Inflammatory intermediaries in inflammatory bowel disease

Int J Colorect Dis (1989)4:75-90

Review

~j ........ ~ j o o l ( oliii ecta Disease

�9 Springer-Verlag 1989

Inflammatory intermediaries in inflammatory bowel disease

K. Lauritsen ~, L.S. Laursen ~, K. Bukhave 2 and J. Rask-Madsen 3

1 Department of Medical Gastroenterology, Odense University Hospital, Odense z Department of Biochemistry and Nutrition, The Technical University of Denmark 3 Section of Gastroenterology, Department of Medicine G, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark

The chronic inflammatory bowel diseases (IBD) include ulcerative colitis (UC) and Crohn's disease (CD). Their causes and precise relations remain obscure which is why these conditions are commonly designated as "nonspecific" or "idiopathic". The current concept of their pathogenesis involves an interaction between host responses, immunologic genetic influences, and environmental agents [1-3]. In the absence of decisive knowledge of the causes of IBD no consistently curative medical treatment exists and the foundation of drug treatment remains empirical. Within the last decade, however, increased research interest has been fo- cused on soluble intermediaries of the inflammatory response with the major object of uncovering specific targets for pharmacologic manipulation.

The inflammatory response

Inflammation is a term used to describe a series of responses of vascularized tissues to injury. The clinical signs of this phenomenon, acknowledged since ancient times (Celsus), can be related to increased local blood flow (calor and rubor), increased vascular permeability and cellular infiltration (tumor), and release of a variety of substances at the site of inflammation that induces pain (dolor). Later, loss

Abbreviations." 5-ASA, 5-aminosalicylic acid; C, complement- derived peptide; CD, Crohn's disease; FMLP, N-formyl- methionyl-leucyl-phenylalanine; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroxypcroxyeicosatetraenoic acid; IBD, inflammatory bowel diseases; IL, interleukin; LT, leucotriene; NSAID, nonsteroidal anti-inflammatory drug; PAF-acether, platelet-activating factor; PG, prostaglandin; ROS, reactive oxygen species; TNF, tumour necrosis factor, TX, thromboxane; UC, ulcerative colitis

of function was added by Virchow as an additional cardinal sign of inflammation (functio laesa).

The mechanisms of inflammation depend great- ly on the site and the stimulus and are influenced by genetic, nutritional, as well as hormonal factors. Furthermore, the processes themselves represent an extensive network of interacting mechanisms, mediators, and cells.

In principle inflammation is a beneficial reaction of tissues to injury, which normally leads to removal of the inciting agent and repair of the in- jured site. In contrast, the uncontrolled inflammatory reactions occuring in IBD may induce perma- nent tissue destruction.

The term "inflammatory intermediary" should, in essence, be reserved for chemical substances, which signal information between different cells involved in the inflammatory process. More than a hundred compounds appear to mediate some aspect or other of the inflammatory process, and this number will increase considerably if the current rate of discovery is maintained [4]. Inflammation thus becomes ever more complex, but over the last few years evidence for an ordered control of the inflammatory response has begun to emerge in the sense that inflammation is one of the integral parts of the body defence mechanisms.

The criteria used to determine whether an endogenous substance serves a primary role in mediat- ing inflammation has been considered by Vane [5]: (i) the substance should be detectable, at the site of inflammation, in amounts adequate to account for the effect under consideration; (ii) the substance, when administered in concentrations of the order of those found in the lesion, should produce the observed effect; (iii) specific blocking agents or antagonists of the effects of the substance should prevent or attenuate the effect; (iv) prevention of release of the substance should abolish or prevent the

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MEMBRANE PHOSPHOLIPID

Phosphollpases (A2, C)

ARACHIDONIC ACID

C y c l o o x y g e d ~ 15-HPETE /Pathway k "Lipo~enase

/ ~ S " - ~ 12-HPETE CYCLIC ENDOPEROXIDEL ~ 5-HPETE

PGI S / I ~ "~ TXA 2 5-HETE " I " PoGD' 1 PGF2" [

OH OH I ~ , PGE 2 ~ OH

6-keto-PGF1. TXB 2 LTB 4

', 15-HETE

, 12-HETE

H O LTA.

jr LTC4'-e.. LTD4-e.. LTE 4

Fig. 1. Biosynthetic pathways ofeicosanoid metabolism leading to formation of prostaglandins (PGs), prostacyclin (PGI2), thromboxanes (TXs), hydroxyperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), and leucotrienes (LTs)

effect; and (v) agents or procedures preventing the breakdown or removal of the substances should prolong or potentiate the effect.

