inflammasome activation and activation of the stimulator of interferon gene pathway (STING).18Cell-free mtDNA (cf-mtDNA) has been shown to be thousands of times higher in trauma patients when compared to normal volunteers. In addition, direct injection of mitochon-dria lysates in an animal model causes remote organ damage, including liver, and lung inflammation.19 These data suggest that with cellular stress or tissue injury, cf-mtDNA released from damaged/stressed mitochondria contribute to the sterile inflammatory response in injured patients. From an evolution-ary perspective, given that eukaryotic mitochondria derive from bacterial origin, it would make sense that they retain bacterial features capable of eliciting a strong response that is typically associated with a pathogen trigger. In addition, the mitochon-drial transcription factor A (TFAM), a highly abundant mito-chondrial protein,


Surgery_Schwartz

Surgery_Schwartz. capable of eliciting a strong response that is typically associated with a pathogen trigger. In addition, the mitochon-drial transcription factor A (TFAM), a highly abundant mito-chondrial protein, is functionally and structurally homologous to HMGB1. It has also been shown be released in high amounts from damaged cells where it acts in conjunction with mtDNA to activate TLR9 signaling.20Following trauma, cf-mtDNA levels appear to be higher in nonsurvivors when compared to survivors and correlate with the development of both SIRS and sepsis post injury.21,22 Cf-mtDNA has also been linked both ex vivo and in vivo to the formation of neutrophil extracellular traps, which are also associated with sterile inflammation and are a possible cause of secondary tissue injury.23,24 Reducing cf-mtDNA, perhaps by targeting enzymes capable of digesting circulating mtDNA is an attractive therapeutic option to prevent development of inflam-matory complications of trauma.25Heat-Shock Proteins as


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Surgery_Schwartz. perhaps by targeting enzymes capable of digesting circulating mtDNA is an attractive therapeutic option to prevent development of inflam-matory complications of trauma.25Heat-Shock Proteins as DAMPs. Heat shock proteins (HSPs) are a large and diverse family of intracellular proteins that are expressed during times of inflammation and oxidative stress or following tissue injury.26 Very highly conserved across species, HSPs function as molecular chaperones to monitor and maintain appropriate protein folding.27 They accomplish this task through the promotion of protein refolding, the targeting of misfolded proteins for degradation, or the sequestering of partially folded proteins for movement to appropriate membrane compartments. HSPs are also capable of binding foreign proteins and thereby function as intracellular chaperones for ligands such as bacterial DNA and endotoxin.HSPs are presumed to protect cells from the effects of traumatic stress and, when released by damaged cells, alert


Surgery_Schwartz

Surgery_Schwartz. function as intracellular chaperones for ligands such as bacterial DNA and endotoxin.HSPs are presumed to protect cells from the effects of traumatic stress and, when released by damaged cells, alert the immune system of the tissue damage by activating both innate and acquired immunity.28 HSPs are also released from intact cells via a nonclassical secretory pathway, both via “secretory lysosomes” as well as the exosomal pathway. For example, HSP70-containing exosomes have been implicated in postshock inflammation.29 Once outside the cell, free HSPs can bind to pattern-recognition receptors (PRR) as well as other cell surface receptors to modu-late the inflammatory response. Recently, the role of free HSP-mediated proinflammatory properties via TLR2 and TLR4 has been questioned, as it has been suggested that the presence of con-taminating endotoxin in bacterially-produced HSP preparations may explain at least some of these inflammatory effect results.30 However, the additional evidence


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Surgery_Schwartz. suggested that the presence of con-taminating endotoxin in bacterially-produced HSP preparations may explain at least some of these inflammatory effect results.30 However, the additional evidence suggests that the immunos-timulatory properties may be dependent on how HSPs arrive outside the cell. In the context of massive cell damage or large exosome release, HSPs may serve as proinflammatory DAMPs. In contrast, HSPs released by active secretion may exert anti-inflammatory immune dampening signals (Table 2-3).31,32 New receptors for HSP have been identified that are members of the sialic acid-binding immunoglobulin-like lectins (siglecs), which may explain these effects. Two members of the family, Siglec-5 and Siglec-14, with similar binding sites for HSP70, exhibit oppo-site intracellular events in response to HSP binding, being either pro-(Siglec-14) or anti-(Siglec-5) inflammatory.33,34From a clinical perspective, extracellular HSPs have been demonstrated to be elevated almost


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Surgery_Schwartz. events in response to HSP binding, being either pro-(Siglec-14) or anti-(Siglec-5) inflammatory.33,34From a clinical perspective, extracellular HSPs have been demonstrated to be elevated almost immediately post injury in polytraumatized patients (up to 10 times normal) with the degree of elevation being correlated with the severity of illness.35 Moreover, in the setting of polytrauma, plasma HSP70 levels have been shown to correlate inversely with HLA-DRA expression, a marker of immunosuppression.36Extracellular Matrix Molecules Act as DAMPs. Recent work has explored the role of extracellular matrix (ECM) proteins in the TLR-mediated inflammatory response that follows tissue injury. These molecules, which are sequestered under normal conditions, can be released in a soluble form with proteolytic digestion of the ECM. Proteoglycans, glycosaminoglycans, and glycoproteins such as fibronectin have all been implicated as key players in the DAMP/TLR interaction. Proteoglycans, in


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Surgery_Schwartz. with proteolytic digestion of the ECM. Proteoglycans, glycosaminoglycans, and glycoproteins such as fibronectin have all been implicated as key players in the DAMP/TLR interaction. Proteoglycans, in particu-lar, have also been shown to activate the intracellular inflamma-somes that trigger sterile inflammation. These molecules, which consist of a protein core with one or more covalently attached glycosaminoglycan chains, can be membrane-bound, secreted, or proteolytically cleaved and shed from the cell surface.Biglycan is one of the first proteoglycans to be described as a TLR ligand.37 It consists of a protein core containing leucine-rich repeat regions, with two glycosaminoglycan (GAG) side-chains (chondroitin sulfate or dermatan sulfate). While biglycan typically exists in a matrix bound form, with tissue injury it is released from the ECM in a soluble form where it interacts with TLR2 or TLR4 to generate an immediate inflammatory response.Various proinflammatory cytokines and


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Surgery_Schwartz. bound form, with tissue injury it is released from the ECM in a soluble form where it interacts with TLR2 or TLR4 to generate an immediate inflammatory response.Various proinflammatory cytokines and chemokines includ-ing tumor necrosis factor (TNF)-α and interleukin (IL)-1β are Brunicardi_Ch02_p0027-p0082.indd 3001/03/19 6:49 PM 31SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2downstream effector molecules of biglycan/TLR2/4 signaling. Among these, the mechanism of biglycan-mediated autonomous synthesis and secretion of mature IL-1β is unique. Usually, release of mature IL-1β from the cell requires two signals: one that is needed to initiate synthesis (TLR2/4-mediated), and the other to process pro-IL-1β to its mature form (inflammasome-mediated). How is it possible for biglycan to provide both signals? Current evidence indicates that when soluble biglycan binds to the TLR, it simultaneously serves as a ligand for a purinergic receptor, which facilitates the


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Surgery_Schwartz. for biglycan to provide both signals? Current evidence indicates that when soluble biglycan binds to the TLR, it simultaneously serves as a ligand for a purinergic receptor, which facilitates the inflammasome activation required for IL-1β processing.38 These data support the idea that DAMP-mediated signals can initiate a robust inflammatory response.S100 Proteins as DAMPs. S100 proteins are a group of cal-cium-binding proteins that participate in the regulation of intra-cellular calcium. There are at least 25 members identified to date, with diverse functions that are cell-type dependent. While regulation and management of calcium storage is a primary function of S100 proteins, additional specialized roles include cytoskeletal organization, protein trafficking and transcriptional regulation. They are loosely grouped according to their func-tional capability: those that work exclusively inside the cell, outside the cell, or in both locations.8,39Similar to both HMGB1 and HSP, S100


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Surgery_Schwartz. They are loosely grouped according to their func-tional capability: those that work exclusively inside the cell, outside the cell, or in both locations.8,39Similar to both HMGB1 and HSP, S100 proteins are released passively from damaged cells as well as actively via nonclassical protein secretion mechanisms. For example, S100A8/A9 (also called calprotectin) is released by activated neutrophils and macrophages, although the exact mechanisms that regulate the active release of S100 proteins remain unclear.40 In addition, S100A8/A9 secretion is linked to the release of neu-trophil extracellular traps (NETs), a fibrillar matrix of DNA and granule proteins that are extruded from activated neutrophils and that serve an antimicrobial function.41 Similar to HMGB1, S100 protein functions can be modulated by their redox state.Extracellular S100A8/A9 functions as an endogenous agonist to bind TLR4 and RAGE, serving as a strong proin-flammatory mediator. It can induce both inflammatory cytokine


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Surgery_Schwartz. by their redox state.Extracellular S100A8/A9 functions as an endogenous agonist to bind TLR4 and RAGE, serving as a strong proin-flammatory mediator. It can induce both inflammatory cytokine production and activation of leukocyte migration, as well as pro-mote apoptosis and autophagy in distinct cell types. S100A8/A9 is increased following polytrauma, and in some studies, higher levels of S100A8/A9 have been correlated with patient survival.9 More recent work in a second severely injured patient cohort (median ISS of 39) showed that the most seriously injured patients demonstrated a significantly smaller overall increase in S100A8/A9 when compared to the other patients, and that lower S100A8/A9 levels were associated with infectious risk.42A second S100 family protein, S100B, is highly expressed in astrocytes and is an important biomarker for traumatic brain injury. When assessed within 3 hours of injury, it is a highly sensitive marker that is helpful in identifying those patients


Surgery_Schwartz

Surgery_Schwartz. expressed in astrocytes and is an important biomarker for traumatic brain injury. When assessed within 3 hours of injury, it is a highly sensitive marker that is helpful in identifying those patients with mild TBI who do not require imaging.43 In a recent cohort of 100 patients with severe traumatic brain injury, serum S100B levels were significantly higher in those patients who had an unfavorable outcome 3 months post injury compared with those who had made a good recovery. The patients who died also had significantly higher S100B levels than the survivors.8,44Heme as DAMP. Heme is the oxygen-binding moiety found in hemoglobin and other hemoproteins in the muscle and mito-chondria. It is a very highly conserved molecule composed of a tetrapyrrole ring surrounding a single iron. When red blood cells are damaged, hemoglobin is released, where it is bound by plasma proteins such as haptoglobin. In turn, the hemoglobin-haptoglobin complexes are scavenged by the reticuloendothelial system


