Overview

Cytokines are small, nonstructural proteins that are key regulators of the human response to noxious stimuli. Cytokines can be proinflammatory, antiinflammatory, or both depending on the timing and context in which they are secreted. Nearly all nucleated cells are capable of both synthesizing and responding to cytokines, but most cytokine genes are not expressed unless stimulated by a specific stressor. This chapter reviews the roles that specific stimuli—including endotoxin, microbial products, and cytokines—play in both the normal physiology and pathophysiology of liver, biliary, and pancreatic disease, with particular attention to surgical problems. Both a metabolic and immunologic organ, the liver has been the subject of extensive research, but cytokines undoubtedly play a role in pancreatic pathophysiology as well. The entire field of cytokine research has continued to grow exponentially since the last version of this text, with several key discoveries that are directly applicable to surgical practice. Exciting areas of therapeutics based on this new knowledge are also discussed.

Microbiologic Recognition and Toll-Like Receptors

In mounting an immune response, the challenge to the host is to identify the presence of an infectious process early and to coordinate its considerable resources to eradicate the pathogen, while minimizing tissue damage and chronic inflammation. The recognition of microbial pathogens and the ability to distinguish self from nonself relies heavily on the innate immunity and cell surface receptors present on antigen-presenting cells in the liver (Akira & Hemmi, 2003). Antigen-rich blood from the portal circulation has the potential to activate either the innate and adaptive immune responses against infections or to maintain immunologic tolerance of harmless antigens (Tacke et al, 2009). In addition, the infiltration of monocytes during liver injury is an important adaptive mechanism leading to chronic infammation (Karlmark et al, 2009). The immune response to a biologic insult is a highly organized process that involves pattern-recognition receptors (PRRs) that identify preserved structures of different pathogens. Toll-like receptors (TLRs) are an important family of at least 10 PRRs that are ubiquitously expressed in humans (Akira & Takeda, 2004). Current evidence demonstrates crucial roles for TLRs in alcoholic liver disease, nonalcoholic steatoheaptitis, hepatitis B, hepatitis C, hepatic fibrosis and cirrhosis, hepatocellular carcinoma, primary biliary cirrhosis, acetaminophen-induced hepatotoxicity, and ischemia-reperfusion (I/R) liver injury, all of which are potential therapeutic targets (Pimental-Nunes et al, 2010; Table 10.1).

Table 10.1 Toll-like Receptors and Their Potential Targets for Gastrointestinal Diseases

Endotoxin Structure and Biologic Activity

Lipopolysaccharides (LPSs) from gram-negative bacteria have a wide range of harmful biologic activities and are therefore also considered endotoxins (Dinarello, 2004). Research over the past decade has elucidated the mechanisms by which the host recognizes endotoxin and the innate immune response is initiated. Much more is known about the cell surface receptors and signaling pathways involved in the host response to endotoxin than is known about several other microbial products and TLRs. Although endotoxin has been implicated in the beneficial and injurious host responses to gram-negative infections, the exact role of endotoxin in the pathogenesis of human disease remains unclear. Similarities in host responses to various microbial products, including LPSs from gram-negative bacteria and the glycoconjugates of gram-positive bacterial and fungal infections, suggest that the host innate immune system has evolved to respond consistently to microbial invasion, regardless of its antigenic source (Fearon & Locksley, 1996).

Endotoxin is the major constituent of the outer cell wall of gram-negative bacteria and can comprise 65% by weight of the total bacterium. Endotoxin is shed spontaneously from the cell walls of living bacteria and is released in copious amounts upon cell death and cell lysis. Endotoxin comprises three major components: an inner lipid A, an intermediate R-core oligosaccharide, and an outer O-polysaccharide (Fig. 10.1). The inner lipid A portion and the R-core oligosaccharide generally are conserved, whereas the structure of the O-polysaccharide is unique for each strain of bacteria. The R-core oligosaccharide is exposed in rough forms of gram-negative bacteria. The internal lipid A moiety is poorly antigenic but seems to be a primary factor in inducing most of the biologic effects of LPS. Binding of endotoxin to serum proteins and its activation of host immunity through its cellular receptor are mediated predominantly via the lipid A moiety; the O-antigen is primarily immunogenic (Ulevitch & Tobias, 1999).