Eicosanoids

Chemistry and cellular origin

These compounds (eicosa derived from the Greek term denoting 20) constitute a group of biologically active lipid mediators derived from 20-carbon "essential" fatty acids. In mammals, the most important is arachidonic acid (5,8,11,14-eicosatetraenoic acid), which is released from cellular phospholipids by the activity of various phospholipases [6, 7] and subsequently metabolized either through the cyclo-oxygenase pathway [8] to prostaglandins (PGs), prostacyclin (PGI2), and thromboxanes (TXs) or through the lipoxygenase pathways [9, 10] to hydroxyperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), and leucotrienes (LTs) (Fig. 1). PGs are designated A through F depending on the exact structure of the cyclopentane ring. The subscript number (for example, 1, 2, or 3) denotes the number of double bonds in the side chain. The Greek

letter that follows (either alpha or beta) describes the stereochemistry at the carbon 9 position. Also for LTs the subscript figure (3, 4, or 5) indicates the number of double bonds, three of which occur at alternate carbon atoms (a conjugated "triene").

Eicosanoid formation in mammalian tissues is ubiquitious, but the profile of eicosanoids varies dependent on species, tissue, and cell population within the tissue. Most studies on the regulation of eicosanoid synthesis have concluded that the availability of arachidonic acid is the limiting factor for the total output of its metabolites [6]. Arachidonic acid may be released by the Ca2+-dependent enzyme, phospholipase A2, from phosphatidylcholine, phosphatidylethanolamine, and phospha- tidylinositol [6,7]. However, release of arachidonic acid can also be derived from inositol phospholipids through alternative reactions catalyzed by phospholipase C and diacylglycerol [11]. Very little is known about the regulation of further steps either in the cyclo-oxygenase or the lipoxygenase pathways. Several mechanisms and routes of inactivation and degradation are available, either locally or in the lungs, but neither oxidation nor substi- tution or uptake by excretory organs are known to be under specific control. On stimulation substantial amounts of eicosanoids may be released, but no indications are presently available for storage and only little is known about the mechanism of excre- tion from the producing cells.

Different cell types have different potentials for arachidonic acid metabolism. Whether receptor- mediated regulation of oxidative metabolism of

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arachidonic acid relates to the distribution and action of membrane phospholipases and lipases or to the presentation of phospholipid substrates or both is not known. Whereas the oxidative metabolism of arachidonic acid to cyclooxygenase products via five or more terminal synthetases [12] with differential cellular prominence is characteristic of most cell types, 5-1ipoxygenation appears to be less widely distributed, mainly to neutrophils, eosinophils, monocytes, macrophages, and mast cells. These cells originate in the bone marrow and probably derive from the same stem-cell. Further, among these cell types relevant to host inflammatory response, the generation of LTs, quantitatively and qualitatively, exibits remarkable cellular speci- ficity [13]. Thus in the stimulated neutrophil, the major route of arachidonic acid metabolism along the 5-1ipoxygenase pathway accounts for production of 5-HETE and LTB 4 [14]. With eosinophils, the preferentially generated compound is LTC 4 [15]. Monocytes and macrophages are able to synthesize both LTB~ and the sulfidopeptide-lipid LTs [12].

Methodological problems

Materials for studying the role of eicosanoids in IBD have comprised not only patients with the disorders and healthy controls, but also a variety of animal models [16, 17]. The pattern of arachidonic acid metabolism in these models (and the effect of medication) varies considerably with the model and the experimental conditions [18-20] and the results are interpreted in the light of the notion that lack of a reliable model for IBD has usually been considered a hall-mark of these disorders. The experimental designs applied include various in vitro methods, such as determination of contents of eicosanoids or activities of enzymes in tissue homo- genates and generation of eicosanoids in organ cultures or in leucocytes derived from blood or intestinal tissues. These approaches may define the capacity of the tissue to produce different eicosanoids, and thus the profile of arachidonic acid metabolites. Ex vivo organ perfusions have also been applied. The in vivo methods include equilibrium and nonequilibrium dialysis against rectal mucosa or faeces. These approaches appear presently to provide a more reliable index of the balance between intestinal mucosal eicosanoid formation and degradation in vivo [21-23], since they limit artificial eicosanoid formation.