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Surgery_Schwartz. cells are damaged, hemoglobin is released, where it is bound by plasma proteins such as haptoglobin. In turn, the hemoglobin-haptoglobin complexes are scavenged by the reticuloendothelial system in the liver and spleen to form bilirubin as the ultimate end product. When the amount of free hemoglobin exceeds the binding capacity of haptoglobin and other specialized binding proteins, it is loosely bound to other plasma proteins where it can be readily oxidized. Ultimately, this can result in the release of the prosthetic heme group from hemoglobin, generating labile heme, which is a pro-oxidant.45In vitro experiments demonstrate that labile heme induces cell activation, via both TLR4-dependent processes and the inflammasome, resulting in cytokine release.46,47 Moreover, Table 2-3The immunomodulatory functions of heat shock proteins (HSPs)CELL LOCATIONRECOGNIZED AS DAMPIMMUNOMODULATORY FUNCTIONHSP90Cytoplasm, endoplasmic reticulumCan function both inside and outside the cellMay act as


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Surgery_Schwartz. functions of heat shock proteins (HSPs)CELL LOCATIONRECOGNIZED AS DAMPIMMUNOMODULATORY FUNCTIONHSP90Cytoplasm, endoplasmic reticulumCan function both inside and outside the cellMay act as DAMP chaperone to activate innate immune responseBinds and optimizes RNA polymerase II action to regulate gene transcriptionStabilizes glucocorticoid receptor in the cytoplasmImportant for processing and membrane expression of TLRChaperones include IKKFacilitates antigen presentation to dendritic cellsHSP70Can function both inside and outside the cellEndoplasmic reticulum homolog is BiPExogenous HSP70 elicits cellular calcium flux, NF-κB activation, cytokine productionCan have anti-inflammatory actions when expression is increasedInhibits TLR-mediated cytokine production via NF-κBReduces dendritic cell capacity for T-cell stimulationBiP sequesters proteins important to the unfolded protein responseHSP60MitochondriaExogenous HSP60 inhibits NF-κB activationPlays a role in intracellular protein


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Surgery_Schwartz. cell capacity for T-cell stimulationBiP sequesters proteins important to the unfolded protein responseHSP60MitochondriaExogenous HSP60 inhibits NF-κB activationPlays a role in intracellular protein traffickingModulates cytokine synthesisBiP = binding immunoglobulin protein; DAMP = damage-associated molecular pattern; IKK = IκB kinase; NF-κB, nuclear factor-κB; TLR = Toll-like receptorBrunicardi_Ch02_p0027-p0082.indd 3101/03/19 6:49 PM 32BASIC CONSIDERATIONSPART Iheme-induced neutrophils activation leads to extracellular traps (NETs) release through a mechanism dependent on reactive oxygen species.48 However, unlike the other DAMPs discussed, labile heme can also have direct cytotoxic effects on cells by a direct interaction with membrane phospholipids and the catal-ysis of membrane lipid peroxidation, leading to programmed cell death. In macrophages, labile heme can induce necroptosis, rather than apoptosis.45DAMPs Are Ligands for Pattern Recognition ReceptorsThe inflammatory


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Surgery_Schwartz. lipid peroxidation, leading to programmed cell death. In macrophages, labile heme can induce necroptosis, rather than apoptosis.45DAMPs Are Ligands for Pattern Recognition ReceptorsThe inflammatory response that occurs following traumatic injury is similar to that observed with pathogen exposure. Not surprisingly, surface and cytoplasmic receptors that medi-ate the innate immune response to microbial infection have also been implicated in the activation of sterile inflamma-tion. In support of this idea, genes have been identified that are dysregulated acutely both in response to a microbial ligand administered to human volunteers and in response to traumatic injury in a large patient population.49 The classes of receptors that are important for sensing damaged cells and cell debris are part of the larger group of germ-line encoded pattern recogni-tion receptors (PRRs). The best described ligands for these receptors are microbial components, the pathogen-associated molecular patterns


Surgery_Schwartz

Surgery_Schwartz. of the larger group of germ-line encoded pattern recogni-tion receptors (PRRs). The best described ligands for these receptors are microbial components, the pathogen-associated molecular patterns (PAMPs). The PRRs of the innate immune system are varied and include Toll-like receptors (TLRs), calcium-dependent (C-type) lectin receptors (CLRs), the nucleotide-binding domain, leucine-rich repeat–containing (NBD-LRR) proteins (NLRs; also nucleotide-binding and oligomerization domain [NOD]-like receptors), receptors for advanced glycation end-products (RAGE), and retinoic acid–inducible gene (RIG)-I-like receptors (RLRs). Following receptor ligation, intracel-lular signaling modulates transcriptional and posttranslational events necessary for host defense by coordinating the synthesis and release of cytokines and chemokines to either initiate or suppress the inflammatory response. The best described of these receptors, the TLRs, NLRs, CLRs and RAGE, are dis-cussed in the following


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Surgery_Schwartz. and release of cytokines and chemokines to either initiate or suppress the inflammatory response. The best described of these receptors, the TLRs, NLRs, CLRs and RAGE, are dis-cussed in the following section.Toll-Like Receptors. The Toll-like receptors are evolutionarily conserved type 1 transmembrane proteins that are the best-char-acterized PRRs in mammalian cells. They were first identified in Drosophila, where a mutation in the Toll gene led to its iden-tification as a key component in their immune defense against fungal infection. The first human TLR, TLR4, was identified shortly thereafter. Now, more than 10 human TLR family mem-bers have been identified, with distinct ligands that include lipid, carbohydrate, peptide, and nucleic-acid components of various pathogens. TLRs are expressed by both immune and nonimmune cells. At first, the expression of TLR was thought to be isolated to professional antigen-presenting cells such as dendritic cells and macrophages. However, mRNA for


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Surgery_Schwartz. by both immune and nonimmune cells. At first, the expression of TLR was thought to be isolated to professional antigen-presenting cells such as dendritic cells and macrophages. However, mRNA for TLR family members have been detected in most cells of myeloid lineage, as well as NK cells.50 In addition, activation of T cells increases their TLR expression and induces their survival and clonal expan-sion. Direct engagement of TLR in Treg cells promotes their expansion and reprograms them to differentiate into T helper cells, which in turn provides help to effector cells. In addition, B cells express a distinct subset of the TLR family that deter-mines their ability to respond to DAMPs; however, the signifi-cance of restricted TLR expression in these cells is not yet clear.All TLRs consist of a ligand-binding domain, char-acterized by multiple leucine-rich repeats (LRRs), and a carboxy-terminal, intracellular Toll/interleukin (IL) 1 recep-tor (TIR) domain. The LRR domains recognize


Surgery_Schwartz

Surgery_Schwartz. of a ligand-binding domain, char-acterized by multiple leucine-rich repeats (LRRs), and a carboxy-terminal, intracellular Toll/interleukin (IL) 1 recep-tor (TIR) domain. The LRR domains recognize bacterial and viral PAMPs in the extracellular environment (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11) or in the endolysosomes (TLR3, TLR7, TLR8, TLR9, and TLR10). While the role of TLRs in sepsis has been well described, more recent data indi-cate that a subset of the TLRs—TLR4 in particular—also rec-ognize DAMPs released from injured cells and tissues.51 Among the DAMP ligands for surface TLR are HMGB1, HSPs, S100 proteins, and several others. Endosomal TLR ligands include mtDNA and other mitochondrial proteins.What we know about TLR signaling events has largely been derived from the TLR-mediated response to bacterial pathogens. However, it is largely accepted that the intracellu-lar adaptors required for signal transmission by TLRs are con-served and utilized for “damage” sensing of


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Surgery_Schwartz. response to bacterial pathogens. However, it is largely accepted that the intracellu-lar adaptors required for signal transmission by TLRs are con-served and utilized for “damage” sensing of endogenous (“self”) ligands as well.52,53 The intracellular domain structure of TLRs is highly conserved and is characterized by a cytoplasmic Toll/IL-1R homology (TIR) domain. Binding of ligand to the recep-tor results in a receptor dimer, either a homodimer (e.g., TLR4/TLR4) or heterodimer (e.g., TLR2/TLR1), which recruits a number of adaptor proteins to the TIR domains through TIR-TIR interaction.54 With one exception (TLR3), the universal adaptor protein central to the TLR signaling complex is myeloid dif-ferentiation factor 88 (MyD88), a member of the interleukin-1 receptor subfamily. MyD88 works through the recruitment of a second TIR-containing adaptor, MyD88 adaptor-like protein (Mal, also termed Toll/interleukin-1 receptor-containing adaptor protein, or TIRAP) in the context of TLR4 and


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Surgery_Schwartz. through the recruitment of a second TIR-containing adaptor, MyD88 adaptor-like protein (Mal, also termed Toll/interleukin-1 receptor-containing adaptor protein, or TIRAP) in the context of TLR4 and TLR2 signaling, which serves as a bridge between MyD88 and activated TLRs to initiate signal transduction. It is interesting that Mal’s adaptor function requires cleavage of the carboxy-terminal portion of the protein by caspase-1, a key effector of the inflammasome.55 This finding suggests an important synergy between TLRs and the inflammasome that may potentiate TLR-mediated signaling.Signaling through the MyD88-dependent pathway occurs once the receptor is ligated at the cell surface.54 Receptor liga-tion, dimerization, and recruitment of the MyD88/Mal com-plex results in the activation of numerous cytoplasmic protein kinases, including IL-1 receptor–associated kinases, resulting in an interaction with tumor necrosis factor receptor–associated factor 6 (TRAF6). TRAF6, an E3 ubiquitin


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Surgery_Schwartz. numerous cytoplasmic protein kinases, including IL-1 receptor–associated kinases, resulting in an interaction with tumor necrosis factor receptor–associated factor 6 (TRAF6). TRAF6, an E3 ubiquitin ligase, forms a com-plex with two other proteins, which together activate the com-plex that subsequently phosphorylates IκB kinase (IKK)-β and the MAP kinases (MAPKs). Ultimately, the phosphorylation of IκB leads to its degradation, which frees NF-κB and allows its translocation to the nucleus and the transcription of NF-κB tar-get genes. Simultaneously, MAP kinase activation is critical for activation of the activator protein-1 (AP-1) transcription factor, and thus production of inflammatory cytokines.Two other TIR domain-containing adaptor proteins, TIR-domain-containing adapter-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM), are important to TLR-signaling events that are involved in the MyD88-independent sig-naling pathways, activated by TLR3 and TLR4. One


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Surgery_Schwartz. interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM), are important to TLR-signaling events that are involved in the MyD88-independent sig-naling pathways, activated by TLR3 and TLR4. One distinction of MyD88-dependent and -independent TLR signaling is that TLR4/TRIF transduction begins after the signaling complex is internalized into endosomes. The MyD88-independent pathway acts through TRIF to activate NF-κB, similar to the MyD88-dependent pathway. However, TRIF can also recruit other sig-naling molecules to phosphorylate interferon-regulatory factor 3 (IRF3), which induces expression of type I IFN genes.5422Brunicardi_Ch02_p0027-p0082.indd 3201/03/19 6:49 PM 33SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2The initiation of transcription by TLR activation leads to the upregulation of a large cohort of target genes that include interferons α and β (IFNα/β), nitric oxide synthase 2 (NOS2A), and tumor necrosis factor (TNF), which play critical roles in