FIGURE 10.1 Schematic representation of the structure of endotoxin.

The host response to endotoxin is immediate (within minutes), is dose dependent, and affects all organ and tissue systems (Table 10.2). High-dose endotoxin exposure in rodents and primates reproduces the toxic effects of gram-negative bacteremia, characterized by hemodynamic collapse, shock, organ failure, and death (Beutler et al, 1985). Low-dose administration of endotoxin to human volunteers (Fong et al, 1990; Michie et al, 1988) produces a variety of constitutional symptoms consistent with a milder infectious process, including fever, myalgia, tachycardia, occasional hypotension, transient leukopenia followed by neutrophilia, and a hepatic acute-phase response (Table 10.3).

Table 10.2 Biologic Actions of Endotoxin

IL, interleukin; TNF, tumor necrosis factor; CSF, colony-stimulating factor; PAF, platelet activating factor

Table 10.3 Biologic Responses to Low-Dose Endotoxin in Human Volunteers

TNF-α, tumor necrosis factor α; IL, interleukin; s-TNFR, soluble TNF receptor

* ≤4 hours’ duration

† 6 to 24 hours’ duration

‡ 6 to 12 hours’ duration

Data from Fong et al, 1990: The acute splanchnic and peripheral tissue metabolic response to endotoxin in humans. J Clin Invest 85:1896-1904.

Although the host responses to endotoxin are likely to be dose dependent, there is now general acceptance that the clinical sequelae associated with systemic endotoxin administration are probably secondary to the host innate immune response rather than a result of direct interactions between endotoxin and cell membranes or secretory proteins (Fearon & Locksley, 1996; Ulevitch & Tobias, 1999). At high doses, endotoxin can produce direct endothelial injury, but most host responses to endotoxin are mediated by complement activation and the release of humoral factors, including proinflammatory cytokines, nitric oxide (NO), and prostaglandins.

Immune Regulation and Response to Endotoxin (SEE CHAPTER 9)

The host defense against many gram-negative bacteria is dependent on the innate immune recognition of endotoxin, and this response must be highly sensitive and self-limited. The sensitivity of this system is crucial for prompt mobilization to combat infection, but a risk of severe immune-mediated pathology exists if this response not self-limited. Optimal sensitivity is achieved by the strictly ordered interactions of endotoxin with different extracellular and cell surface proteins, including lipopolysaccharide (LPS)-binding protein (LBP), cluster of differentiation (CD)14, MD-2, and Toll-like receptor 4 (TLR-4) (Gioannini & Weiss, 2007). The presence of endotoxin in the plasma and lymphatic system is initially recognized by serum proteins and lipoproteins, and this recognition initiates the activation of the innate immune response (Beutler et al, 2003). Redundancy is the hallmark of this activation process, as proteins, glycoproteins, lipids, and nucleic acids of prokaryotic origins are recognized by different TLRs and other components of innate immunity. For the most part, the innate immune system relies on cell surface receptors and hepatic secretory proteins, primarily opsonins, to recognize carbohydrate and lipid, protein, and DNA structures indicative of a microbial infection.

Different constituents of specific microbial products, including endotoxin, are recognized by different aspects of the innate immune system (Ulevitch & Tobias, 1999). The first step in the recognition of endotoxin by the innate immune system is its binding to a hepatic secretory protein, LBP, to form the LPS-LBP complex (Fig. 10.2). LBP is a member of a group of homologous lipid-binding proteins that function as lipid-transfer proteins (Tobias et al, 1986, 1989; Wright et al, 1989). Secreted by the liver and found in human blood in 2 to 20 mg/mL concentrations, LBP binds avidly to the lipid A moiety of endotoxin and mediates its transfer to the cell surface endotoxin receptor, CD14. Although CD14 functions as a ligand-binding protein for endotoxin-LBP complexes, CD14 is bound to the cell membrane through a glycosyl–phosphatidylinositol anchor, and it lacks an intracellular domain capable of transducing a signal. LPS-LBP complex binding to CD14 alone is insufficient to transduce a signal.

