Cytokines and acute pancreatitis

BEST

Baillière’s Clinical Gastroenterology Vol. 13, No. 2, pp 265–289, 1999

B A I L L I È R E ’ S

5

PRACTICE & RESEARCH

Cytokines and acute pancreatitis Mark Brady

BSc

Steve Christmas

PhD

Senior Lecturer in Immunology

Robert Sutton

PhD, FRCS

Consultant Surgeon and Senior Lecturer, Royal Liverpool University Hospital

John Neoptolemos

MA, MD, FRCS

Professor of Surgery and Head, Department of Surgery

John Slavin

MS, FRCS

Senior Lecturer and Honorary Consultant Department of Surgery, University of Liverpool, 5th Floor UCD Building, Royal Liverpool University Hospital, Daulby Street, Liverpool L69 3GA, UK

Cytokines have been shown to play a pivotal role in multiple organ dysfunction, a major cause of death in severe acute pancreatitis. Moreover, the two-hit hypothesis of the cytokine-induced systemic inflammatory response syndrome explains the variable individual response to severe acute pancreatitis and the impact of secondary events such as sepsis or therapeutic intervention. Many experimental anti-cytokine therapies have been administered following induction of experimental pancreatitis, and have proved to be therapeutic. Patients with severe pancreatitis present early because of pain. Clearly then a window for therapeutic intervention is available between onset of symptoms and peak pro-inflammatory cytokine expression. It is this fundamental observation that convinces many in the field that the treatment of AP will be one of the first clinical successes for novel drugs or therapy that seek to modulate the inflammatory response. Key words: cytokines; therapy; leukocytes; acute pancreatitis; interleukin-1, tumour necrosis factor; systemic inflammatory response syndrome.

Acute pancreatitis (AP) is a common disease. The incidence in the United Kingdom is about 30 cases per 100 000 population and has been increasing over recent years (Sinclair et al, 1997). In the majority of patients the condition is mild, but about 25% of patients suffer a severe attack and between 30 and 50% of these will die (Wilson et al, 1998). The usual cause of death is multiple organ failure secondary to systemic leukocyte activation. Acute pancreatitis is thought to begin as an autodigestive process within the gland. Although there are many causes, most cases are secondary to bilary disease or excess alcohol consumption. The exact mechanisms 1521–6918/99/020265 + 25 $12.00/00

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by which diverse aetiological factors induce an attack are still unclear, but once the disease process is initiated common inflammatory and repair pathways are invoked (Wilson et al, 1998). Many types of cell, including leukocytes, endothelial cells and fibroblasts, are involved in the initial local inflammatory response, the subsequent repair response and systemic response to pancreatic damage. Cytokines acting in a paracrine and autocrine fashion to control cellular function play a pivotal role. In this chapter we will describe the evidence implicating individual cytokines. Their value as prognostic markers or as therapeutic targets will then be explored in some detail.

CYTOKINES Tumour necrosis factor-␣ Although many types of cell can synthesize tumour necrosis factor-α (TNF-α), this cytokine is derived predominantly from activated macrophages (Hohmann et al, 1989). TNF-α was originally identified as a serum factor able to cause necrosis in certain solid tumours, but it was soon realised that it had many other actions. The mature form has 157 amino acids. Two receptors, p55 and p75, are found on the surface of most cells (Hohmann et al, 1989). TNF-β, a lymphocyte-derived cytokine with a similar structure, binds to the same receptor and has very similar effects (Tracey and Cerami, 1993). Binding of TNF-α to its receptor results in hydrolysis and release of the extracellular domain. Increased levels of circulating receptor have been shown to occur following endotoxaemia (Spinas et al, 1992). Release of soluble tumour necrosis factor receptor (TNFR) into the peripheral circulation may be a mechanism by which cellular responsiveness is down-regulated (Aderka et al, 1992). Activated neutrophils and lymphocytes also release soluble TNF receptors and this may represent a further method of neutralizing excess TNF activity (Porteu and Nathan, 1990). Injection of TNF-α into experimental animals causes a syndrome that is indistinguishable from septic shock. It is now thought that TNF-α is one of the major mediators of shock; circulating levels are elevated in a number of conditions that lead to shock, including AP. Levels correlate with outcome in a number of clinical and experimental studies (Strieter et al, 1993). Intrapancreatic and serum TNF-α is detectable 1 hour following induction of pancreatitis and increases rapidly over the following 6 hours. Immunohistochemical staining shows infiltration into the pancreas of macrophages that stain heavily for TNF-α. The overall rise in both tissue and serum TNF-α concentrations correlates directly with the severity of pancreatic damage and inflammation (Norman et al, 1995a). Both receptors, p55 and p75, are expressed within the pancreas. Cultured pancreatic acinar cells also have been shown to produce, release, and respond (by apoptosis) to TNF-α (Gukovskaya et al, 1997). TNF-α expression in AP is a primary response and is not the result of endotoxaemia because identical changes can be seen in germ-free animals (Hughes et al, 1995a). TNF-α production is also seen at distant sites, but later (Hughes et al, 1995b). Circulating levels of TNF-α are not as reliable an indicator of disease severity as those of other cytokines; the liver rapidly clears TNF before it reaches the general circulation, and this may explain why it is often difficult to detect TNF in the serum of