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Surgery_Schwartz. leads to the upregulation of a large cohort of target genes that include interferons α and β (IFNα/β), nitric oxide synthase 2 (NOS2A), and tumor necrosis factor (TNF), which play critical roles in initiating innate immune responses to cellular injury and stress. Given the importance of TLR triggering of the innate immune response to immune homeostasis, it is no surprise that the pro-cess is tightly regulated. TLR signaling is controlled at multiple levels, both posttranscriptionally via ubiquitination, phosphory-lation, and micro RNA actions that affect mRNA stability, and by the localization of the TLRs and their signaling complexes within the cell.TLR expression is significantly increased following blunt traumatic injury.50,51 A recent study of patients undergoing “high-risk” surgical procedures examined immune parameters, includ-ing TLR expression, that were associated with the development of SIRS. The investigators demonstrated that patients who developed postoperative SIRS


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Surgery_Schwartz. procedures examined immune parameters, includ-ing TLR expression, that were associated with the development of SIRS. The investigators demonstrated that patients who developed postoperative SIRS exhibited increased TLR4 and TLR5 expres-sion on a subgroup of CD14+ monocytes when compared to those patients with an uneventful recovery.56 Moreover, the upregulation of TLR in these patients was associated with increased expression of IL-6. Interestingly, the authors hypothesize that preoperatively, a subset of monocytes may already be primed to act in this way and thus may identify a vulnerable patient group.Nucleotide-Binding Oligomerization Domain (NOD)-like Receptor (NLR) Family. The nucleotide-binding oligo-merization domain-like receptors (NLR) are a large family of proteins composed of intracellular PRRs that sense both endog-enous (DAMPs) and exogenous (PAMPs) molecules to trigger innate immune activation. The best characterized of the NLRs is the NLR family pyrin domain-containing


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Surgery_Schwartz. PRRs that sense both endog-enous (DAMPs) and exogenous (PAMPs) molecules to trigger innate immune activation. The best characterized of the NLRs is the NLR family pyrin domain-containing 3 (NLRP3), which is highly expressed in peripheral blood leukocytes. It forms the key “sensing” component of the larger, multiprotein inflamma-some complex, which is composed of NLRP3; the adapter pro-tein apoptosis-associated speck-like protein containing a CARD (ASC); and the effector protein, caspase 1.57 Activation of the NLRP3 inflammasome is tightly regulated, both transcription-ally and at the posttranslational level. An initial priming event (typically via TLR/nuclear factor [NF]-κB signals) upregulates NLRP3 expression. The receptor then resides in the cytoplasm in an inactive form due to an internal interaction between two adjacent domains. When phagocytosed DAMPs are sensed by NLRP3, this second event releases the self-repression. The pro-tein can then oligomerize and recruit other complex


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Surgery_Schwartz. interaction between two adjacent domains. When phagocytosed DAMPs are sensed by NLRP3, this second event releases the self-repression. The pro-tein can then oligomerize and recruit other complex members. The net result is the auto-activation of pro-caspase 1 to caspase 1. This event is pivotal to all known inflammasome signaling pathways.57,58 The caspase-1 products assemble to form the IL-1 converting enzyme (ICE), which cleaves the proforms of IL-1β, IL-18, and IL-33 to form their active, mature forms required for secretion from the cell.59The inflammasome-activated cytokines, IL-1β and IL-18, are potent proinflammatory molecules that promote key immune responses essential to host defense. Both IL-1β and IL-18 lack a signal sequence, which is usually necessary for the secretion of cellular proteins. More than 20 proteins in addition to IL-1β and IL-18 undergo unconventional protein secretion independent of the ER and Golgi, including a number of the DAMP molecules.60 Currently, the


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Surgery_Schwartz. proteins. More than 20 proteins in addition to IL-1β and IL-18 undergo unconventional protein secretion independent of the ER and Golgi, including a number of the DAMP molecules.60 Currently, the mechanisms responsible for unconventional protein secretion are not understood; however, the process is also evident in yeast under conditions of cellular stress. It makes evolutionary sense that a mechanism for rapid secretion of stored proteins essential to the stress response is highly conserved.Evidence suggests that genetic variations in the NLRP3 gene might affect the magnitude of immune inflammatory responses following trauma. Single nucleotide polymor-phisms within the NLRP3 gene were found to be associated with increased risk of sepsis and MODS in patients with major trauma.61 In an animal model of burn injury, early inflamma-some activation has been detected in a variety of immune cells (NK cells, CD4/ CD8 T cells, and B cells), as determined by the assessment of caspase 1 cleavage


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Surgery_Schwartz. model of burn injury, early inflamma-some activation has been detected in a variety of immune cells (NK cells, CD4/ CD8 T cells, and B cells), as determined by the assessment of caspase 1 cleavage by flow cytometry.62 Further, inhibition of caspase 1 activity in vivo results in increased burn mortality, suggesting that inflammasome activation may play an unanticipated protective role in the host response to injury that may be linked to increased production of specific cytokines.CNS trauma induces inflammasome activation in the ner-vous system. Moreover, exosomes containing inflammasome protein cargo are secreted into cerebral spinal fluid and can be detected in patients with TBI.63 In an animal model of TBI, controlled cortical impact, exosomes containing inflammasome proteins are detected in the serum and appear to be linked to TBI-related acute lung injury.64C-Type Lectin and Lectin-Like Receptors. Macrophages and dendritic cells possess receptors that detect molecules released from


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Surgery_Schwartz. in the serum and appear to be linked to TBI-related acute lung injury.64C-Type Lectin and Lectin-Like Receptors. Macrophages and dendritic cells possess receptors that detect molecules released from damaged or dying cells in order to retrieve and process antigens for T cell presentation. A key family of receptors that directs this process is the C-type lectin (CLR) and C-type lec-tin-like (CTLR) receptor family that includes the selectin and the mannose receptor families. CLR and CTLR bind carbohy-drates in both a calcium-dependent (CLR) and -independent (CTLR) fashion. Best described for their sensing of PAMPs, the CLRs can also act to promote the endocytosis and clear-ance of cell debris, which can be processed and presented to T cells.65,66 CTLR receptor recognition of DAMPs of intracellular origin, such as F-actin and the ribonucleoprotein SAP-130, can trigger multiple signaling pathways leading to NF-κB, type I interferon (IFN), and/or inflammasome activation. Expression of the


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Surgery_Schwartz. origin, such as F-actin and the ribonucleoprotein SAP-130, can trigger multiple signaling pathways leading to NF-κB, type I interferon (IFN), and/or inflammasome activation. Expression of the CTLR, MINCLE (macrophage-inducible C-type lectin), is increased after exposure to proinflammatory stimuli or cell stress. When MINCLE senses self-damage in association with ischemia-reperfusion injury, it promotes proinflammatory cyto-kine, chemokine, and nitric oxide production.67Receptor for Advanced Glycation End Products (RAGE). Another key player in the sterile inflammatory response to injury is the transmembrane receptor, the receptor for advanced glycation endproducts, or RAGE. Highly conserved across spe-cies, RAGE is a member of the immunoglobulin superfamily that is constitutively expressed at high levels in the lung, with low/absent expression in other adult cell types. However, pro-inflammatory stimuli and the presence of RAGE ligands can increase RAGE expression on immune cells such


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Surgery_Schwartz. at high levels in the lung, with low/absent expression in other adult cell types. However, pro-inflammatory stimuli and the presence of RAGE ligands can increase RAGE expression on immune cells such as neutrophils, macrophages, and lymphocytes.68 RAGE also exists as a solu-ble form (sRAGE) composed only of the extracellular domain, which can bind to and sequester RAGE ligands, without conse-quent signaling events.RAGE binds diverse ligands, including HMGB1 and S100, as well as components of the extracellular matrix such as collagen. As a receptor, RAGE recognizes the three-dimensional structure of its ligands that allow it to bind a diverse reper-toire of molecules, independent of their amino acid sequence. Brunicardi_Ch02_p0027-p0082.indd 3301/03/19 6:49 PM 34BASIC CONSIDERATIONSPART ISignaling via RAGE is mediated via multiple pathways lead-ing to transcriptional activation and release of proinflammatory mediators.69 Animal models have linked RAGE to acute lung injury in


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Surgery_Schwartz. via RAGE is mediated via multiple pathways lead-ing to transcriptional activation and release of proinflammatory mediators.69 Animal models have linked RAGE to acute lung injury in ischemia-reperfusion models.70 In clinical studies, high sRAGE levels have be linked to prolonged mechanical ventila-tion post lung transplant as well as worse outcomes following TBI-associated acute lung injury.42 These events likely represent a role for an HMGB1-RAGE axis in these pathologic processes.Soluble Pattern Recognition Molecules: The Pentraxins.68 Soluble pattern recognition molecules (PRMs) are a molecu-larly diverse group of molecules that share a conserved mode of action defined by complement activation, agglutination and neutralization, and opsonization. The best described of the PRMs are the pentraxins. PRMs can be synthesized at sites of injury and inflammation by macrophages and dendritic cells, while neutrophils can store PRMs and release them rapidly fol-lowing activation. In addition,


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Surgery_Schwartz. PRMs can be synthesized at sites of injury and inflammation by macrophages and dendritic cells, while neutrophils can store PRMs and release them rapidly fol-lowing activation. In addition, epithelial tissues (the liver in par-ticular) serve as a reservoir source for systemic mass release. The short pentraxin, C-reactive protein (CRP), was the first PRM to be identified. Serum amyloid protein (SAP), which has 51% sequence similarity to human CRP, also contains the pentraxin molecular signature. CRP and SAP plasma levels are low (≤3 mg/L) under normal circumstances. However, CRP is synthesized by the liver in response to interleukin-6, increasing serum levels more than a 1000-fold. Thus, CRP is considered part of the acute-phase protein response in humans. For this reason, C-reactive protein has been studied as a marker of the proinflammatory response in many clinical settings, includ-ing appendicitis, vasculitis, and ulcerative colitis. CRP and SAP are ancient immune molecules that


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Surgery_Schwartz. has been studied as a marker of the proinflammatory response in many clinical settings, includ-ing appendicitis, vasculitis, and ulcerative colitis. CRP and SAP are ancient immune molecules that share many functional properties with antibodies: they bind bacterial polysaccharides, ECM components, apoptotic cells, and nuclear materials, as well as all three classes of Fcγ receptors (FcγR). Both molecules also participate in the activation and regulation of complement pathways. In this way, short pentraxins can link immune cells to the complement system.71Finally, there is significant data to support a role for pen-traxin 3 (PTX3), a long pentraxin family member, in the “sterile” inflammatory response associated with cellular stress. While CRP is produced solely in the liver, PTX3 is produced by vari-ous cells in peripheral tissues, including immune cells. PTX3 plasma concentrations increase rapidly in various inflammatory conditions, including sepsis. Further, in a recent prospective