FIGURE 10.2 Endotoxin-binding proteins and signal transduction. Endotoxin in body fluids is rapidly recognized by endotoxin-binding proteins and lipoproteins. The balance between lipopolysaccharide binding protein (LBP) and bactericidal permeability–increasing protein (BPI) or high-density lipoprotein or both determines whether endotoxin in body fluids is detected by macrophages and other effector cells of the innate immune system. LBP-endotoxin complexes bind to CD14 and interact with Toll-like receptor-4 (TLR-4) and MD-2 membrane proteins, which activates intracellular myeloid differentiation response gene (88) (MyD88) dependent and independent pathways that ultimately lead to nuclear factor (NF)-κB activation and type I interferon production. IkB, inhibitor of NF-κB; IkK, inhibitor of NF-κB kinase; IRAK, interleukin-1 receptor–associated kinase; LPS, lipopolysaccharide; NIK, NF-κB–inducing kinase; NEMO, NF-κB essential modulator; TRAF, tumor necrosis factor receptor–associated factor; TRAM, Toll receptor–associated molecule; TRIF, Toll receptor–associated activator of interferon.

The signal-transducing component of the CD14 bipartite complex protein has been identified as a member of the family of TLRs, specifically TLR-4 (Beutler, 2002). Although TLR-4 is expressed in several cells of healthy liver, signs of inflammation are absent because of low expression levels of TLR-4 and modulation of TLR-4 signaling. Mounting evidence suggests that altered LPS–TLR-4 signaling plays a role in the pathogenesis of chronic liver disease (Soares, 2010). TLR-4 seems to interact with CD14, the LBP-endotoxin complex, and a third protein, MD-2 (Viriyakosol et al, 2001).

Defining the complex role of CD14 in endotoxin-induced activation of TLR-4 is a challenge, and whether this role is obligatory remains unclear (Beutler et al, 2006; Jiang et al, 2005). However, the activation of TLR-4 by endotoxin does appear to require the simultaneous binding of endotoxin and TLR-4 by MD-2 (Gioannini et al, 2004). TLR-4 signaling can occur via a Toll-like and interleukin (IL)-1 receptor (TIR) domain and the formation of a scaffold composed of members of myeloid differentiation factor 88 (MyD88) adaptor proteins. At the same time, TLR-4 signaling also occurs through a MyD88-independent pathway that involves the adaptor protein TIR domain–containing adaptor inducing interferon-β (TRIF). This latter pathway seems to be more essential for the expression of type I interferons (IFNs) and IFN-dependent proteins in response to endotoxin exposure (Yamamoto et al, 2003). Although it is presently unknown how CD14-dependent signaling is integrated from the cell surface via TLR-4, the confirmation of TLR-4 as the signaling complex is important, because it identifies a signal transduction pathway and potential therapeutic targets.

The presence of LBP in the plasma increases the sensitivity of cells bearing CD14 to endotoxin by at least 1000-fold. From a teleologic standpoint, this has distinct advantages, because endotoxin can be detected, and innate immunity activated early, in response to gram-negative infection. However, increasing the sensitivity of the innate immune response to endotoxin can have devastating pathologic consequences, as may occur during sepsis or when gastrointestinal integrity is lost. These pathologic consequences include loss of vascular integrity, shock, organ failure, and death. Hosts have evolved several mechanisms to reduce the likelihood of an exaggerated endotoxin-induced signal.

High-density lipoproteins can bind endotoxin in serum and may play a role in protecting against endotoxemia (Cue et al, 1994). An additional endogenous mechanism to suppress endotoxin responsiveness is through the release of an LBP-homologous protein, bactericidal/permeability-increasing protein (BPI; see Fig. 10.2). BPI is a 55- to 60-kD protein structurally similar to LBP, which binds endotoxin through its lipid A moiety. However, the resulting BPI-endotoxin complexes do not bind to CD14 or transduce a signal (Elsbach, 1998). BPI acts as an endogenous endotoxin inhibitor and has shown some modest beneficial effects in patients with meningococcal sepsis (Levin et al, 2000). In addition, BPI released from activated neutrophils can bind directly to endotoxin being expressed on the surface of gram-negative bacteria, and it is growth arresting and cytolytic for the bacteria (von der Mohlen et al, 1996).