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patients with AP (Grewal et al, 1994a). The presence of soluble TNF receptors in the circulation may therefore provide a better indication of the severity of disease, and indeed predicted organ failure in patients with AP in one study even when TNF levels were not detectable (de Beaux et al, 1996). Interleukin-1 Interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine. Like TNF-α it is derived predominantly from macrophages (Fink and Norman, 1997). IL-1β activates neutrophils and induces up-regulation of adhesion molecules on both leukocytes and endothelium. Injection of IL-1β into experimental animals produces fever, hypotension, tachycardia and anorexia. The changes are remarkably similar to those seen with TNF-α, and it is hard to distinguish the effects of these two mediators. Up-regulation of IL-1β occurs when AP is induced in germ-free animals, suggesting that its upregulation is a direct consequence of pancreatitis rather than endotoxin. Large amounts of a specific interleukin-1 receptor antagonist (IL-1ra) are also produced within the pancreatic parenchyma. Changes are indicative of the severity of AP and are not model-dependent (Fink and Norman, 1997). Combined infusions of TNF-α and IL-1β have synergistic actions (Okusawa et al, 1988; Mizel, 1990; Fisher et al, 1994; Tanaka et al, 1995). As well as stimulating their own production and that of each other, IL-1β and TNF-α also play an important role in inducing other regulatory genes. Production of IL-1β is accompanied by the induction of its receptors as well as the interleukin-1 converting enzyme (ICE), which is responsible for cleaving pro-IL-1β to the active form (Fink and Norman, 1995, 1997). Both are also capable of increasing the expression of cholecystokinin (CCK) receptors on the surface of acinar cells (Viguerie et al, 1994). During an attack of AP, the concentration of IL-1β and TNF-α in pancreatic tissue is several fold higher than in serum (Grewal et al, 1994a). Using transgenic knockout (–/–) mice deficient in either IL-1 type 1 receptors, TNF type 1 receptors, or both IL-1 and TNF type 1 receptors, it has been shown that IL-1β and TNF-α make an equivalent contribution to the severity of an attack. Preventing the activity of both cytokines concurrently has no additional effect on the degree of pancreatitis but does attenuate the systemic stress response and is associated with an additional but modest decrease in mortality (Denham et al, 1997a). Although IL-1β and TNF-α are both produced in the pancreas during the development of AP (Norman et al, 1994) and are responsible for many of the detrimental effects of the disease they do not appear to play a causal role. Treatment of isolated acinar cells directly with IL-1 or TNF does not result in co-localization of zymogen granules and lysosomes or the activation or release of enzymes (Fink et al, 1997), and perfusion of isolated human pancreas with IL-1 and TNF does not induce AP (Denham et al, 1998). Clearly, these cytokines are involved in the subsequent inflammatory cascade. Interleukin-6 A wide range of cells, including monocytes/macrophages, endothelial cells, fibroblasts and smooth muscle cells, produce IL-6. Endotoxin, IL-1β and TNF-α stimulate synthesis and release of IL-6. IL-6 binds to a cell surface receptor which triggers the association of a non-ligand binding molecule, gp 130 (Taga et al, 1989), although relatively little is known about the downstream signalling pathway. In volunteers to

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whom endotoxin was administered, IL-6 levels were raised by 1 hour (Fong et al, 1989). Administration of IL-6 induces pyrexia. The importance of IL-6 in the acute phase response has been confirmed by the observation that it stimulates synthesis of acute phase proteins (including C-reactive protein, CRP) from hepatocytes in vitro and in vivo (Geiger et al, 1988; Castell et al, 1989) and that levels of IL-6 peak about 24 hours before those of acute phase proteins (Messmann et al, 1997). Raised levels of IL-6 have been described in a number of acute conditions such as following burns (Nijsten et al, 1991), major surgery (Ohzato et al, 1992), and sepsis (Damas et al, 1992). In one small study, interleukin levels were found to be higher in patients with a variety of primary pathologies who went on to develop multiple organ failure (Borrelli et al, 1996). Interleukin-6 levels are also raised in patients with acute pancreatitis and correlate with severity of disease (Leser et al, 1991; Gross et al, 1993; Heath et al, 1993; Viedma et al, 1992). Interleukin-10 Interleukin-10 (IL-10) was originally identified as a mediator in the Th2-driven immune response (Howard et al, 1992). It is an anti-inflammatory cytokine which modulates the expression of the early pro-inflammatory cytokines, down-regulating TNF-α and IL-1 as well as members of the CC and CXC chemokine families (Fiorentino et al, 1991; Kasama et al, 1994). It also stimulates production of the naturally occurring IL-1 receptor antagonist and release of the soluble p75 TNF receptor (Seitz et al, 1995). IL-10 is elevated in animal models of endotoxaemia and inhibits the release of proinflammatory cytokines from monocytes/macrophages, thus preventing subsequent tissue damage (Howard et al, 1993; Smith et al, 1994; Standiford et al, 1995; van der Poll et al, 1997). In human patients with acute pancreatitis IL-10 has a protective role. In healthy patients levels of IL-10 are not measurable in serum whereas levels are markedly raised within the first 24 hours of an attack followed by a steady decline in the following days. During the first 24 hours serum IL-10 levels are higher in those patients with mild as opposed to severe forms of the disease (Pezzilli et al, 1997). Platelet activating factor Platelet activating factor (PAF) (1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a low-molecular-weight phospholipid which acts via specific cell surface receptors that have been identified on numerous cells and tissues, including platelets, leukocytes and endothelial cells. PAF has numerous pro-inflammatory effects; intravascular administration produces a syndrome characterized by widespread leukocyte activation that has many similarities to the systemic inflammatory response syndrome (SIRS) (Humphrey et al, 1982; Wallace et al, 1987). PAF binds to a G-protein-coupled transmembrane receptor; intracellular signalling is by activation of intracellular enzymes such as phospholipase (PL) A2 and phospholipase C (PLC). Two pathways for PAF synthesis exist. In the ‘remodelling pathway’ membrane phospholipid is converted to lyso-PAF by the action of membrane-associated PLA2 (mPLA2). This is then acetylated to yield the active PAF by acetyltransferase using acetyl co-enzyme A as the substrate. The reverse reaction is catalysed by PAF acetylhydrolase. Thus a dynamic equilibrium exists between active and inactive PAF. PLA2 is also required for eicosanoid synthesis. De novo synthesis of PAF is catalysed by cholinephosphotransferase from 1-o-alkyl-2-acetyl-sn-glycerol. PAF appears to be a