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Surgery_Schwartz. by vari-ous cells in peripheral tissues, including immune cells. PTX3 plasma concentrations increase rapidly in various inflammatory conditions, including sepsis. Further, in a recent prospective study of polytraumatized patients, serum PTX3 concentrations were highly elevated, peaking at 24 hours. Further, PTX3 con-centrations at admission were associated with injury severity, while higher PTX3 serum concentrations 24 hours after admis-sion correlated with lower probability for survival.72CENTRAL NERVOUS SYSTEM REGULATION OF INFLAMMATION IN RESPONSE TO INJURYThe central nervous system (CNS) communicates with the body through ordered systems of sensory and motor neurons, which receive and integrate information to generate a coordinated response. Rather than being an immune-privileged organ, recent work indicates that the CNS receives information with regard to injury-induced inflammation both via soluble media-tors as well as direct neural projections that transmit informa-tion to


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Surgery_Schwartz. recent work indicates that the CNS receives information with regard to injury-induced inflammation both via soluble media-tors as well as direct neural projections that transmit informa-tion to regulatory areas in the brain (Fig. 2-2). How does the TNFIL-1Central nervous systemACTHglucocorticoidsSensory vagusSympatheticParasympathetic(Motor vagus)AcetylcholineInjuryinflammationInflammatorycascadeInjury siteEPI, NOREPIFigure 2-2. Neural circuit relaying messages of localized injury to the brain (nucleus tractus solitarius). The brain follows with a hor-mone release (adrenocorticotropic hormone [ACTH], glucocorticoids) into the systemic circulation and by sympathetic response. The vagal response rapidly induces acetylcholine release directed at the site of injury to curtail the inflammatory response elicited by the activated immunocytes. This vagal response occurs in real time and is site specific. EPI = epinephrine; IL-1 = interleukin-1; NOREPI = norepinephrine; TNF = tumor necrosis


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Surgery_Schwartz. response elicited by the activated immunocytes. This vagal response occurs in real time and is site specific. EPI = epinephrine; IL-1 = interleukin-1; NOREPI = norepinephrine; TNF = tumor necrosis factor. (Adapted with permission from Tracey KJ: The inflammatory reflex, Nature. 2002 Dec 19-26;420(6917):853-859.)Brunicardi_Ch02_p0027-p0082.indd 3401/03/19 6:49 PM 35SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2CNS sense inflammation? DAMPs and inflammatory molecules convey stimulatory signals to the CNS via multiples routes. For example, soluble inflammatory signaling mol-ecules from the periphery can reach neurons and glial cells directly through the fenestrated endothelium of the circumven-tricular organs (CVO) or via a leaky blood-brain barrier in path-ological settings following a traumatic brain injury.73 In addition, inflammatory stimuli can interact with receptors located on the brain endothelial cells to generate a variety of proinflammatory mediators


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Surgery_Schwartz. settings following a traumatic brain injury.73 In addition, inflammatory stimuli can interact with receptors located on the brain endothelial cells to generate a variety of proinflammatory mediators (cytokines, chemokines, adhesion molecules, proteins of the complement system, and immune receptors) that directly impact the brain parenchyma. Not surprising, this response is countered by potent anti-inflammatory signaling, a portion of which is provided by the HPA axis and the release of systemic glucocorticoids. Inflammatory stimuli in the CNS result in behavioral changes, such as increased sleep, lethargy, reduced appetite, and the most common feature of infection, fever.Information regarding peripheral inflammation and tis-sue damage can also be signaled to the brain via afferent neu-ral fibers, particularly those of the vagus nerve.74 These afferent fibers can interconnect with neurons that project to the hypo-thalamus to modulate the HPA axis. In addition, afferent vagal nerve


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Surgery_Schwartz. fibers, particularly those of the vagus nerve.74 These afferent fibers can interconnect with neurons that project to the hypo-thalamus to modulate the HPA axis. In addition, afferent vagal nerve impulses modulate cells in the brain stem, at the dorsal motor nucleus of the vagus, from which efferent pregangli-onic parasympathetic originate. Axons from these cells, which comprise the visceromotor component of the vagus nerve, form an “inflammatory reflex” that feeds back to the periphery to regulate inflammatory signaling events.75 Mechanistic insight into the “inflammatory reflex” was provided by the observa-tion in several experimental model systems, that vagal stimu-lation reduced proinflammatory cytokine production from the spleen.75,76 This effect was dependent on both vagal efferent signals and on splenic catecholaminergic nerve fibers that origi-nated in the celiac plexus and terminated in the T cell–rich area of the spleen. The vagal efferent fibers that terminated within the


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Surgery_Schwartz. signals and on splenic catecholaminergic nerve fibers that origi-nated in the celiac plexus and terminated in the T cell–rich area of the spleen. The vagal efferent fibers that terminated within the celiac ganglion were found to synapse on the cell bodies of the catecholaminergic splenic nerves. Vagal stimulation resulted in the firing of these adrenergic nerves, resulting in the activation of β2-adrenergic receptors on a subset of acetylcholine (ACh)-producing T cells. The ACh released from this T cell popula-tion targets α-7 nicotinic ACh receptors (a7nAChR) expressed by splenic macrophages.77 Macrophage ACh receptor ligation blocks cell activation, inhibiting cytokine production and shifting the macrophages towards an M2 anti-inflammatory phenotype. Moreover, ACh-receptor binding inhibits intracellular signaling including the nuclear translocation of NF-κB and the activation of the inflammasome. In a rat model of hemorrhagic shock with reperfusion, vagal nerve stimulation post


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Surgery_Schwartz. intracellular signaling including the nuclear translocation of NF-κB and the activation of the inflammasome. In a rat model of hemorrhagic shock with reperfusion, vagal nerve stimulation post injury resulted in a decrease in the inflammatory response to hemorrhage.78Neuroendocrine Response to InjuryTraumatic injury results in complex neuroendocrine signaling from the brain that serves to enhance immune defense and rap-idly mobilize substrates necessary to meet essential energy and structural needs. The two principle neuroendocrine pathways that orchestrate the host response are the hypothalamic-pitu-itary-adrenal (HPA) axis, which results in the release of gluco-corticoid hormones, and the sympathetic nervous system, which results in release of the catecholamines, epinephrine (EPI), and norepinephrine (EPI). Virtually every hormone of the HPA axis influences the physiologic response to injury and stress (Table 2-4), but some with direct influence on the inflammatory 3Table 2-4Hormones


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Surgery_Schwartz. (EPI). Virtually every hormone of the HPA axis influences the physiologic response to injury and stress (Table 2-4), but some with direct influence on the inflammatory 3Table 2-4Hormones regulated by the hypothalamus, pituitary, and autonomic systemHypothalamic RegulationCorticotropin-releasing hormoneThyrotropin-releasing hormoneGrowth hormone–releasing hormoneLuteinizing hormone–releasing hormoneAnterior Pituitary RegulationAdrenocorticotropic hormoneCortisolThyroid-stimulating hormoneThyroxineTriiodothyronineGrowth hormoneGonadotrophinsSex hormonesInsulin-like growth factorSomatostatinProlactinEndorphinsPosterior Pituitary RegulationVasopressinOxytocinAutonomic SystemNorepinephrineEpinephrineAldosteroneRenin-Angiotensin SystemInsulinGlucagonEnkephalinsresponse or immediate clinical impact are highlighted here, including growth hormone (GH), macrophage inhibitory factor (MIF), aldosterone, and insulin.The Hypothalamic-Pituitary-Adrenal Axis. One of the main mechanisms by which the


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Surgery_Schwartz. are highlighted here, including growth hormone (GH), macrophage inhibitory factor (MIF), aldosterone, and insulin.The Hypothalamic-Pituitary-Adrenal Axis. One of the main mechanisms by which the brain responds to injury-associated stress is through activation of the hypothalamic-pituitary-adrenal (HPA) axis. Following injury, corticotrophin-releasing hormone (CRH) is secreted from the paraventricular nucleus (PVN) of the hypothalamus. This action is mediated in part by circulating cytokines produced as a result of the innate immune response to injury. These include tumor necrosis factor-α (TNF-α) IL-1β, IL-6, and the type I interferons (IFN-α/β). Cytokines that are produced as a result of the adaptive immune response (IL-2 and IFN-γ) are also capable of increasing cortisol release. Direct neu-ral input via afferent vagal fibers that interconnect with neurons projecting to the hypothalamus can also trigger CRH release. CRH acts on the anterior pituitary to stimulate the secretion of


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Surgery_Schwartz. neu-ral input via afferent vagal fibers that interconnect with neurons projecting to the hypothalamus can also trigger CRH release. CRH acts on the anterior pituitary to stimulate the secretion of adrenocorticotropin hormone (ACTH) into the systemic circula-tion. Interestingly, the cytokines that act on the hypothalamus are also capable of stimulating ACTH release from the anterior pituitary so that marked elevations in ACTH and in cortisol can occur that are proportional in magnitude to the injury severity. Additionally, pain, anxiety, vasopressin, angiotensin II, chole-cystokinin, vasoactive intestinal peptide, and catecholamines all contribute to ACTH release in the injured patient.Brunicardi_Ch02_p0027-p0082.indd 3501/03/19 6:49 PM 36BASIC CONSIDERATIONSPART IACTH acts on the zona fasciculate of the adrenal glands to synthesize and secrete glucocorticoids (Fig. 2-3). Cortisol is the major glucocorticoid in humans and is essential for survival during significant physiologic


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Surgery_Schwartz. fasciculate of the adrenal glands to synthesize and secrete glucocorticoids (Fig. 2-3). Cortisol is the major glucocorticoid in humans and is essential for survival during significant physiologic stress. The resulting increase in cortisol levels following trauma have several important anti-inflammatory actions.Cortisol elicits its many actions through a cytosolic recep-tor, the glucocorticoid receptor (GR). Because it is lipid soluble, cortisol can diffuse through the plasma membrane to interact with its receptor, which is sequestered in the cytoplasm in a com-plex with heat shock proteins (Fig. 2-4). Upon ligand binding, the GR is activated and can employ a number of mechanisms to modulate proinflammatory gene transcription and signaling events, with a “net” anti-inflammatory effect.79 For example, the activated GR complex can interact with transcription factors to sequester them in the cytoplasm, promote their degradation, or inhibit them through other mechanisms. Affected target