Currently available analytical methods are bioassays, thin-layer chromatography with auto- radiography, radioimmunoassays, high pressure

liquid chromatography, and gas chromatography- mass spectrometry [8-10, 21].

Formation of eicosanoids in IBD

Since Gould [24] first reported raised concentrations of PG-like material in stools from patients with an acute attack of UC, in vitro experiments with fresh biopsies of colonic mucosa obtained from patients with IBD have demonstrated PGE2- , LTB4- , and 5-HETE concentrations three, six and ten times higher than in normal tissues [25, 26], in addition to increased activities of PG synthetase [27]. The amounts of PGE z, PGF2~, 6-keto-PGFl~ (that is, the stable breakdown product of PGI2) , TXB 2 (that is, the stable breakdown product of T X A 2 ) , 12-HETE, 15-HETE, LTB4, and LTC4 generated by cultures of rectal mucosa have also been reported abnormally increased [25, 26, 28-301.

Similarly, the accumulation o fPGE 2 and TXB 2 in the medium of cultured peripheral blood mononuclear cells [31] and intestinal mononuclear cells [32] from patients with CD, but not in the medium of cells from patients with UC [31, 32], has been reported significantly increased compared with that measured in the medium of cells from controls. By contrast, neutrophils activated with the calcium ionophore A23187 [33] appear to have increased capacity for release of LTBa in both groups of patients, but not if activated during phagocytosis of artificial immune complexes [34]. Also the formation of LTB 4 and LTC 4 in organ cultures obtained from rectal biopsies of patients with CD is increased compared to control cultures [30, 35] and the ileal mucosa from a small group of patients with CD has shown a trend toward increased phos- p h o l i p a s e A 2 activity, even in unaffected areas [36].

Measurements of tissue concentrations of eicosanoids are suspect, however, because these compounds are generally not stored. The effect of ma- nipulating (for example, by biopsy) could be to activate membrane bound phospho!ipases with subsequent artificial eicosanoid formation [20-22, 37, 38], which would also reflect increased arachidonic acid contents in the phospholipids as observed in patients with UC [39]. Similar limitations apply to tissue culture techniques [23]. Also the conclusions based on profiles of products obtained in response to ionophore activation should be tem- pered due to the limited data available for cells in response to transmembrane physiologic stimuli [13]. Finally, the findings that intestinal phagocytes appear to behave differently from similar cells in

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peripheral blood originating in patients with IBD have emphasized the limitation of data obtained from studies of phagocytes in blood [40].

Measurements of blood or plasma levels of eicosanoids as well as urinary excretions are inappropriate for estimation of local production. Also results of plasma determinations of eicosanoids are often artifactual because synthesis may occur during bloodletting. Consequently, plasma concentrations may vary by orders of magnitude [21, 41].

In vivo formation of eicosanoids has been esti- mated, therefore, by measuring the rate of PGE 2 accumulating in dialysis bags placed in the empty rectum [42-46] or the equilibrium concentrations of PGE 2 and LTB 4 [47-53] by use of the same technique or the original method [54] of in vivo dialysis of faeces [48, 51, 52, 55]. The named methods have demonstrated that the luminal concentrations of eicosanoids are always raised in active disease, but decrease toward normal levels in inactive disease. Also profiles of eicosanoids have been determined by equilibrium in vivo dialysis of the rectum in consecutive untreated patients with IBD and Clostridium difficile associated colitis [50]. PGE2, PGF2~ and TXB2, but not 6-keto-PGFl~, were abnormally high in all types of inflammation, whereas extreme rises in PGE 2 and TXB/ levels were observed in patients with UC. By contrast, the luminal concentrations of LTB4 were significantly increased only in the group of patients with UC, although abnormally high values were observed in single cases of Crohn's colitis and Clostridium difficile colitis with rectal ulceration [50].