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particularly important mediator in AP. Isolated pancreatic acini have been shown to synthesize PAF, and pancreatic tissue concentrations rise during the course of an attack (Zhou et al, 1993). Blood and pulmonary tissue levels also rise co-ordinately, indicating that PAF is a key mediator of the systemic inflammatory response (Kald et al, 1993; Zhou et al, 1993). In animal models intraperitoneal or intravascular injection can bring about or increase the severity of AP (Emanuelli et al, 1989; Konturek et al, 1992; Emanuelli et al, 1994; Yotsumoto et al, 1994). Chemokines The chemokines are a family of small (8–10 kDa), inducible, secreted cytokines with chemotactic and activating effects on leukocyte subsets. They provide a key stimulus for directing leukocytes to areas of injury. They can be broadly subdivided on a structural basis into the CXC subfamily in which the first two of four conserved cysteine residues are separated by another amino acid, and the CC subfamily in which the first two cysteine residues are adjacent (Table 1). The genes for the human CXC chemokines have been mapped to chromosome 4q while those for the CC chemokines are clustered on chromosome 11q. The CXC chemokines can be further subdivided into two groups; those containing the conserved amino acid sequence ELR immediately preceding the first cysteine residue at the N-terminus and those lacking this motif. The structural classification of the chemokines also determines their biological activity. Those CXC chemokines possessing the ELR motif, including IL-8, the prototype CXC chemokine, are potent neutrophil chemoattractants and activators while those lacking the motif have little if any neutrophil activating activity (Murphy, 1994; Yan et al, 1994). The CC chemokines, of which monocyte chemotactic protein (MCP)-1 is the prototype, predominantly affect monocytes.

Table 1. The chemokine superfamily. CXC chemokines ELR IL-8 Gro-α ENA-78

non-ELR

CC chemokines

PF-4 IP-10

MCP-1/2/3 MIP-1α/β RANTES Eotaxin

IL-8 = interleukin-8; Gro-α = growth-related gene-α; ENA-78 = epithelial-derived neutrophil attractant-78; PF-4 = platelet factor-4; IP-10 = interferon-inducible protein-10; MCP-1/2/3 = monocyte chemotactic protein1/2/3; MIP-1α/β = macrophage inflammatory protein1α/β; RANTES = regulated on activation, normal T cell expressed and secreted.

The chemokines bind to G-protein-coupled transmembrane receptors of the rhodopsin superfamily. Ligand binding leads to increased intracellular calcium and activation of protein kinase C (PKC). Chemokine receptors can be broadly subdivided into those that bind a single chemokine or those that bind a number of chemokines of either CXC or CC type (Table 2). For example the CXC-CKR2 receptor is specific

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Table 2. Human chemokine receptors (CKR), ligand binding specificities and cellular expression. Chemokine receptor

Ligand(s)

Cellular expression

Specific CXC-CKR2 CC-CKR2

IL-8 MCP-1, MCP-3

Neutrophils Monocytes

Shared CXC-CKR1 CC-CKR1 CC-CKR3 CC-CKR4 CC-CKR5

IL-8, Gro-α, NAP-2, ENA-78 MIP-1α, MIP-1β, RANTES, MCP-1, MCP-3 Eotaxin, RANTES, MIP-1α, MIP-1β MCP-1, MIP-1α, RANTES MIP-1α, MIP-1β, RANTES

Neutrophils Monocytes, eosinophils Eosinophils, monocytes, basophils Monocytes Monocytes, T cells

Non-signalling Duffy antigen

IL-8, Gro-α, RANTES, MCP-1, MIP-1α

Red blood cells

for IL-8 and binds it with high affinity while the CXC-CKR1 receptor binds IL-8 with high affinity but also a small number of other CXC chemokines, including growthrelated (Gro)-α and neutrophil-activating peptide (NAP)-2 (Murphy, 1994; Ben-Baruch et al, 1995). A third promiscuous chemokine receptor which binds several CC and CXC chemokines has also been identified on the surface of red blood cells. This receptor has no signal transducing activity and may act as a sink mopping up excess free chemokine and preventing inappropriate activation of circulating leukocytes (Wu et al, 1993; Horuk, 1994). Interleukin-8 IL-8 is a potent neutrophil chemoattractant and activating factor (Baggiolini et al, 1989). It is secreted by a wide variety of cell types, including monocytes/macrophages, neutrophils, endothelial cells, fibroblasts, epithelial cells, T cells, NK cells, keratinocytes and chondrocytes, either in a constitutive manner or following stimulation by proinflammatory mediators (Braun et al, 1993; Jeannin et al, 1994; Saito et al, 1994). Like most chemokines, IL-8 has a C-terminal heparin-binding site that allows it to become immobilized within the endothelial glycocalyx in the solid phase, thus presenting a stable gradient to passing leukocytes (Rot, 1992; Webb et al, 1993; Adams and Shaw, 1994; Clark-Lewis et al, 1994; Witt and Lander, 1994). Interleukin-8 is now known to be an important mediator in many inflammatory diseases (Saito et al, 1994). In patients with systemic inflammatory conditions such as sepsis/SIRS, circulating levels are raised and predict morbidity and mortality (Hack et al, 1992; Fujishima et al, 1996). In contrast to TNF-α and IL-1β, injection of IL-8 does not however induce a shock-like state. Levels of IL-8 are raised in AP and correlate with levels of neutrophil elastase, a marker of neutrophil activation (Gross et al, 1992; McKay et al, 1996). Other chemokines Although the importance of chemokines in inflammatory conditions is now well recognized, very little work has currently been undertaken to evaluate their role in AP. MCP-1 mRNA, along with mobl, a CXC rat chemokine, has been shown to be elevated within 1 hour of induction of AP in the rat by caerulein hyperstimulation