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Surgery_Schwartz. For example, the activated GR complex can interact with transcription factors to sequester them in the cytoplasm, promote their degradation, or inhibit them through other mechanisms. Affected target genes include proinflammatory cytokines, growth factors, adhesion molecules, and nitric oxide. In addition, glucocorticoids can negatively affect the access of the transcription factor, nuclear factor-κB (NF-κB), to the promoter regions of its target genes via a mechanism that involves histone deacetylase 2. In this way, glucocorticoids can inhibit a major mechanism by which TLR ligation induces proinflammatory gene expression.80 The GR complex can also bind to specific nucleotide sequences (termed glucocorticoid response elements) to promote the 17-˜-OH-progesterone11-DeoxycortisolCortisolCholesterolACTHPregnenolone17-˜-OH-PregnenoloneDehydroepiandrosteroneAndrostenedioneTestosteroneEstradiolSex


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Surgery_Schwartz. response elements) to promote the 17-˜-OH-progesterone11-DeoxycortisolCortisolCholesterolACTHPregnenolone17-˜-OH-PregnenoloneDehydroepiandrosteroneAndrostenedioneTestosteroneEstradiolSex steroidsGlucocorticoid11-DeoxycorticosteroneCorticosteroneAldosteroneMineralocorticoidProgesteroneFigure 2-3. Steroid synthesis from cholesterol. Adrenocorticotropic hormone (ACTH) is a principal regulator of steroid synthesis. The end products are mineralocorticoids, glucocorticoids, and sex steroids.HSPRProtein synthesisCytoplasmic membraneDNAmRNASRSSSSSSHSPRSNucleusFigure 2-4. Simplified schematic of steroid transport into the nucleus. Steroid molecules (S) diffuse readily across cytoplasmic membranes. Intracellularly, the receptors (R) are rendered inactive by being coupled to heat shock protein (HSP). When S and R bind, HSP dissoci-ates, and the S-R complex enters the nucleus, where the S-R complex induces DNA transcription, resulting in protein synthesis. mRNA = messenger


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Surgery_Schwartz. shock protein (HSP). When S and R bind, HSP dissoci-ates, and the S-R complex enters the nucleus, where the S-R complex induces DNA transcription, resulting in protein synthesis. mRNA = messenger RNA.Brunicardi_Ch02_p0027-p0082.indd 3601/03/19 6:49 PM 37SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2transcription of genes, which have anti-inflammatory functions. These include interleukin-10 and interleukin-1-receptor antago-nists. Further, GR complex activation can indirectly influence TLR activity via an interaction with signaling pathways such as the mitogen-activated protein kinase and transforming growth factor–activated kinase-1 (TAK1) pathways. Finally, a recent report demonstrated that GCs target suppressor of cytokine signaling 1 (SOCS1) and type 1 interferons to regulate TLR-induced signaling events.81Adrenal insufficiency represents a clinical syndrome high-lighted largely by inadequate amounts of circulating cortisol and aldosterone. Classically, adrenal


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Surgery_Schwartz. regulate TLR-induced signaling events.81Adrenal insufficiency represents a clinical syndrome high-lighted largely by inadequate amounts of circulating cortisol and aldosterone. Classically, adrenal insufficiency is described in patients with atrophic adrenal glands caused by exogenous steroid administration who undergo a stressor such as surgery. These patients subsequently manifest signs and symptoms such as tachycardia, hypotension, weakness, nausea, vomiting, and fever. However, it is now apparent that severe traumatic injury associated with an extended proinflammatory response can increase the risk of critical illness–related corticosteroid insuf-ficiency, or CIRCI.In the postinjury setting, CIRCI describes a phenomenon in which an exaggerated proinflammatory response is associated with a blunted adrenocortical response.82 Factors that have been linked to CIRCI include dysregulation of the HPA axis with altered adrenal synthesis of cortisol, altered cortisol metabo-lism, and


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Surgery_Schwartz. with a blunted adrenocortical response.82 Factors that have been linked to CIRCI include dysregulation of the HPA axis with altered adrenal synthesis of cortisol, altered cortisol metabo-lism, and tissue resistance to corticosteroids with inadequate glucocorticoid receptor activity. As a consequence, cortisol levels prove insufficient for the severity of stress. Investigators have determined that CIRCI in trauma patients occurs more fre-quently than previously thought.83 In one recent study, CIRCI occurred in 38 of 70 patients with multiple injuries. In most cases, the diagnosis was made within the first 48 hours follow-ing injury.84 Laboratory findings in adrenal insufficiency include hypoglycemia from decreased gluconeogenesis, hyponatremia from impaired renal tubular sodium resorption, and hyperka-lemia from diminished kaliuresis. Recommended guidelines to diagnose CIRCI include measuring delta cortisol (change in baseline cortisol at 60 min of <9 µg/dL) after cosyntropin (250 µg)


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Surgery_Schwartz. and hyperka-lemia from diminished kaliuresis. Recommended guidelines to diagnose CIRCI include measuring delta cortisol (change in baseline cortisol at 60 min of <9 µg/dL) after cosyntropin (250 µg) administration and a random plasma cortisol of <10 µg/dL. Treatment strategies remain controversial in the set-ting of trauma.85,86Macrophage Migration Inhibitory Factor Modulates Cortisol Function. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed by a variety of cells and tissues, including the anterior pituitary, macrophages and T lymphocytes. MIF is also classified as an atypical chemo-kine that binds to the CXC4 receptor.87 Several important func-tions of MIF in innate and adaptive immune responses and in inflammation have been described supporting the idea that MIF may function to counteract the anti-inflammatory activity of glucocorticoids.88 For example, MIF has been reported to play a central role in the exacerbation of inflammation associated


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Surgery_Schwartz. idea that MIF may function to counteract the anti-inflammatory activity of glucocorticoids.88 For example, MIF has been reported to play a central role in the exacerbation of inflammation associated with acute lung injury, where it has been detected in the affected lungs and in alveolar macrophages. MIF has also been reported to upregulate the expression of Toll-like receptor 4 (TLR4) in macrophages,89 and an early increase in plasma MIF has been detected in severely injured patients and was found to corre-late with NF-κB translocation and respiratory burst in PMNs derived from severely injured patients. Further, nonsurvivors were shown to have higher serum MIF concentrations early after injury than survivors.90 These data suggest that targeting MIF with available small molecule inhibitors may be a novel therapeutic strategy for preventing early PMN activation and subsequent organ failure in severely injured patients.Growth Hormone, Insulin-Like Growth Factor, and Ghrelin. Growth


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Surgery_Schwartz. may be a novel therapeutic strategy for preventing early PMN activation and subsequent organ failure in severely injured patients.Growth Hormone, Insulin-Like Growth Factor, and Ghrelin. Growth hormone (GH) is a neurohormone expressed primarily by the pituitary gland that has both metabolic and immunomodulatory effects. GH promotes both protein synthe-sis and insulin resistance while enhancing the mobilization of fat stores. GH secretion is upregulated by hypothalamic GH–releasing hormone and downregulated by somatostatin. GH pri-marily exerts its downstream effects through direct interaction with GH receptors and through the enhanced hepatic synthesis of insulin-like growth factor-1 (IGF-1), an anabolic growth fac-tor that is known to improve the metabolic rate, gut mucosal function, and protein loss after traumatic injury. Less than 5% of IGF-1 circulates free in the plasma, with the remainder bound principally to one of six IGF-binding proteins (IGFBPs), the majority to IGFBP-3.


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Surgery_Schwartz. protein loss after traumatic injury. Less than 5% of IGF-1 circulates free in the plasma, with the remainder bound principally to one of six IGF-binding proteins (IGFBPs), the majority to IGFBP-3. In the liver, IGF-1 stimulates protein syn-thesis and glycogenesis; in adipose tissue, it increases glucose uptake and lipid utilization; and in skeletal muscles, it mediates glucose uptake and protein synthesis. In addition to its effects on cellular metabolism, GH enhances phagocytic activity of immu-nocytes through increased lysosomal superoxide production. It also increases the proliferation of T-cell populations.91The catabolic state that follows severe injury has been linked to the suppression of the growth hormone-IGF-IGFBP axis, as critical illness is associated with decreased circulat-ing IGF-1 levels. Not surprising, the administration of exog-enous recombinant human GH (rhGH) has been studied in a prospective, randomized trial of critically ill patients where it was associated


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Surgery_Schwartz. IGF-1 levels. Not surprising, the administration of exog-enous recombinant human GH (rhGH) has been studied in a prospective, randomized trial of critically ill patients where it was associated with increased mortality, prolonged ventilator dependence, and increased susceptibility to infection.92 More recently, circulating GH levels were examined on admission in 103 consecutive critically ill adult patients. In this study, cir-culating GH levels were increased by about sevenfold in the 24 nonsurvivors when compared with survivors, and they were an independent predictor of mortality, along with APACHE II/SAPS II scores. In distinct contrast, the effect of rhGH adminis-tration in severely burned children, both acutely and following prolonged treatment, has been proven to be beneficial. Pediatric burn patients receiving rhGH demonstrated markedly improved growth and lean body mass, while hypermetabolism was signifi-cantly attenuated.93 This finding was associated with significant


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Surgery_Schwartz. Pediatric burn patients receiving rhGH demonstrated markedly improved growth and lean body mass, while hypermetabolism was signifi-cantly attenuated.93 This finding was associated with significant increases in serum GH, IGF-I, and IGFBP-3.Ghrelin, a natural ligand for the GH-secretagogue receptor 1a (GHS-R1a), is an appetite stimulant that is secreted by the stomach. GHS-R1a is expressed in a variety of tissues in differ-ent concentrations including the immune cells, Band T-cells, and neutrophils. Ghrelin seems to play a role in promoting GH secretion, and in glucose homeostasis, lipid metabolism, and immune function. In a rodent gut ischemia/reperfusion model, ghrelin administration inhibits proinflammatory cytokine release, reduces neutrophil infiltration, ameliorates intestinal barrier dysfunction, attenuates organ injury, and improves sur-vival. It is interesting that this effect was dependent on an intact vagus nerve, and that intracerebroventricular injection of ghre-lin was


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Surgery_Schwartz. dysfunction, attenuates organ injury, and improves sur-vival. It is interesting that this effect was dependent on an intact vagus nerve, and that intracerebroventricular injection of ghre-lin was also protective.94 These data suggest that the effect of ghrelin is mediated via the central nervous system, most likely through the “cholinergic anti-inflammatory pathway.” High ghrelin levels were demonstrated in critically ill patients as compared to healthy controls, independent of the presence of Brunicardi_Ch02_p0027-p0082.indd 3701/03/19 6:49 PM 38BASIC CONSIDERATIONSPART Iinflammatory markers. Moreover, the high ghrelin levels were a positive predictor of ICU-survival in septic patients, match-ing previous results from animal models. Based on these data, ghrelin seems to exert anti-inflammatory effects that are medi-ated by diverse pathways. Recent work has linked ghrelin to a novel pathway mediated by upregulation of uncoupling protein 2 (UCP2) particularly in the setting of