BPI is stored in the granules of neutrophils and is released on neutrophil activation and degranulation. In a healthy adult, BPI levels in the serum are very low (15 to 50 ng/mL), but concentrations increase in response to an inflammatory or endotoxemic challenge (Calvano et al, 1994). In contrast, LBP concentrations are several logs higher in patients in the surgical ICU (2 to 20 mg/mL) and increase only modestly in response to inflammation. During infection, the ratio of LBP to BPI concentrations in the plasma approaches 1000:1, and this ratio favors endotoxin signaling. In closed-space infections infiltrated with neutrophils, however, BPI exceeds LBP concentrations, and BPI concentrations are directly proportional to neutrophil counts. These findings suggest that in the normal healthy adult and in patients with sepsis, the relationship between plasma LBP and BPI concentrations favors the recognition of endotoxin and activation of the innate immune response. In local infectious sites, where neutrophil and inflammatory cell recruitment has occurred, the increased BPI response is presumably compensatory and aimed at the continued activation of the immune system and reduction of bacterial growth.

Endotoxin signaling via the CD14 receptor and TLR-4 involves a novel IL-1 receptor-associated kinase, which activates a secondary kinase, NF-κB–inducing kinase (NIK) (Yang et al, 1999). Activation of this cascade rapidly induces the phosphorylation of additional kinases, which results in NF-κB translocation and the transcription of NF-κB–dependent genes (see Fig. 10.2). Genes containing NF-κB response elements are numerous and include most of the proinflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-1, IL-6, IL-8, IL-12, IL-18, and IFN-γ (Blackwell & Christman, 1997).

Most of the systemic inflammatory responses to endotoxin are mediated by the release of proinflammatory cytokines and other humoral factors, predominantly TNF-α, IL-1, and IL-6 and, to a lesser extent, Fas ligand (FasL) (Suffredini et al, 1999). These proinflammatory cytokines not only globally regulate the inflammatory response to endotoxin, they also play a crucial role in reprogramming the metabolic and protein synthetic responses by the liver. As we will discuss in greater detail, TNF-α and IL-6 in particular play unique roles in regulating not only the hepatocyte acute-phase response to endotoxin but also hepatocyte proliferation versus apoptosis in liver regeneration (Michalopoulos & DeFrances, 1997; see Chapter 5), viral hepatitis (Hayashi & Mita, 1997; see Chapter 64), and toxic liver injury (Batey et al, 1999; Bradham et al, 1998; McClain et al, 1999). In contrast, FasL is a potent inducer of hepatocyte apoptosis and has been implicated in the pathogenesis of viral hepatitis (Kondo et al, 1997; Hayashi & Mita, 1997).

Tumor Necrosis Factor Superfamily

Since the discovery of TNF-α 35 years ago, the TNF superfamily has grown to comprise at least 20 related proteins that signal through greater than 30 receptors (Grewal, 2009). Members of the TNF superfamily are primarily homotrimeric proteins, with the exception of lymphotoxin, and they exist primarily in a membrane-associated form (Bazzoni & Beutler, 1996). As a general rule, members of the TNF family are primarily involved in the regulation of cell proliferation and apoptosis, although several of the members—including TNF-α, TNF-β, FasL, CD30L, and CD40L—also have proinflammatory properties.

TNF-α is synthesized as a bioactive cell-associated protein that is primarily involved in juxtacrine signaling of cytotoxicity. This 26-kD intermediate is enzymatically cleaved by a matrix metalloproteinase to a 17-kD secreted form that acts in a paracrine or endocrine fashion (Fig. 10.3). TNF-α originally was characterized as a factor that produced necrosis of solid tumors in vivo (Carswell et al, 1975). It subsequently was recognized that TNF-α modulates growth, differentiation, and metabolism in a variety of cell types; it can produce cachexia by stimulating lipolysis, inhibiting lipoprotein lipase activity in adipocytes, and stimulating hepatic lipogenesis; and it can initiate apoptosis in hepatocytes and lymphoid cells (Table 10.4; Ksontini et al, 1998).