Eicosanoids and the activity o f IBD

Positive correlations between the luminal concentration of PGE 2 or LTB 4 and disease activity as judged by clinical, endoscopic, or histologic grad- ings have been demonstrated by means of the method of equilibrium in vivo dialysis of the rectum in patients with distal UC [47]. A decrease in raised concentrations of the named eicosanoids toward the levels observed in healthy controls occurred only in patients who responded to treatment. Fur- thermore, pretreatment PGE 2 and LTB 4 levels seemed to be significantly higher in patients not responding to therapy than in those who improved [47], Approximately 15 % of patients with UC con- fined to the rectum and sigmoid colon are resistant to most forms of drug treatment [56]. If pretreatment levels of PGE2 and LTB4 reflect local disease activity, they may prove more useful predictors of

the outcome of medical treatment in these patients than clinical indices of disease activity [48, 51].

Finally, it has been suggested that a rise in PGE2 concentrations in rectal dialysis fluid from patients with UC in complete remission precedes the development of clinical symptoms in those who later suffer a relapse [52]. In vivo dialysis of the rectum for determination of luminal PGE 2 concentrations may, therefore, improve decision-making in symptom-free patients, who wish to stop sulphasalazine medication, but relatively frequent measurements are desirable [52].

Potential role in inflammation

The role of eicosanoids in various aspects of intestinal function, such as transmucosal ion transport, mucosal blood flow, microvasculature, and motility, in addition to mucus composition and secretion has been reviewed elsewhere [57-60]. The present paper will consider only the potential role of eicosanoids in inflammation.

Prostaglandins enhance vasodilation and oedema formation, acting synergistically with other mediators of inflammation (for example, histamine and bradykinin), whereas LTs promote leucocyte migration and stimulate aggregation and degranulation of neutrophils and the release of lysosomal enzymes and superoxide production [14, 61-66]. Hence, LTB 4 is the most potent chemotactic (i.e., stimulating directional movements of neutrophils) and chemokinetic (i.e., stimulating random move- ment of neutrophils) eicosanoid and its activity ex- ceeds that of other eicosanoids 100-fold [67]. The cell motion and secretory activities of LTB 4 could be due to changes in calcium and sodium flux across the cell membranes of neutrophils [68]. LTB 4 induces neutrophil chemotaxis in vitro (69) at concentrations of 3 ng/ml with a maximum response at 30 ng/ml - comparable to concentrations observed in rectal dialysates from patients with UC [50]. Also 5-HETE is a weak chemotactic agent, whereas LTC4, LTD 4 and LTE4 (collectively accounting for the biological activity of "slow-reacting substance of anaphylaxis") have vasoconstrictor activity and contribute to oedema by increasing capillary permeability.

There is increasing evidence that platelets, in addition to their function in haemostasis, play a role in inflammation by exhibiting a platelet- leucocyte cell-to-cell interaction [70]. Indeed, the platelet eicosanoid, 12-HPETE, formed during platelet aggregation leads to activation of human neutrophil 5-1ipoxygenase up to levels obtained upon

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stimulation of the cells with the calcium ionophore A 23187 [12]. Considering the findings of markedly enhanced in vivo LTB 4 formation in UC [47] to- gether with the known biological effects of the compound (especially its chemotactic and chemokinetic properties) is suggestive of a triggering role of this substance in the inflammatory process. Hypothet- ically, tissue macrophages and neutrophils present in the intestinal mucosa would be activated by phagocytic and chemotactic stimuli, such as the complement-derived peptide C5a [71], and by mediators released by aggregating platelets, in addition to various kinins [72]. LTB 4 would then be released and act as a chemoattractant to stimulate further the migration of neutrophils, thus accelerating the development of the inflammatory reaction. The essential function of LTB 4 would, therefore, be to amplify or stabilize the chemotactic gradients [68, 73].

Platelet-activating factor (PAF-acether)

PAF-acether is a phospholipid mediator [74] formed by eosinophils, macrophages, neutrophils, platelets, and vessel endothelium. Like eicosanoids, but unlike other potential mediators of inflammation, PAF-acether is not stored as such in the cells, but is present as a precursor phospholipid (1-0- alkyl- 2 (R) acyl- glycero- 3- phosphatidylcholine) in cell membranes. It is released by de-acylation to lyso-PAF, following activation of phospholipase A 2 and subsequent acetylation by an acetyl trans- ferase. PAF-acether is catabolized by at least two different acetyl hydrolases, a cytosolic and a plasma enzyme [75], the latter being a very effective means of removing circulating PAF-acether.