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Surgery_Schwartz. effects that are medi-ated by diverse pathways. Recent work has linked ghrelin to a novel pathway mediated by upregulation of uncoupling protein 2 (UCP2) particularly in the setting of traumatic brain injury.95The Role of Catecholamines in Postinjury Inflammation. Injury-induced activation of the sympathetic nervous system results in secretion of acetylcholine from the preganglionic sympathetic fibers innervating the adrenal medulla. The adre-nal medulla is a special case of autonomic innervation and is considered a modified postganglionic neuron. Thus, acetyl-choline signaling to the resident chromaffin cells ensures that a surge of epinephrine (EPI) and norepinephrine (NE) release into the circulation takes place in a ratio that is tightly regulated by both central and peripheral mechanisms. Circulating levels of EPI and NE are threeto fourfold elevated, an effect that per-sists for an extended time. The release of EPI can be modulated by transcriptional regulation of


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Surgery_Schwartz. mechanisms. Circulating levels of EPI and NE are threeto fourfold elevated, an effect that per-sists for an extended time. The release of EPI can be modulated by transcriptional regulation of phenylethanolamine N-methyl-transferase (PNMT), which catalyzes the last step of the cat-echolamine biosynthesis pathway methylating NE to form EPI. PNMT transcription, a key step in the regulation of epinephrine production, is activated in response to stress and tissue hypoxia by hypoxia-inducible factor 1α (HIF1A).Catecholamine release almost immediately prepares the body for the “fight or flight” response with well-described effects on the cardiovascular and pulmonary systems, and on metabolism. These include increased heart rate, myocardial contractility, conduction velocity, and blood pressure; the redi-rection of blood flow to skeletal muscle; increased cellular metabolism throughout the body; and mobilization of glucose from the liver via glycogenolysis, gluconeogenesis, lipolysis, and


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Surgery_Schwartz. the redi-rection of blood flow to skeletal muscle; increased cellular metabolism throughout the body; and mobilization of glucose from the liver via glycogenolysis, gluconeogenesis, lipolysis, and ketogenesis. To compound the resulting hyperglycemia, insulin release is decreased mainly through the stimulation of α-adrenergic pancreatic receptors. Hyperglycemia, as will be discussed, contributes to the proinflammatory response and to further mitochondrial dysfunction.The goal of this well-orchestrated catecholamine response is to reestablish and maintain the systems’ homeostasis, includ-ing the innate immune system. Circulating catecholamines can directly influence inflammatory cytokine production.96 Data indicate that basal EPI levels condition the activity and respon-siveness of cytokine-secreting cells, which may explain large inter-individual variability in innate cytokine profiles observed following injury. Epinephrine infusion at higher doses has been found to inhibit production


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Surgery_Schwartz. cells, which may explain large inter-individual variability in innate cytokine profiles observed following injury. Epinephrine infusion at higher doses has been found to inhibit production of tumor necrosis factor (TNF)-alpha in vivo and to enhance the production of the anti-inflammatory cytokine interleukin IL-10.97 Additionally, in vitro studies indi-cate that stress levels of glucocorticoids and epinephrine, acting in concert, can inhibit production of IL-12, a potent stimulator of Th1 responses. Further, they have been shown in vitro to decrease Th1 cytokine production and increase Th2 cytokine production to a significantly greater degree compared to either adrenal hormone alone. Thus, catecholamines secreted from the adrenal, specifically epinephrine, play a role in both innate proinflammatory cytokine regulation, as well as adaptive Th responses, and may act in concert with cortisol during the injury response to modulate cytokine activity.98How are these effects explained? It is


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Surgery_Schwartz. cytokine regulation, as well as adaptive Th responses, and may act in concert with cortisol during the injury response to modulate cytokine activity.98How are these effects explained? It is well established that a variety of human immune cells (e.g., mononuclear cells, macrophages, and granulocytes) express adrenergic receptors that are members of the family of G-protein coupled recep-tors that act through the activation of intracellular second mes-sengers such as cAMP and calcium ions influx (discussed in more detail in the following section). These second messengers can regulate a variety of immune cell functions, including the release of inflammatory cytokines and chemokines.The sympathetic nervous system also has direct immune-modulatory properties via its innervation of lymphoid tissues that contain resting and activated immune cells. The close prox-imity of sympathetic nerve terminals to immune cells responding to antigens (e.g., in the spleen) allows for a high concentration of


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Surgery_Schwartz. that contain resting and activated immune cells. The close prox-imity of sympathetic nerve terminals to immune cells responding to antigens (e.g., in the spleen) allows for a high concentration of norepinephrine to be localized within the microenvironment of antigen-activated immune cells. Norepinephrine can then interact with b2-adrenergic receptors expressed by CD4+ T and B lymphocytes, many of which also express α2-adrenergic receptors. Additionally, endogenous catecholamine expression has been detected in these cells (both CD4+ CD25+ T cells and phagocytes) as has the machinery for catecholamine synthesis. For example, monocytes contain inducible mRNA for the catecholamine-generating enzymes, tyrosine-hydroxylase, and dopamine-b-hydroxylase, and there is data to suggest that cells can regulate their own catecholamine synthesis in response to extracellular cues. Immune cell release of NE provides a way in which cells may exert additional regulation of inflammatory cell activation.


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Surgery_Schwartz. regulate their own catecholamine synthesis in response to extracellular cues. Immune cell release of NE provides a way in which cells may exert additional regulation of inflammatory cell activation. For example, mature dendritic cells express both functional αand b-adrenergic receptor (AR) types, as do monocytes and monocyte-derived macrophages, whereas B cells and Th1 cells express b2-AR exclusively.99 Exposure of PBMCs to NE triggers a distinct genetic profile that indicates a modulation of Th cell function. Thus, stimulation of AR results in varied signaling events to regulate both immune cell pheno-type as well as mature cell function.100Aldosterone. Aldosterone is a mineralocorticoid released by the zona glomerulosa of the adrenal cortex. It binds to the mineralocorticoid receptor (MR) of principal cells in the col-lecting duct of the kidney where it can stimulate expression of genes involved in sodium reabsorption and potassium excretion to regulate extracellular volume and


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Surgery_Schwartz. (MR) of principal cells in the col-lecting duct of the kidney where it can stimulate expression of genes involved in sodium reabsorption and potassium excretion to regulate extracellular volume and blood pressure. Mineralo-corticoid receptors (MR) have also been shown to have effects on cell metabolism and immunity. For example, recent studies show aldosterone interferes with insulin signaling pathways and reduces expression of the insulin-sensitizing factors, adiponec-tin and peroxisome proliferator activated receptor−γ (PPAR-γ), which contribute to insulin resistance. In the immune system, monocytes, lymphocytes, dendritic cells, and neutrophils have all been shown to possess a MR that binds aldosterone with high specificity, regulating sodium and potassium flux, as well as plasminogen activator inhibitor-1 and p22 phox expression in these cells.101 In dendritic cells, MR activation by aldoste-rone induces the secretion of proinflammatory cytokines. Fur-ther, aldosterone inhibits


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Surgery_Schwartz. activator inhibitor-1 and p22 phox expression in these cells.101 In dendritic cells, MR activation by aldoste-rone induces the secretion of proinflammatory cytokines. Fur-ther, aldosterone inhibits cytokine-mediated NF-κB activation in neutrophils, which also possess a functional MR.Insulin. Hyperglycemia and insulin resistance are hallmarks of injury and critical illness due to the catabolic effects of circu-lating mediators, including catecholamines, cortisol, glucagon, and growth hormone. The increase in these circulating progly-cemic factors, particularly epinephrine, induces glycogenoly-sis, lipolysis, and increased lactate production independent of available oxygen in a process that is termed “aerobic glycoly-sis.” Although there is an increase in insulin production at the Brunicardi_Ch02_p0027-p0082.indd 3801/03/19 6:49 PM 39SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2same time, severe stress is frequently associated with insulin resistance, leading to


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Surgery_Schwartz. 3801/03/19 6:49 PM 39SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2same time, severe stress is frequently associated with insulin resistance, leading to decreased glucose uptake in the liver and the periphery contributing to acute hyperglycemia. Insulin is a hormone secreted by the pancreas, which mediates an overall host anabolic state through hepatic glycogenesis and glycolysis, peripheral glucose uptake, lipogenesis, and protein synthesis.102The insulin receptor (IR) is widely expressed and con-sists of two isoforms, which can form homoor hetero-dimers with insulin binding. Dimerization leads to receptor autophos-phorylation and activation of intrinsic tyrosine kinase activity. Downstream signaling events are dependent on the recruitment of the adaptor proteins, insulin receptor substrate (IRS-1), and Shc to the IR. Systemic insulin resistance likely results from proinflammatory signals, which modulate the phosphorylation of IRS-1 to affect its


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Surgery_Schwartz. proteins, insulin receptor substrate (IRS-1), and Shc to the IR. Systemic insulin resistance likely results from proinflammatory signals, which modulate the phosphorylation of IRS-1 to affect its function.Hyperglycemia during critical illness is predictive of increased mortality in critically ill trauma patients.103 It can modulate the inflammatory response by altering leukocyte func-tions and the resulting decreases in phagocytosis, chemotaxis, adhesion, and respiratory burst activities are associated with an increased risk for infection. In addition, glucose administration results in a rapid increase in NF-κB activation and proinflamma-tory cytokine production. Insulin therapy to manage hypergly-cemia has grown in favor and has been shown to be associated with both decreased mortality and a reduction in infectious complications in select patient populations. However, the trend towards tight glycemic control in the intensive care unit failed to show benefit when examined in several


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Surgery_Schwartz. and a reduction in infectious complications in select patient populations. However, the trend towards tight glycemic control in the intensive care unit failed to show benefit when examined in several reviews.104 Thus, the ideal blood glucose range within which to maintain critically ill patients and to avoid hypoglycemia has yet to be determined.THE CELLULAR STRESS RESPONSESReactive Oxygen Species and the Oxidative Stress ResponseReactive oxygen and nitrogen species (ROS, RNS, respectively) are small molecules that are highly reactive due to the presence of unpaired outer orbit electrons. They can cause cellular injury to host cells and invading pathogens through the oxidation of cell membrane substrates, cellular proteins, and DNA. ROS has also been shown to have important roles as signaling messengers, particularly in the immune system.105,106Oxygen radicals (superoxide anion, hydroxyl radical, hydrogen peroxide) are produced as a by-product of oxygen metabolism. The main areas of