FIGURE 10.3 Processing of tumor necrosis factor (TNF)-α, which is initially synthesized as a 26-kD membrane-associated protein that is processed by TNF-α converting enzyme (TACE) to a secreted 17-kD homotrimer. Cell-associated and secreted molecules can bind and transduce a signal through two TNF receptors (TNFRs; p55 [type I] and p75 [type II]). mTNF, membrane TNF; sTNF, soluble TNF.

Table 10.4 Biologic Activities of Tumor Necrosis Factor-α

Vascular Endothelial Cells

Fibroblasts

Adipocytes

Endocrine System

Central Nervous System

Cardiovascular

Gastrointestinal

Metabolic

Inflammatory

FSH, follicle-stimulating hormone; G-CSF, granulocyte colony-stimulating factor; GH, growth hormone; GM-CSF, granulocyte-macrophage colony-stimulating factor; ICAM, intercellular adhesion molecule; IL, interleukin; LIF, leukemia inhibitory factor; TSH, thyroid-stimulating hormone; VCAM, vascular cell adhesion molecule

TNF-α is a powerful inducer of the inflammatory response both directly and through stimulation of numerous downstream proinflammatory mediators. Secondary mediators that are known to be induced by systemically administered TNF-α include cytokines (IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, type I and type II IFN, transforming growth factor-β, leukemia inhibitory factor [LIF], and macrophage migration inhibitory factor [MIF]), hormones (cortisol, epinephrine, glucagon, insulin, and norepinephrine), and assorted other molecules (acute-phase proteins, IL-1 receptor antagonist [IL-1Ra], leukotrienes, oxygen free radicals, NO, platelet-activating factor, and prostaglandins; Tracey & Cerami, 1994). Other principal biologic effects of TNF-α are listed in Table 10.4 (see Chapter 9).

Not only is TNF-α involved in tissue inflammation, but there is a growing recognition that it is also a prominent ligand for the activation of programmed cell death. Apoptosis occurs naturally during growth and development, but it may also result from certain pathologic conditions in which local and systemic production of TNF-α is increased. Hepatocytes are particularly sensitive to TNF-α–induced apoptosis, especially during simultaneous transcriptional inhibition (Leist et al, 1994).

TNF-α has been administered systemically in low doses to human volunteers and regionally in higher doses to sarcomas and melanomas. The physiologic responses to low-dose (50 mg/m²) TNF-α administration were remarkably similar to the responses seen with low-dose endotoxin administration: fever and constitutional symptoms of pain, headache, myalgia, and nausea. Hematologically, the volunteers developed a rapid leukopenia, followed by neutrophilia with sustained lymphopenia and monocytopenia (van der Poll et al, 1992). Plasma IL-6 levels increased 40-fold, and the volunteers developed an acute-phase protein response. Prostaglandin (6-keto prostaglandin [PG] F_1a) production also was markedly increased. Metabolically, the patients exhibited increased lipolysis and glucose turnover (van der Poll et al, 1991b). There also were significant effects on the vascular endothelium. Within 1 hour, TNF-α administration induced activation of the fibrinolytic pathway, followed by activation of the coagulation cascade (van der Poll et al, 1991a).

At higher doses of TNF-α, as achieved with regional perfusion of limbs, the systemic release of large quantities of protein occasionally occurs, and the effects on the vascular endothelium are profound. Under these conditions, activation of the endothelium is evident by increased release of soluble selectins and integrins, and hemodynamic instability is common (Aderka et al, 1998; Zwaveling et al, 1996); therefore continuous monitoring for systemic leakage is required.