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(Grady et al, 1997). This certainly suggests that the chemokines are involved in the earliest stages of this condition. It has been shown in previous studies, however, that high levels of MCP-1 can be found in the healthy pancreas while levels are lower in patients with chronic pancreatitis (Walz et al, 1997). It has been shown that MCP-1 has a protective role in severe sepsis modulating the expression of inflammatory and anti-inflammatory cytokines. Plasma MCP-1 levels rise with time following administration of lipopolysaccharide (LPS) to CD-1 mice. Administration of antiMCP-1 antibodies resulted in increased mortality with corresponding rises in peak TNF-α, whereas administration of MCP-1 had a protective effect. Whether MCP-1 plays a similar role in AP remains to be determined (Zisman et al, 1997). MIP-1α levels are elevated in sepsis (Fujishima et al, 1996) and early production of MIP-1α is associated with poor outcome in infant respiratory distress syndrome (Murch et al, 1996). Transforming growth factor-␤ Transforming growth factor-β (TGF-β) is a 25-kDa homodimeric peptide (Massague, 1990). Three isoforms of human TGF-β showing significant sequence homology and similar biological activity have been identified (Massague, 1990). Most of the published literature relates to TGF-β1. TGF-β receptors are widely distributed and found on essentially all cell types. TGF-β is produced by numerous cells, including activated macrophages, fibroblasts and lymphocytes; platelets are also a rich source. It is released as an inactive peptide bound to its pro-peptide and requires activation either by proteolysis or low pH (Grainger et al, 1995). TGF-β is chemotactic for both macrophages and fibroblasts at femtomolar concentrations (Massague, 1990). TGF-β is a potent stimulator of fibroblast collagen and matrix production. It also decreases synthesis of collagenase while increasing production of collagenase inhibitors such as tissue inhibitors of metallo-proteinases (TIMPs). The overall effect is to stimulate formation of scar tissue (Slavin, 1996). TGF-β has pro-inflammatory effects but is also immunosuppressive (Whal et al, 1988; McCartney-Francis and Wahl, 1994). Injected subcutaneously, TGF-β stimulates an inflammatory infiltrate and induces angiogenesis and fibrosis (Roberts et al, 1986). Its various biological effects suggest that it plays an essential role in promoting the process of fibrotic repair (Slavin, 1996). In experimental models of AP, TGF-β1, 2 and 3 have each been identified during the early and later stages of acute pancreatitis. Expression is maximal at sites of collagen deposition (Kimura et al, 1995; Konturek et al, 1997; Riesle et al, 1997), suggesting a role in the control of post-pancreatitis repair. Exogenous TGF-β is able to promote chronic fibrosis after necrotizing acute pancreatitis induced by caerulein in mice. It has been suggested that the pattern of pancreatic fibrosis seen in this model is similar to that in chronic pancreatitis (van Laethem et al, 1996).

PATHOPHYSIOLOGY Initiating events Although there is considerable argument about the exact mechanisms by which an attack is induced, it is now thought that conversion of trypsinogen to active trypsin

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within the pancreatic acinar cell is a critical event. A number of mechanisms operate to prevent premature activation of trypsin and other digestive enzymes. Trypsin is packaged in discrete zymogen granules immediately after synthesis and these contain a specific pancreatic trypsin inhibitor. Proteases with trypsin lytic activity are also present, and trypsin itself has intrinsic autocatalytic activity. Only if these defence mechanisms are overwhelmed will an attack of acute pancreatitis occur (Steer, 1992; Hofbauer et al, 1998). If intracellular activation does occur then autodigestion begins, with subsequent acinar cell damage and release of active digestive enzymes. The role of the leukocyte Depletion of circulating leukocytes decreases both the magnitude of the local pancreatic damage and the systemic inflammatory response (Fujimoto et al, 1997). That leukocytes contribute to local damage is not in doubt, but the mechanisms by which pancreatic damage, enzyme activation and leukocyte migration and activation are linked remain incompletely characterized. Trypsin and other pancreatic enzymes have proteolytic activity and can cleave components of the complement system, producing the chemoattractants C3a and C5a. Levels of these are raised in experimental pancreatitis and may cause priming of circulating neutrophils and perhaps migration of activated neutrophils into the pancreas (Acioli et al, 1997). Lysis and activation of other inflammatory mediators such as the kinin/kallikrein systems or the production of 5-lipoxygenase metabolites likely contributes to the overall inflammatory stimulus (Folch et al, 1998). Leukocyte adhesion to endothelium, migration into inflamed tissues and subsequent leukocyte activation is controlled in part by chemokines. This process occurs in four clearly defined steps (Adams and Nash, 1996) (Figure 1):

Figure 1. Leukocyte recruitment into inflamed tissue. (1) Leukocyte rolling is mediated by weak selectinmediated adhesive interactions with the endothelium. (2) Chemokine binding to its receptor triggers conformational activation of integrins. (3) Strong, stabilized adhesion to the endothelium is mediated by integrins. (4) Diapedesis and migration into tissue. Adapted from Adams and Nash (1996, British Journal of Anaesthesia 77: 17–31).

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leukocyte rolling mediated by weak adhesive interactions with the endothelium; triggering; strong, integrin-mediated adhesion; leukocyte migration.

The first step in this process is mediated by the selectins, a family of lectin-like adhesion molecules. L-selectin is widely expressed on the surface of leukocytes and binds to carbohydrate moieties on the surface of endothelial cells. E-selectin and P-selectin are expressed on the surface of endothelial cells following cytokine activation (e.g. IL-1, TNF-α) and bind to oligosaccharide receptors on neutrophils. E-selectin and P-selectin are both long molecules which project into the lumen of the vessel allowing them to interact with circulating leukocytes. These allow weak tethering interactions between the leukocyte and the underlying endothelium. These interactions are dynamic and allow new contacts to be formed at the front of the rolling leukocyte while those at the back are broken. In the absence of a further triggering mechanism, or prolonged selectin–carbohydrate interaction, leukocytes are swept away in the circulation. Weak tethering of the leukocyte and the presence of a chemokine gradient triggers the transition from selectin-mediated adhesion to strong integrin-mediated adhesion and arrest of the rolling leukocyte (Adams and Nash, 1996) (Figure 2). The chemokines contain glycosaminoglycan binding motifs allowing them to bind to proteoglycans within the endothelial glycocalyx which then acts as a scaffolding presenting chemokine ligands to their receptors on passing leukocytes (Tanaka et al, 1993; Ebnet et al, 1996). Different chemokines have different affinities for proteoglycans, and as these may also be tissue-specific this may confer a degree of selectivity to leukocyte recruitment (Witt and Lander, 1994). The notion of chemokines fixed in a solid phase would be consistent with leukocytes migrating in the direction of a chemokine gradient. Without