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Surgery_Schwartz. messengers, particularly in the immune system.105,106Oxygen radicals (superoxide anion, hydroxyl radical, hydrogen peroxide) are produced as a by-product of oxygen metabolism. The main areas of ROS production are oxida-tive processes involving the mitochondrial electron transport chain as well as those mediated by NADPH oxidases (NOX), a large class of ROS producing enzymes. Additional metabolic enzymes such as lipoxygenases, cytochrome P-450 and b5, and cyclooxygenases also produce ROS as by-products of their reactions.107 The synthesis of ROS is regulated at several check-points and via complex signaling mechanisms, including Ca2+ signaling, phosphorylation, and small G protein activation, which influence both the recruitment of the molecules required for NOX function and the synthesis of ROS in the mitochondria. Not surprisingly, NOX activation is triggered by a number of inflammatory mediators (e.g., TNF, chemokines, lysophospho-lipids, complement, and leukotrienes).Host cells are


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Surgery_Schwartz. ROS in the mitochondria. Not surprisingly, NOX activation is triggered by a number of inflammatory mediators (e.g., TNF, chemokines, lysophospho-lipids, complement, and leukotrienes).Host cells are protected from the damaging effects of ROS through a number of mechanisms. The best described of these is via the upregulation and/or activation of endogenous antioxidant enzymes such as superoxide dismutases, catalases, and glutaredoxins. Pyruvate kinase also provides negative feed-back for ROS synthesis as do molecules that react nonenzy-matically with ROS. Under normal physiologic conditions, ROS production is balanced effectively by these antioxidative strate-gies. As a consequence, ROS can act as signaling molecules through their ability to modulate cysteine residues by oxidation, and thus influence the functionality of target proteins.108 ROS can also contribute to transcriptional activity both indirectly through its effects on transcription factor lifespan, and directly through the


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Surgery_Schwartz. influence the functionality of target proteins.108 ROS can also contribute to transcriptional activity both indirectly through its effects on transcription factor lifespan, and directly through the oxidation of DNA.The role for ROS has been well described in phagocytes, which utilize these small molecules for pathogen killing. A sec-ond important role for ROS is in the regulation of the inflamma-some. As discussed previously, the inflammasome mediates the activation of inflammatory capsases leading to the production and secretion of mature cytokines in macrophages.109 Impor-tantly, the best described inflammasome, NLRP3, is redox sen-sitive. Increased intracellular ROS enables the assembly of the protein complex.110 ROS also appears to be involved in adap-tive immunity by influencing immune cell response.106 ROS can alter thiol group oxidative states on the cell surface and, in turn, affect cell signaling. Moreover, intracellular ROS can inhibit DNA transcription. ROS has been


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Surgery_Schwartz. immune cell response.106 ROS can alter thiol group oxidative states on the cell surface and, in turn, affect cell signaling. Moreover, intracellular ROS can inhibit DNA transcription. ROS has been described as a prime source of phosphatase activation in both B and T lymphocytes, which can regulate the function of key receptors and intracellu-lar signaling molecules in these cells by affecting phosphoryla-tion events. Finally, large amounts of ROS cannot only suppress cell function, but also can result in cell death.111The Unfolded Protein ResponseSecreted, membrane-bound, and organelle-specific proteins fold in the lumen of the endoplasmic reticulum (ER) where they also receive their posttranslational modifications. Cellular stress dis-rupts the quality control required for this process leading to the accumulation of misfolded or unfolded proteins. These occur-rences are sensed by a highly conserved array of signaling pro-teins in the ER that try to reestablish appropriate folding,


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Surgery_Schwartz. leading to the accumulation of misfolded or unfolded proteins. These occur-rences are sensed by a highly conserved array of signaling pro-teins in the ER that try to reestablish appropriate folding, while at the same time decreasing protein synthesis.112 The important proteins involved in this process include inositol requiring enzyme 1 (IRE1), protein kinase RNA (PKR)–like ER kinase (PERK), and activating transcription factor 6 (ATF6). Together, these proteins form a complex that generates the unfolded pro-tein response (UPR). The UPR is a mechanism by which ER distress signals are sent to the nucleus to modulate transcription in an attempt to restore homeostasis. While obviously important to secretory epithelial cells, the UPR is also important to cells of the immune system.113Significant protein misfolding results in an alarm signal that, if not addressed, can result in cell death. Genes activated in the UPR result not only in the inhibition of translation, but also other


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Surgery_Schwartz. protein misfolding results in an alarm signal that, if not addressed, can result in cell death. Genes activated in the UPR result not only in the inhibition of translation, but also other potentially immunomodulatory events including induction of the acute phase response, activation of NF-κB, and the generation of antibody-producing B cells.114 Activation of the UPR is also an alternative mechanism for activation of the inflammasome115 and can increase proinflammatory cytokine production.116Markers of ER stress during critical illness have been demonstrated conclusively in burn patients,114,117 and in animal models they have been detected following hemorrhagic shock, correlating with the degree of organ dysfunction. Burn injury in particular leads to the marked reduction in ER calcium levels 4Brunicardi_Ch02_p0027-p0082.indd 3901/03/19 6:49 PM 40BASIC CONSIDERATIONSPART Iand activation of UPR sensing proteins. Moreover, recent data in a series of burn patients strongly links the


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Surgery_Schwartz. 4Brunicardi_Ch02_p0027-p0082.indd 3901/03/19 6:49 PM 40BASIC CONSIDERATIONSPART Iand activation of UPR sensing proteins. Moreover, recent data in a series of burn patients strongly links the UPR to insulin resistance and hyperglycemia in these patients.117 Thus, a better understanding of the UPR, which is triggered by severe inflam-mation, may allow the identification of novel therapeutic targets for injury-associated insulin resistance.118Fibroblast growth factor-21 (FGF21), a recently identi-fied hormone that regulates systemic metabolic homeostasis, is upregulated following mitochondrial damage and may be part of an integrated stress response that includes ER stress and the UPR.119 In animal models, induction of ER stress with chemical ER stressors results in increased FGF21 expression. A recent study examining FGF21 in critically ill patients demonstrated that serum FGF21 concentrations were eight-fold higher in the critically ill patients as compared with the matched


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Surgery_Schwartz. expression. A recent study examining FGF21 in critically ill patients demonstrated that serum FGF21 concentrations were eight-fold higher in the critically ill patients as compared with the matched controls, regardless of the presence of sepsis. While FGF21 concentrations gradually decreased over time, they remained highly elevated at all studied time points and cor-related with patient mortality.120 These data support the idea that the UPR may play an important role in the response to severe injury.AutophagyUnder normal circumstances, cells need to have a way of dis-posing of damaged organelles and debris aggregates that are too large to be managed by proteosomal degradation. In order to accomplish this housekeeping task, cells utilize a process referred to as “macroautophagy” (autophagy), which is thought to have originated as a stress response.121 The steps of autoph-agy include the engulfment of cytoplasm/organelle by an “isola-tion membrane,” which is also called a phagophore.


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Surgery_Schwartz. which is thought to have originated as a stress response.121 The steps of autoph-agy include the engulfment of cytoplasm/organelle by an “isola-tion membrane,” which is also called a phagophore. The edges of the phagophore then fuse to form the autophagosome, a dou-ble-membraned vesicle that sequesters the cytoplasmic material and is a characteristic feature of autophagy. The autophagosome then fuses with a lysosome to form an autolysosome, where the contents, together with the inner membrane, are degraded. This process is controlled by numerous autophagy-specific genes and by the specific kinase, mammalian target of rapamycin (mTOR).As noted previously, autophagy is a normal cellular pro-cess that occurs in quiescent cells for cellular maintenance. However, under conditions of hypoxia and low cellular energy, autophagy is induced in an attempt to provide additional nutri-ents for energy production. The induction of autophagy pro-motes a shift from aerobic respiration to glycolysis


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Surgery_Schwartz. and low cellular energy, autophagy is induced in an attempt to provide additional nutri-ents for energy production. The induction of autophagy pro-motes a shift from aerobic respiration to glycolysis and allows cellular components of the autophagosome to be hydrolyzed to energy substrates. Increased levels of autophagy are typical in activated immune cells and are a mechanism for the disposal of ROS and phagocytosed debris.Recent data support the idea that autophagy plays an important role in the immune response.122 Autophagy is stimu-lated by Th1 cytokines and with activation of TLR in macro-phages but is inhibited by Th2 cytokines. It is also recognized as an important regulator of cytokine secretion, particularly those cytokines of the IL-1 family that are dependent on inflamma-some processing for activation. For example, autophagosomes can sequester and degrade pro-IL-1β and inflammasome com-ponents. In animal models of sepsis, inhibition of autophagy results in increased


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Surgery_Schwartz. processing for activation. For example, autophagosomes can sequester and degrade pro-IL-1β and inflammasome com-ponents. In animal models of sepsis, inhibition of autophagy results in increased proinflammatory cytokine levels that corre-late with increased mortality.123 These data suggest that autoph-agy is a protective mechanism whereby the cell can regulate the levels of cytokine production.ApoptosisApoptosis (regulated cell death) is an energy-dependent, orga-nized mechanism for clearing senescent or dysfunctional cells, including macrophages, neutrophils, and lymphocytes, without promoting an inflammatory response. This contrasts with cel-lular necrosis, which results in a disorganized sequence of intra-cellular molecular releases with subsequent immune activation and inflammatory response. Systemic inflammation modulates apoptotic signaling in active immunocytes, which subsequently influences the inflammatory response through the loss of effec-tor cells.Apoptosis proceeds


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Surgery_Schwartz. response. Systemic inflammation modulates apoptotic signaling in active immunocytes, which subsequently influences the inflammatory response through the loss of effec-tor cells.Apoptosis proceeds primarily through two pathways: the extrinsic pathway and the intrinsic pathway. The extrinsic path-way is activated through the binding of death receptors (e.g., Fas, TNFR), which leads to the recruitment of Fas-associated death domain protein and subsequent activation of caspase 3 (Fig. 2-5). On activation, caspases are the effectors of apoptotic signaling because they mediate the organized breakdown of nuclear DNA. The intrinsic pathway proceeds through protein mediators (e.g., Bcl-2, Bcl-2-associated death promoter, Bcl-2–associated X protein, Bim) that influence mitochondrial mem-brane permeability. Increased membrane permeability leads to the release of mitochondrial cytochrome C, which ultimately activates caspase 3 and thus induces apoptosis. These pathways do not function in a


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Surgery_Schwartz. permeability. Increased membrane permeability leads to the release of mitochondrial cytochrome C, which ultimately activates caspase 3 and thus induces apoptosis. These pathways do not function in a completely autonomous manner because there is significant interaction and crosstalk between mediators of both extrinsic and intrinsic pathways. Apoptosis is modulated by several regulatory factors, including inhibitor of apoptosis proteins and regulatory caspases (e.g., caspases 1, 8, 10).Apoptosis during sepsis may influence the ultimate com-petency of the acquired immune response. In a murine model of peritoneal sepsis, increased lymphocyte apoptosis was associ-ated with mortality, which may be due to a resultant decrease in IFN-γ release. In postmortem analysis of patients who expired from overwhelming sepsis, there was an increase in lymphocyte apoptosis, whereas macrophage apoptosis did not appear to be affected. Clinical trials have observed an association between the degree of