Interleukin-1 Family

The IL-1 family of ligands and cytokines are similar to the TNF superfamily in that they are also primarily associated with inflammation. IL-1 possesses several biologic properties that result in increased expression of downstream proinflammatory genes. The most salient and relevant is the ability of IL-1 to initiate and sustain the expression of cyclooxygenase type 2 and inducible NO synthase (Dinarello, 1996). This property accounts for the large amount of PGE₂ and NO produced by cells exposed to IL-1 or in subjects injected with IL-1. Other important proinflammatory properties of IL-1 are its ability to increase IL-8 synthesis and to express adhesion molecules on endothelial cell surfaces, which accounts for the infiltration of inflammatory cells into the extravascular space. IL-1 also potentiates the biologic activities of TNF-α, and modest doses of the two in combination can be lethal in experimental animals.

The original IL-1 superfamily consisted of two members: IL-1α and IL-1β. Currently, the IL-1 superfamily also contains IL-1Ra, IL-16, IL-17, IL-18, IL-33, and at least six other homologues in the IL-1 family (Schmitz et al, 2005). IL-1α and IL-1β are agonists, and IL-1Ra is a specific receptor antagonist for the IL-1 receptor family. The naturally occurring IL-1Ra seems to be unique in cytokine biology (Arend, 1993). The intron–exon organization of three primary IL-1 genes suggests the duplication of a common gene some 350 million years ago. Processing of IL-1α or IL-1β to “mature” forms requires specific cellular proteases. In contrast, IL-1Ra evolved with a signal peptide and is readily transported out of the cell and is termed secreted IL-1Ra.

Even under conditions of cell stimulation, human blood monocytes do not process or readily secrete mature IL-1α, which is probably only released by dying cells, as it is a key factor released by necrotic cells that promotes inflammation (Chen, 2007). The IL-1α precursor proIL-1α is synthesized in association with cytoskeletal structures (microtubules), which is in contrast to most proteins translated in the endoplasmic reticulum. ProIL-1α is fully active as a precursor and remains intracellular. The opposite is the case with the IL-1β precursor proIL-1β, which is not fully active, and a considerable amount is secreted after cleavage by a specific, intracellular cysteine protease, IL-1β–converting enzyme, or caspase-1. Caspase-1 plays a central role in the biology of IL-1β, IL-18, and IL-33 and has become a therapeutic target for modulating IL-1–based diseases (Martinon & Tschopp, 2004). IL-33 is an activator of Th2 cells and has recently been shown to play a protective role in the development of atherosclerosis (Miller et al, 2008; Sanada et al, 2007) and is thought to be released by cell death (Carriere et al, 2007).

Nearly all microbes and microbial products induce production of the three IL-1 family proteins, but stimulants of nonmicrobial origin also can elicit transcription and synthesis. Stimulants such as the complement component C5a, hypoxia, adherence to surfaces, and clotting of blood induce the synthesis of large amounts of IL-1 mRNA in monocytic cells without significant translation into the IL-1 protein.

Although animal experiments revealed that IL-1 was proinflammatory, a great deal of information has been gathered from studies in which humans have been injected with recombinant IL-1α or IL-1β. Humans have received IL-1α infusions as part of clinical trials following bone marrow transplantation because of its hematopoietic properties. Although the duration of leukopenia and thrombocytopenia were reduced, patients had serious signs of systemic inflammation that included fever, hypotension, and flulike symptoms, similar to endotoxemia (Smith et al, 1993). Interestingly, the exquisite sensitivity of humans to IL-1 given systemically was not appreciated from animal studies.

Interleukin-6

IL-6 is another pleiotropic cytokine produced by a wide variety of cells, including T cells, B cells, endothelial cells, fibroblasts, monocytes, and macrophages. The biologic activities of IL-6 include immune regulation, hematopoiesis, inflammation, and oncogenesis (see Chapter 9). The IL-6 gene has been mapped to chromosome 7, and its product varies from 21 to 28 KD, depending on posttranslational modifications. IL-6 belongs to a much larger superfamily of related cytokines, including leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, cardiotrophin-1, and IL-11. All members of this superfamily share a modest degree of structural homology, but more importantly, all these related cytokines use a common signal transduction pathway through heterocomplexes composed of gp130. Each ligand has its own receptor, which combines in a duplex to transduce its signal through the JAK/Stat signaling cascade.