Figure 2. Mediators of leukocyte–endothelial cell adhesion. Selectins on the surface of the leukocyte and endothelium bind to carbohydrate (CHO) moieities on the opposite cell. Chemokine ligand bound to proteoglycan (PG) binds to a seven-pass transmembrane receptor (7TMR) on the leukocyte, bringing about conformational activation of the β2 integrin Mac-1 on the surface via G-protein activation. This mediates strong binding to intracellular adhesion molecule (ICAM)-1 on the surface of the endothelial cell. Adapted from Adams and Nash (1996, British Journal of Anaesthesia 77: 17–31).

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a solid phase free chemokine released into the lumen would be instantly dispersed in the circulation, preventing the formation of a stable gradient and requiring continual release of chemokine. Occupation of a chemokine receptor on a leukocyte triggers a conformational activation of leukocyte integrins (Springer, 1994; Butcher and Picker, 1996). Whereas selectins can mediate activation-independent adhesion, the integrins require conformational activation for strong adhesion. The β2 integrin, Mac-1 is the predominant integrin expressed on the surface of neutrophils and interacts with the endothelial counter-receptors intercellular adhesion molecule (ICAM)-1 and ICAM-2. Interleukin-8 is known to promote this interaction and stabilize adhesion (Baggiolini et al, 1994). Following arrest of the leukocyte and strong adhesion further cytoskeletal changes occur with flattening of the leukocyte and ‘crawling’ along the endothelium in the direction of the chemokine gradient. Finally the leukocyte migrates between the endothelial cells in a process called diapedesis and enters the tissues. There it undergoes a respiratory burst with release of proteolytic enzymes, reactive oxygen species and other mediators capable of destroying tissue and microbial invaders and recruiting further leukocytes. Release of chemokines or other inflammatory cytokines, particularly IL-1β, TNF-α or PAF, can lead to activation of leukocytes within the general circulation. Leukocyte tethering, margination, migration and activation can then occur at distant sites and lead to widespread organ damage. These leukocyte-mediated mechanisms of tissue damage are particularly important in patients with severe pancreatitis and will be discussed in some depth. Systemic inflammatory response syndrome A clinical syndrome secondary to the systemic effects of infection has been recognized for many decades; conflicting terms have been used to describe this condition and include sepsis, septic shock and septicaemia. Subsequently it was realized that many patients with the features of sepsis had no identifiable focus of infection. To resolve the confusion in the use of nomenclature a collaborative conference sponsored by the American College of Chest Physicians and Society of Critical Care Medicine was held in 1992 (Bone et al, 1992). Sepsis is defined as a SIRS response in which there is an identifiable focus of infection. The systemic inflammatory response syndrome is an entirely normal response to injury. Many infective and non-infective causes other than AP are now recognized (Figure 3). As discussed above, marked systemic leukocyte activation (cytokine mediated) as a consequence of an aggressive SIRS can lead to distant organ damage and the onset of multiple system organ failure (MSOF). Acute pancreatitis provides an ideal model for studying the inflammatory pathways involved in SIRS/sepsis (Wilson et al, 1998), because pain is often the initial feature in AP and patients present early before the systemic features become marked (Norman, 1998). Additionally, there exists a therapeutic window in patients with severe AP during which specific anticytokine therapy may reduce later systemic organ failure (Figure 4). Patients who die from AP can be considered in two groups. Most deaths, about 60%, occur within the first week. These patients suffer a severe initial attack and develop an exaggerated systemic inflammatory response with the development of MSOF and death. Patients with a severe attack who survive beyond this period often go on to develop extensive retroperitoneal pancreatic necrosis. Persisting infection in

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Figure 3. Causes of the systemic inflammatory response syndrome (SIRS).

necrotic tissue leads to sepsis, a persisting systemic inflammatory response and MSOF and accounts for the 40% of patients who die late (Wilson et al, 1998) (Figure 5). Organ failure The first sign of MSOF in AP is often impaired lung function. The process affecting the lung is, of course, occurring simultaneously in other organs. Ashbaugh et al (1967) first described the adult respiratory distress syndrome (ARDS). ARDS is an inflammatory condition that affects the lungs, which become oedematous and congested leading to collapse of the smaller airways, increased lung compliance and respiratory failure. It has many causes, including major surgery, trauma, multiple transfusions and AP. In severe AP the very first changes can be seen a few hours after the onset of an attack. As a consequence of the SIRS response, leukocytes become activated within the general circulation and some then lodge within the pulmonary microcirculation. As the condition develops, leukocytes migrate into the pulmonary interstitium. Increased endothelial permeability leads to tissue oedema. Leukocyte-derived free radicals are thought to mediate much of the local damage seen in ARDS (Murakami et al, 1995). Other organs affected by an overactive inflammatory response include the kidneys and liver. Translocation of endotoxin and bacteria may occur through a compromised bowel wall and persisting endotoxaemia may aggravate the inflammatory response (Kivilaakso et al, 1984). Systemic leukocyte activation and increased endothelial permeability is, at least initially, a consequence of the inflammatory mediators released by the pancreas into the systemic circulation. Those directly implicated include IL-1β, TNF-α, IL-6, PAF and IL-8 (Parsons et al, 1992; Strieter et al, 1993; Dinarello, 1994; McKay et al, 1996; Norman, 1998). Teasing apart the exact contribution that each cytokine makes can be difficult as there appears to be a great deal of redundancy, but IL-1β, PAF and TNF-α appear to be particularly important as direct mediators of organ damage. Once the lungs, or other tissues, become inflamed, leukocytes within these

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M. Brady et al ENZYMES a) MILD ACUTE PANCREATITIS

'CYTOKINES'

12 hrs

24 hrs

48 hrs

5 days

b) SEVERE ACUTE PANCREATITIS

'INTERVENTION'

12 hrs

24 hrs

48 hrs

5 days

Figure 4. Cytokine flux, enzyme levels and organ failure in patients with (a) mild AP, and (b) severe AP. Note the possible attenuation of cytokine levels in severe AP following specific anti-cytokine therapy, and the corresponding effect on multiple system organ failure (MSOF).