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Surgery_Schwartz. from overwhelming sepsis, there was an increase in lymphocyte apoptosis, whereas macrophage apoptosis did not appear to be affected. Clinical trials have observed an association between the degree of lymphopenia and disease severity in sepsis. In addition, after the phagocytosis of apoptotic cells by macro-phages, anti-inflammatory mediators such as IL-10 are released that may exacerbate immune suppression during sepsis. Neutro-phil apoptosis is inhibited by inflammatory products, including TNF, IL-1, IL-3, IL-6, GM-CSF, and IFN-γ. This retardation in regulated cell death may prolong and exacerbate secondary injury through neutrophil free radical release as the clearance of senescent cells is delayed.124NecroptosisCellular necrosis refers to the premature uncontrolled death of cells in living tissue typically caused by accidental exposure to external factors, such as ischemia, inflammation or trauma, which result in extreme cellular stress. Necrosis is character-ized by the loss of


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Surgery_Schwartz. living tissue typically caused by accidental exposure to external factors, such as ischemia, inflammation or trauma, which result in extreme cellular stress. Necrosis is character-ized by the loss of plasma membrane integrity and cellular col-lapse with extrusion of cytoplasmic contents, but the cell nuclei typically remain intact. Recent data have defined a process by which necrosis occurs through a series of well-described steps that are dependent on a signaling pathway that involves the receptor-interacting protein kinase (RIPK) complex. Termed “necroptosis,” it occurs in response to specific stimuli, such as TNFand TLR-mediated signals.125 For example, ligation of the tumor necrosis factor receptor 1 (TNFR1) under conditions in which caspase-8 is inactivated (e.g., by pharmacological Brunicardi_Ch02_p0027-p0082.indd 4001/03/19 6:49 PM 41SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2agents) results in the over-generation of ROS and a metabolic collapse. The net


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Surgery_Schwartz. Brunicardi_Ch02_p0027-p0082.indd 4001/03/19 6:49 PM 41SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2agents) results in the over-generation of ROS and a metabolic collapse. The net result is programmed necrosis (necroptosis). The effect of cell death by necroptosis on the immune response is not yet known. However, it is likely that the “DAMP” sig-nature that occurs in response to necroptotic cell death is an important contributor to the systemic inflammatory response. Evidence to support this concept was provided by investiga-tors who examined the role of necroptosis in murine model of sepsis. They demonstrated that Ripk3−/− mice were capable of recovering body temperature better, exhibited lower circu-lating DAMP levels, and survived at higher rates than their WT littermates.126 These data suggest that the cellular damage that occurs with programmed necrosis exacerbates the sepsis-associated systemic inflammatory response.PyroptosisPyroptosis is a form of regulated


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Surgery_Schwartz. These data suggest that the cellular damage that occurs with programmed necrosis exacerbates the sepsis-associated systemic inflammatory response.PyroptosisPyroptosis is a form of regulated cell death that is dependent on the activity of the proinflammatory caspase enzymes associ-ated with the inflammasome and is thus an inflammatory form of cell death.127 Pyropotosis shares some features with apopo-tosis, including DNA fragmentation and positive annexin V staining, among others. However, it is associated with the acti-vation of caspase-1 and the formation of caspase-1–dependent pores that allow early permeabilization of the cell membrane, electrolyte movement into the cells, and, finally, osmotic lysis of the cell.128 As a form of cell death, pyroptosis seems to be largely observed in macrophages, dendritic cells, and neutro-phils, although it has been documented in other cells as well, especially if they express high levels of caspase-1. As noted, pyroptosis is linked to activation


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Surgery_Schwartz. dendritic cells, and neutro-phils, although it has been documented in other cells as well, especially if they express high levels of caspase-1. As noted, pyroptosis is linked to activation of the inflammasome, which can occur in response to diverse cell alarm signals, including DAMPs. Not surprising, the mechanism of cell death leads to the release of additional intracellular DAMPs, including HMGB1 and S100 proteins.A recent study examined pyroptosis in peripheral blood mononuclear cells in a cohort of 60 trauma patients.129 The investigators found that the percentages of pyroptotic PBMCs were significantly higher in trauma patients than those in healthy CD95TNFR-1(p55)TNFR-2(p75)FADDDDDDDDDEDDDDEDDDDEDDEDFADDCaspase 8Caspase 2CaspaseCascadeApoptosisNIKI-˜B/NF-˜BNF-˜BMEKK1JNKc-JunRIPRAIDDTRAF2IAPTRADDDDDDDDTRADDDEDDDDDFADDRecruitedTRAF1TRAF2IAPFigure 2-5. Signaling pathway for tumor necrosis factor receptor 1 (TNFR-1) (55 kDa) and TNFR-2 (75 kDa) occurs by the recruitment of several


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Surgery_Schwartz. 2-5. Signaling pathway for tumor necrosis factor receptor 1 (TNFR-1) (55 kDa) and TNFR-2 (75 kDa) occurs by the recruitment of several adapter proteins to the intracellular receptor complex. Optimal signaling activity requires receptor trimerization. TNFR-1 initially recruits TNFR-associated death domain (TRADD) and induces apoptosis through the actions of proteolytic enzymes known as caspases, a pathway shared by another receptor known as CD95 (Fas). CD95 and TNFR-1 possess similar intracellular sequences known as death domains (DDs), and both recruit the same adapter proteins known as Fas-associated death domains (FADDs) before activating caspase 8. TNFR-1 also induces apoptosis by activating caspase 2 through the recruitment of receptor-interacting protein (RIP). RIP also has a func-tional component that can initiate nuclear factor-κB (NF-κB) and c-Jun activation, both favoring cell survival and proinflammatory functions. TNFR-2 lacks a DD component but recruits adapter proteins


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Surgery_Schwartz. component that can initiate nuclear factor-κB (NF-κB) and c-Jun activation, both favoring cell survival and proinflammatory functions. TNFR-2 lacks a DD component but recruits adapter proteins known as TNFR-associated factors 1 and 2 (TRAF1, TRAF2) that interact with RIP to mediate NF-κB and c-Jun activation. TRAF2 also recruits additional proteins that are antiapoptotic, known as inhibitor of apoptosis proteins (IAPs). DED = death effector domain; I-κB = inhibitor of κB; I-κB/NF-κB = inactive complex of NF-κB that becomes activated when the I-κB portion is cleaved; JNK = c-Jun N-terminal kinase; MEKK1 = mitogen-activated protein/extracellular regulatory protein kinase kinase kinase-1; NIK = NF-κB–inducing kinase; RAIDD = RIP-associated interleukin-1b-converting enzyme and ced-homologue-1–like protein with death domain, which activates proapoptotic caspases. (Adapted with permission from Vincent JL: Marshall JC, Cohen J: Update in Intensive Care and Emergency Medicine: Vol. 31: Immune


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Surgery_Schwartz. protein with death domain, which activates proapoptotic caspases. (Adapted with permission from Vincent JL: Marshall JC, Cohen J: Update in Intensive Care and Emergency Medicine: Vol. 31: Immune Response in Critical Illness. Berlin: Springer-Verlag; 2002.)Brunicardi_Ch02_p0027-p0082.indd 4101/03/19 6:49 PM 42BASIC CONSIDERATIONSPART Icontrols and correlated with injury severity. Moreover, increase in pyroptotic PBMCs significantly correlated with elevated cytokine levels (IL-10, IL-18, and MCP-1) and was a strong predictor for the development of sepsis.MEDIATORS OF INFLAMMATIONCytokinesCytokines are a class of protein signaling compounds that are essential for both innate and adaptive immune responses. Cytokines mediate a broad sequence of cellular responses, including cell migration, DNA replication, cell turnover, and immunocyte proliferation (Table 2-5). When function-ing locally at the site of injury and infection, cytokines mediate the eradication of invading microorganisms


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Surgery_Schwartz. DNA replication, cell turnover, and immunocyte proliferation (Table 2-5). When function-ing locally at the site of injury and infection, cytokines mediate the eradication of invading microorganisms and also promote wound healing. However, an exaggerated proinflammatory cyto-kine response to inflammatory stimuli may result in hemody-namic instability (i.e., septic shock) and metabolic derangements (i.e., muscle wasting). Anti-inflammatory cytokines also are released, at least in part, as an opposing influence on the proin-flammatory cascade. These anti-inflammatory mediators may also result in immunocyte dysfunction and host immunosuppres-sion. Cytokine signaling after an inflammatory stimulus can best be represented as a finely tuned balance of opposing influences and should not be oversimplified as a “black and white” proin-flammatory/anti-inflammatory response. A brief discussion of the important cytokine molecules is included below.Tumor Necrosis Factor-α. Tumor necrosis factor-α


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Surgery_Schwartz. as a “black and white” proin-flammatory/anti-inflammatory response. A brief discussion of the important cytokine molecules is included below.Tumor Necrosis Factor-α. Tumor necrosis factor-α (TNF-α) is a potent inflammatory mediator that is rapidly mobilized in response to stressors such as injury and infection. It is primar-ily synthesized by immune cells, such as macrophages, den-dritic cells, and T lymphocytes, and is generated in a precursor form that is expressed as a trimer on the surface of activated cells. After being processed by the metalloproteinase, TNF-α-converting enzyme (TACE, also known as ADAMS 17), a smaller, soluble form of TNF is released, which mediates its bio-logical activities through types 1 and 2 TNF receptors (TNFR-1; TNFR-2).130 Transmembrane TNF-α also binds to TNFR-1 and -2, but its biological activities are likely mediated through TNFR-2. While the two receptors share homology in their ligand-binding regions, there are distinct differences that regu-late


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Surgery_Schwartz. TNFR-1 and -2, but its biological activities are likely mediated through TNFR-2. While the two receptors share homology in their ligand-binding regions, there are distinct differences that regu-late their biologic function. For example, TNFR-1 is expressed by a wide variety of cells, but it is typically sequestered in the Golgi. Following appropriate cell signaling, TNFR-1 is mobi-lized to the cell surface, where it sensitizes cells to TNF or it can be cleaved from the surface in the form of a soluble recep-tor that can neutralize TNF.131 In contrast, TNFR-2 expression is confined principally to immune cells where it resides in the plasma membrane. Both TNF receptors are capable of binding intracellular adaptor proteins that lead to activation of complex signaling processes and mediate the effects of TNF.Although the circulating half-life of soluble TNF is brief, it acts upon almost every differentiated cell type, eliciting a wide range of important cellular responses. Moreover, it is