Formation of PAF-acether accompanies the stimulation of most inflammatory cells as well as platelets and vascular endothelial cells. Further- more, PAF-acether activates eosinophils, neutrophils and platelets [76]. The pharmacological actions include a variety of in vivo effects related to inflammation, particularly to immediate hypersensitivity [75, 77]. Increased formation of PAF- acether determined by bioassay and characterized by its migration on thin layer chromatography has been reported in incubated colonic mucosa obtained from patients with UC [78], in addition to tissue levels in various models of colitis [79]. Similar methodological constraints as described for eicosanoids must be acknowledged in interpreting these findings.

Different PAF-acether antagonists have been synthesized [75] but their role in experimental and

human colitis has not been worked out. In any case, the burst in PAF-acether antagonists will help to unravel the situation and is likely to provide new tools and more important, possible new remedies.

Biogenie amines (histamine and 5-hydroxytryptamine)

Histamine (2-(4-imidazolyl)ethylamine) and 5-hydroxytryptamine are naturally occurring amines stored within the body and capable of eliciting major changes in a variety of bodily functions [80-82]. Currently, it appears that 5-hydroxytryptamine is a mediator of acute inflammation in rodents only [83]. Histamine is formed by decarboxylation of histidine and is stored primarily in mast cells, although histidine decarboxylase activity has been found in most tissues.

Release of histamine from mast cells immedi- ately after injury has been reported in response to a number of stimuli ranging from anaphylaxis to both physical and chemical insults. Local administration of histamine elicits classical vascular responses of acute inflammation, such as increase in blood flow and vascular permeability, oedema formation, and pain and itching. The histamine H 1 receptors are considered to be responsible for the pro-inflammatory activities. By contrast, sustained release of histamine in chronic inflammation, such as in IBD, has not been proven, but histamine may serve as a co-mediator to PGs in potentiating an increase in vascular permeability. Studies with histamine antagonists are consistent with the role of histamine in the early phase of acute inflammation and not in more sustained inflammation.

Kinins

Kinins are a group of small peptides formed in blood and biological fluids by the actions of kallikreins (kininogenases) on alpha 2 globulins, the kininogens [84 86]. In general, the concentration of kinins in circulating blood and tissue is low since the kallikreins are coupled to inhibitors and kept as inactive precursors, the prekallikreins. When an appropriate physiological or pathological stimulus activates the prekallikreins, the nonapeptide bradykinin is formed in the blood by the action of plasma kallikreins on high molecular weight kininogens (plasma pathway). These proteins are also required for the intrinsic blood coagulation cascade [86]. Similarly, the decapeptide kallidin (lysyl bradykinin) is released in tissues by the actions of tis-

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Surface HMW-Kininogen HF ~ aHF

IT I [ HMW_Kininogen

Prekallikrein ~ Kallikrein

HMW-Kininogen

Factor XI "Pro"-Kallikrein

Intracellular Enzymes Plasmin

~ P a s m a Kallikrein

Tissue Kallikrein Factor XIA ~, LMW-Kininogen

]1 Coagulation ~ Lysyl Bradykinin I, [ Bradykinin ] (Kallidin)

Carboxypeptidase N IAminopeptidas e (CPN)

Des_ArgO_Bradykini n ~ CPN I Bradykinin] Angiotensin Converting Enzyme

l[Otherf P asma Proteases

Inactive Peptides, Amino Acids

Fig. 2. Mechanisms of bradykinin formation by plasma and tissue pathways. HF, Hageman factor (coagulation factor XII); aHF, activated Hageman factor; HMW, high molecular weight; LMW, low molecular weight

sue kallikreins on low molecular weight kininogens (tissue pathway) (Fig. 2). Inactivation of the kinins occurs locally and in the lungs and the kidneys.

Only in recent years, with improved assay tech- nologies and an increased awareness of the mechanisms regulating kinin levels, meaningful direct evidence to support a role for the kinin system in human inflammatory disorders has begun to accu- mulate. The kinins exert a variety of biological actions by activating at least two different receptor types. B 1 and B 2 receptors for kinins have been identified with agonists and antagonists in classical pharmacological assays [84]. The existence of the two receptor types has been confirmed with binding assays, using labelled bradykinin and des Arg 9 bradykinin, its octapeptide metabolite following C-terminal arginine removal [87].