Cytokines and acute pancreatitis Phase I 1st week

PANCREATITIS

SIRS

MSOF Phase II 2–4 weeks

PANCREATIC NECROSIS

Phase III 4–6 weeks

PSEUDOCYST PANCREATIC ABSCESS

277

INFECTION

DEATH

SEPSIS

Figure 5. Causes of multiple system organ failure (MSOF) and death in acute pancreatitis.

areas will constitute a further source of inflammatory mediators (Norman et al, 1997a). It is likely that there are other, as yet unidentified mediators promoting these events (Denham et al, 1997b). Two-hit hypothesis Patients with a severe attack who survive the initial insult often die following a relatively minor second event that would not normally be life-threatening. The two-hit hypothesis helps to explain this phenomenon (Moore and Moore, 1995) (Figure 6). This hypothesis suggests that an initial overactive SIRS somehow primes the inflammatory response. If no further insults occur then recovery is possible. If there is a second hit, even if this is quite a minor event such as a line infection or chest infection, it will lead to an exaggerated secondary inflammatory response and death. Thus, in patients with severe pancreatitis every effort should be made to avoid a second hit, and invasive procedures should be kept to a minimum in the absence of a clear clinical indication (Ogawa, 1998).

Pancreatitis (First insult) Local injury

Systemic Inflammatory Response Syndrome

Not Primed MSOF

RECOVERY Primed (Second insult)

MSOF DEATH Cannot resuscitate Figure 6. ‘Two hit’ hypothesis.

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Reparative phase In mild AP there is minimal tissue damage and there is resolution of the inflammatory process with relatively little tissue damage. Tissue from those with severe AP contains areas of both oedema and necrosis. Veins and venules are often affected with granulocytic infiltration, thrombosis, necrosis, rupture and haemorrhage. Arterioles are less often affected, but when affected panlobular necrosis results. Expanding fat necrosis may lead to focal duct and perilobular acinar cell necrosis. Repair of necrotic tissue is mostly by fibrosis with deposition of collagens type 1 and 3 by fibroblasts. TGF-β1, synthesized by recruited mononuclear cells, is thought to be the major stimulus for fibrotic repair (Menke et al, 1997; Muller-Pillasch et al, 1997). Immunosuppression Immunosuppression is seen after major surgery and trauma (Meakins et al, 1997). In patients in whom it is more marked there is increased morbidity and mortality (Johnson et al, 1979; Christou, 1986). In patients with AP, delayed-type hypersensitivity reactions are impaired and there are an increased number of complications in those with anergy (Garcia-Sabrido et al, 1989). Sepsis, the major cause of late death in AP, may be accentuated by immunosuppression. CD4+ cells are the major source of IL-2, which is required for a normal immunological response. A reduction in systemic and circulating levels of IL-2 may in part explain the immunosuppression that is seen following a severe attack of pancreatitis (Curley et al, 1993; Pezzilli et al, 1994). Murine diet-induced AP is associated with impaired immune function and increased susceptibility to sepsis and may be a valuable tool for the investigation of the immune response in AP (Curley et al, 1996). IL-10, TGF-β and certain chemokines have the capacity to reduce the magnitude of the inflammatory response. It is likely that in individuals who are systemically unwell there is a dynamic balance between pro- and anti-inflammatory cytokines. The exact effect on and relevance of specific immunity to this balance is not yet clear.

PREDICTION OF SEVERE DISEASE Optimal management of a patient with AP requires an early accurate assessment of disease severity. Monitoring and supportive care can then be targeted at patients with severe disease. Equally, eligibility for entry into clinical trials can be determined so that results from different studies can be compared. Specialized clinical scoring systems such as the Ranson or Glasgow systems, or general systems such as Acute Physiology and Chronic Health Evaluation II (APACHE 2), are in widespread use. If the magnitude of the systemic inflammatory response is the major factor determining systemic damage and the development of organ failure, then the levels of the inflammatory mediators promoting this response should be good indicators of the severity of disease. Patients with organ failure or more severe disease do indeed seem to have raised levels of several cytokines (Borrelli et al, 1996; de Beaux et al, 1996). Early work focused on the role of non-specific markers of inflammation such as C-reactive protein (CRP). Acute phase proteins are synthesized in the liver in response to stress and include alpha-1-antitrypsin, haptoglobin and CRP. The best known is CRP, which binds to bacterial cell walls before activating complement. CRP levels are

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raised in AP and correlate with severity of disease (Wilson et al, 1989; Vesentini et al, 1993). Production of CRP is integrally linked to circulating levels of IL-6, as IL-6 induces transcription of the human CRP gene within the liver. Significant correlation has been demonstrated between plasma concentrations of CRP and IL-6 in both sepsis and AP (Leese et al, 1988; Wilson et al, 1989; Leser et al, 1991; Heath et al, 1993; Vesentini et al, 1993). The rise in CRP follows that of IL-6 with a delay of about 24 hours (Messmann et al, 1997). IL-6 levels are also a good measure of the severity of disease (Leser et al, 1991; Damas et al, 1992; Gross et al, 1993; Heath et al, 1993; Viedma et al, 1992, 1994). Although circulating levels of TNF-α are raised in patients with severe pancreatitis (Exley et al, 1992) levels are a very poor indictor of the severity of disease. This is because raised levels are present only intermittently, and excess TNF-α is soon mopped up by circulating soluble receptor and the liver (Paajanen et al, 1995). Levels of circulating soluble receptors, sTNFR55 and sTNFR75, are a better predictor of organ failure and are probably a better indication of TNF-α production (de Beaux et al, 1996). IL-1β activity is similarly rapidly neutralized (Norman et al, 1995b). Interleukin-8, assayed during the first 24 hours of an attack is a better marker than CRP for evaluating the severity of AP (Pezzilli et al, 1995). Levels are higher in patients with infected necrotic tissue than in those with sterile necrotic tissue and after surgical treatment of infected necrotic tissue median IL-8 values continue to be significantly higher in patients with persisting pancreatic sepsis (Rau et al, 1997). IL-10 is not normally measurable in the blood of healthy individuals. Following an attack of pancreatitis, elevation of IL-10 on the first day of the illness is more marked in patients with mild AP than in those with the severe form of the disease. Low values of serum IL-10 in severe AP suggests that there may be impairment of the immune/inflammatory response in these patients (Pezzilli et al, 1997). Serum HGF/scatter factor has recently been shown to be raised in patients with AP; its sensitivity and diagnostic accuracy were similar to those of other non-specific markers of the inflammatory response (Ueda et al, 1997).

ANTI-CYTOKINE THERAPY Most patients with AP die from MSOF as a consequence of systemic leukocyte activation. Depletion of circulating leukocytes with anti-neutrophil serum reduces the damage and prolongs survival time in experimental models of AP (Han et al, 1996). Antibodies that prevent the adhesion, or migration, of neutrophils, or the use of a neutrophil elastase inhibitor (Guo et al, 1995) reduce the severity of pulmonary damage in AP (Inoue et al, 1995, 1996). More specific antagonists or modifiers of the inflammatory response might be useful for the treatment of AP. The importance of TNF-α and IL-1β as mediators of MSOF was realized nearly a decade ago. Numerous investigators have employed specific antagonists with considerable success in experimental models of sepsis/SIRS, including AP. These treatments can be administered at the time of induction, so-called prophylactic therapy, or once the disease process has been induced, so-called therapeutic therapy. Both methods of administration appear to be effective in AP. The naturally occurring interleukin-1 receptor antagonist (IL-Ira) has been assessed in several experimental animal models of AP with promising results (Norman et al, 1995b,c; Tanaka et al,

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1995; Fink et al, 1997) (Table 3). Treatment with IL-Ira decreased local pancreatic inflammation, as assessed by biochemical and histological criteria, and decreased systemic complications. Polyclonal antibodies (PAbs) against TNF-α have similar effects (Grewal et al, 1994b; Hughes et al, 1996a,b) (Table 4). Delayed therapy with soluble type I TNF receptor (TNFbp) improved survival. Strategies that interfere with cytokine gene transcription, translation, intracellular processing and cytokine release rather than antagonising their effects have also met with modest success in experimental models (Hughes et al, 1996c; Denham et al, 1997c; Dunn et al, 1997; Norman et al, 1997b).

Table 3. Anti-IL-1 therapies used in experimental acute pancreatitis. Reference

Model

Intervention

Results

Norman et al (1995b)

Caerulein, mouse

Prophylactic/therapeutic IL-Ira

Reduced IL-6, TNF-α, wet weight and lipase (all P <0.01). Reduced oedema, necrosis, inflammation (all P <0.05) and serum amylase (P < 0.05)

Norman et al (1995c)

CDE diet, mouse

Prophylactic/therapeutic IL-1ra

Reduced pancreatic wet weight, serum amylase and lipase (all P <0.05). Reduced pancreatic damage, cytokine rise (both P <0.05) and mortality (P < 0.001)

Tanaka et al (1995)

Deoxycholate infusion, rat

Prophylactic IL-1ra

Reduced mortality and lung inflammation

Fink et al (1997)

Caerulein/CDE diet, rat

Prophylactic IL-1ra

Reduced pancreatic amylase and tissue necrosis

Norman et al (1997b)

Bile infusion, rat

Prophylactic ICE inactivator

Reduced IL-1β and TNF-α processing and secretion (all P < 0.001). Reduced necrosis, oedema, inflammation, wet weight (all P < 0.05), amylase and lipase (both P < 0.01)

Table 4. Anti-TNF-α therapies used in experimental acute pancreatitis. Reference

Model

Intervention

Results

Grewal et al (1994b)

Bile infusion, rat

Prophylactic TNF PAb

Reduced serum TNF-α, amylase (both P <0.001) and overall severity

Hughes et al (1996a)

Bile infusion, rat

Prophylactic TNF PAb

Reduced severity, increased survival

Hughes et al (1996b)

Bile infusion, rat

Prophylactic TNF PAb

Reduced early rise in TNF. Increased survival, reduced severity

Norman et al (1996)

CDE diet, mouse

Prophylactic/therapeutic soluble TNF receptor

Reduced oedema, serum amylase, lipase,IL-1β and IL-6 (all P < 0.05). Reduced mortality

Hughes et al (1996c)

Bile infusion, rat

Prophylactic Ca2+ channel Reduced serum TNF-α, histological blockade scores. Increased survival