Antagonists for kinins active on B1 receptors have been identified and further developed to fairly potent, specific and competitive blockers [88]. B 2 receptor antagonists have also been found [89] and they are specific, but rather weak and nonselective, since they also block B 1 receptors. B 2 receptors mediate a large number of rapidly occurring biological effects, particularly the symptoms and signs of inflammation [87], while B 1 receptors appear to be involved in some retarded, long lasting effects of kinins, such as collagen synthesis and cell multipli- cation [90].

Kinins are among the naturally occurring agents involved in inflammatory reactions, for example, vasodilation, increase of vascular permeability, venoconstriction, and mobilization of blood and tissue cells. The kinins generated locally contribute to the acute and possibly the chronic phase of the inflammatory reaction by producing vasodi-

lation, local oedema, and pain. Kinins may also modulate migration of blood and tissue leucocytes that take part in the inflammatory process.

Several of the biological effects of the kinins are mediated by eicosanoids [84]. Hence kinins are among the most potent activators of PG formation [87] and the major actions, for instance the endothelium mediated vasodilation, the smooth muscle contraction or relaxation, and the production of pain are associated with release of PGs. In models of IBD increased mucosal generation of kinins is associated with the induction of a special subtype of bradykinin receptors in inflamed colonic tissue [91], which might contribute to the increased PG formation by bradykinin-induced release of arachidonic acid [92, 93]. Kinin-induced PGE 2 seems to be derived principally from cells in the lamina pro- pria and not from the epithelial cells [94]. In the healthy colonic mucosa this eicosanoid release appears to mediate the bradykinin-stimulated secretion [92]. In an ex vivo model of colitis, increased vascular resistance has been observed following bradykinin infusion and was associated with an exaggerated generation of TXs. The increase in per- fusion pressure was blunted, but not abolished, by concomitant infusion of selective TX inhibitors [93]. Although the exaggerated eicosanoid release may function to modulate colonic blood flow, and contribute to manifestations of inflammation [17], the relevance of these observations for IBD is spec- ulative.

Complement-derived peptides

The complement system of blood plasma and ex- travascular tissue fluid plays an important role in

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many immune defence reactions and absence of a functional complement system reduces many inflammatory reactions [95]. The system consists of about 20 different proteins [96-98] which, once the system is activated, interact in an orderly, sequen- tial and regulated series of reactions [99]. Comple- ment activation promotes acute inflammation, re- cruitment of leucocytes, and killing of pathogens by phagocytosis, lysis, or release of toxic products [71, 96, 100]. The central event in complement activation is cleavage of C3 with the liberation of C3a and its metabolite C3a des Arg. This is accom- plished by activation of either of two pathways (Fig. 3), the classical and alternative pathways, to produce C3 convertase. The classical pathway is activated by IgG or IgM antibodies, once they have formed a complex with the antigen. The alternative pathway [101] is typically activated by polysaccharides. The large fragment of C3 cleavage, C3b, binds covalently to the activator and promotes immune adherence and phagocytosis. Addition of C3b to either of the C3 convertases results in the formation of a C5 convertase which cleaves C5 into C5a and C5b. This is the last enzymatic step in the complement cascade, because the larger fragment, C5b, and later components, C6-C9, form the lytic complex (membrane attack complex) by a series of hydrophobic interactions [98]. C5a plays no further direct role in the complement cascade, but contri- butes to the inflammatory response. It is rapidly converted by a carboxypeptidase, found in plasma and extracellular tissue fluid, to C5a des Arg by loss of the carboxyl terminal arginine.

C3a and C5a are anaphylatoxins, because they release histamine and stimulate smooth muscle contraction [96]. C5a has probably the most important biological activities [71]. More important, however, the C5-derived peptides have potent effects on leucocytes in vitro and promote neutrophil-endothelial interactions in vivo, which lead to neutrophil accumulation and associated oedema formation [100]. In addition to initiating neutrophil-endothelial cell interactions, C5a and C5 des Arg may also contribute to inflammation by stimulating the release of other mediators. Apart from the release of histamine, C5a appears to release interleukin-1 [102], PGs [103], LTs [104], and PAF- acether [105]. Thus, inflammatory responses begun by complement activation may be prolonged and potentiated by the actions of other intermediaries released by C5a.