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Treatment with IL-1β and TNF-α antagonists in patients with sepsis has been investigated in a number of clinical trials. The results, however, have been less encouraging (Fisher et al, 1994; Abraham et al, 1998). Most of these trials contained patients with sepsis from many causes. Such heterogeneous patient populations confuse the interpretation of data. No trials have been performed exclusively using patients with severe AP; it is thus hard to draw specific conclusions about the value of anti-TNF-α or anti-IL-1β therapy in this condition. Like IL-1β and TNF-α, PAF is believed to be a key mediator in AP. A wealth of PAF antagonists have become available in recent years and have been evaluated in both animal experimental models and human trials with varying success (Table 5). Prophylactic treatment with a number of these antagonists causes a reduction in local inflammation and acinar cell necrosis (Fujimura et al, 1992; Tomaszewska et al, 1992; Formela et al, 1994; Galloway and Kingsnorth, 1996; Sandoval et al, 1996). In a recent study, however, lexipafant failed to reverse the effects of AP, induced by infusion of glycodeoxycholate and enterokinase in to the bile duct in combination with a supramaximal dose of caerulein administered intravascularly, on survival or local inflammation (Rivera et al, 1998). Therapy was commenced immediately following induction of AP. The degree of pancreatitis induced in this model is very severe, and it is possible that pro-inflammatory signals are so strong that they completely overwhelm any beneficial effect. Table 5. Anti-PAF therapies used in experimental acute pancreatitis. Reference

Model

Intervention

Results

Fujimura et al (1992)

Caerulein, rat

Prophylactic PAF antagonist, CV-6209

Reduced serum pancreatic enzymes, wet weight, oedema and inflammation

Tomaszewska et al (1992)

Caerulein/PAF, rat

Prophylactic PAF antagonist, TCV-309

Reduced caerulein AP and prevented PAF AP

Formela et al (1994)

Ischaemic AP, rat

Therapeutic PAF antagonist, BB-882

Reduced severity. Reduced serum amylase and histological score (both P < 0.001)

Galloway and Kingsnorth (1996)

Ischaemic AP, rat

Therapeutic PAF antagonist, BB-882

Reduced lung capillary permeability (P < 0.01)

Sandoval et al (1996)

Caerulein, rat

Prophylactic PAF antagonist, BN52021

Reduced inflammation and acinar cell damage

Rivera et al (1998)

Bile acid/enterokinase infusion plus caerulein, rat

Therapeutic PAF antagonist, lexipafant

No significant reduction in mortality. No significant difference in survival time or histological score

These encouraging early results led to the establishment of a small pilot study to investigate the effect of the PAF antagonist lexipafant in humans (Kingsnorth et al, 1995). Eighty-three patients were randomized to receive either an infusion of lexipafant 60 mg per day for 3 days (42 patients), or placebo (41 patients). Severity of disease was assessed by the APACHE II system. There was a significant improvement in the organ failure score in the treatment group, although there was no difference in mortality. A second study from Glasgow has confirmed these findings (McKay et al, 1997).

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These two studies were followed by a large multi-centre trial of lexipafant, the results of which have recently been reported. Two hundred and ninety patients with severe pancreatitis (APACHE-II>6) were recruited to the trial. Patients were randomized to receive placebo or lexipafant (100 mg/24 hours for up to 7 days). Seven patients did not satisfy the inclusion criteria and were excluded from the final analysis. At 3 days organ failure scores were significantly better in patients treated with lexipafant. If the results of those patients treated within 48 hours of the onset of an attack are analysed, then a significant reduction in mortality is seen. One of the findings was the reduction in the incidence of acute pseudocyst from 14% in the placebo group to 5% in the lexipafant group (Table 6) (Kingsnorth, 1997). These results are promising and suggest that the PAF antagonist lexipafant may be of use in the treatment of AP. A further large multi-centre international trial to assess the effect of different dosages of lexipafant is currently in progress and will probably finish towards the end of 1998.

Table 6. Summary of results from clinical trial of lexipafant. . Mortality (intent to treat) Mortality (attributable) Mortality (intent to treat) treated within 48 hours of onset Mortality (attributable) treated within 48 hours of onset Organ failure scores change from baseline at day 3 median (interquartile range) Local complications (necrosis, pseudocysts or abscess) Pseudocysts

Placebo

Lexipafant

Probability

24/139 (17.3%) 21/136 (15.4%) 20/98 (20.4%)

18/151 (11.9%) 14/147 (9.5%) 11/107 (10.3%)

0.196 0.13? 0.04?

17/95 (17.9%)

8/104 (7.7%)

0.03?

0 (–1–0)

–1 (–1–0)

0.04?

41/138 (29.7%)

30/148 (20.3%)

0.065

19/138 (13.8%)

8/148 (5.4%)

0.02?

Reproduced from Kingsnorth (1997, Gastroenterology 112: A453) with permission.

IL-10 inhibits release of IL-1β, IL-6 and TNF-α, and this decreases the severity of the inflammatory response. Several studies have shown a protective effect following administration of IL-10 in models of sepsis (Howard et al, 1993; Smith et al, 1994; Rongione et al, 1997a; van der Poll et al, 1997). Administration of IL-10 in experimental AP leads to a reduction in both the local inflammatory response and subsequent mortality (van Laethem et al, 1995; Kusske et al, 1996; Rongione et al, 1997b). The chemokines represent another therapeutic target. Recent work has demonstrated that specific anti IL-8 or MCP-1 strategies can reduce inflammation in both localized and systemic inflammatory conditions (Sekido et al, 1993; Harada et al, 1994; Yokoi et al, 1997). Strategies include the use of blocking antibodies and receptor antagonists (Sekido et al, 1993). The promiscuous nature of chemokine receptors and the redundancy of function shown by various chemokines suggests that the use of specific receptor antagonists will be a particularly fruitful approach. It was shown recently in knockout mice, that deletion of the MIP-1α/RANTES receptor decreased the pulmonary damage seen in severe acute pancreatitis (Gerard et al, 1997). Gene therapy is one final exciting option. In a mouse model of

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caerulein-induced pancreatitis, transfection with a cationic liposome plasmid vector containing human recombinant IL-10 24 hours prior to induction, was successful in reducing severity by biochemical and histological criteria. These effects were presumably achieved through attenuating the early rise in TNF-α and IL-1β (Norman et al, 1997c). These studies confirm the key role of cytokines in eliciting the full inflammatory response in severe AP and their potential as therapeutic targets.

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