Although there are few studies showing an effect of complement-derived peptides on the gastrointestinal tract, the biological properties of C5a and C3a suggest that these compounds play a role in

Classical Pathway IgG / IgM-Antigen

Complexes -o ~

Ciqrs ' .

C4b2~o ' .

C3

C5

Alternative Pathway Biological Effect Surface Contact

(Polysaccharides) Z

oO. " D

."~ C3bBh

- ~ J= C3a. .

~ C3b . . . . . . . . . . . . . . . ~ . . . . . . ~ [ Phagocytosis ]

C4b2*b3b C313b3b . . . .,~

v [ 1 �9 C5a . . . . . . . . . . . ~ I A c u t e I n f l a m m a t i o n [

C5b

C7

C8

C9

C5b6789 . . . . . . . . . . . . . . . . . ~ I - ' - - I

Fig, 3. Activation of complement. The central event, cleavage of the third component to produce C3a and C3b, is mediated by either of two distinct pathways, known as the classical and alternative pathways. Proteolytic activity is denoted by *

IBD. Complement activation is normally an appropriate and beneficial reaction to inflammation. In theory, it may be desirable to be able to inhibit complement activation in inappropriate inflammatory states like IBD, though a beneficial effect is by no means certain. Alternatively, the effects of a C5a receptor antagonist may be beneficial, but such compounds are not available at present. The pharmacological manipulation of the complement system has been reviewed elsewhere [106].

Lymphokines

Lymphokines or 'cytokines' are polypeptides that act on leucocytes and other tissue targets. These compounds activate, modulate, and control various aspects of body defence and repair. The group includes the interleukins (ILs) and tumour necrosis factor (TNF). Interleukin-1 (IL-1), produced by macrophages in response to a large number of stimuli [107], activates lymphocytes and also induces several components of the acute-phase response to infection and injury. Its most dramatic biological property is its ability to induce eicosanoid formation [108]. Interleukin-2 (IL-2), produced by T lymphocytes, is a growth factor for helper, suppressor,

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and cytotoxic T lymphocytes. In contrast to IL-1, IL-2 is a single substance, a true growth factor with seemingly the single purpose of expanding T-cell populations [109], whereas IL-1 is a family of polypeptides that mediates several components of the acute inflammation, in addition to affecting a number of nonleucocytic targets, such as the liver, pan- creas, bone, muscle, synovial fibroblasts, and brain [108, 110].

Two forms of IL-I have been cloned, IL-l-beta from human monocytes [111] and IL-l-alpha from a murine macrophage line [112]. The two forms had originally been identified as pI 7 (beta) and pI 5 (alpha) on isoelectric focusing [107]. The two forms of IL-1 are initially synthesized as 31 kiloDalton precursor peptides and share only small stretches of amino acid homology. Depending on the stimulus, intracellular levels of precursor IL-1 can be high, whereas some cell activators result in large amounts of processed IL-1. Precursor IL-1 is cleaved into a 17.5 kiloDalton peptide, which is the predominant extracellular form. Another macrophage product - TNF - has been cloned and shown to be identical to a substance termed cachectin, which mediates cachexia associated with various diseases [113]. Many of the biological effects of IL-1 and TNF overlap, and the compounds often act synergistically [107].

IL-1 induces eicosanoids and lymphocyte activation as well as many different biological activities including fever, acute-phase protein synthesis, fi- broblast proliferation, and other defence and repair functions [107, 108, 110]. Most if not all of these effects are mediated by an IL-1 stimulated increase in phospholipase A 2 activity and subsequent release and metabolism of arachidonic acid from cellular phospholipids in the target cells. Hence the eicosanoids provide the second signal for IL-1 induced cell activation [108].

It is conceivable that IL-1 plays a role in the inflammation in IBD [114]. Whereas the peripheral blood IL-2 pathway appears normal in patients with IBD [115] increased serum concentrations of IL-1 have been reported in some patients with UC and CD [116] and intestinal mucosal levels [117, 118] and production of IL-I in colonic tissue organ culture [118] are increased in patients with IBD compared with controls.

Chemotactic oligopeptides

As already described a number of substances are chemotactic for leucocytes, for example LTs and

C5a. Another class of chemoattractants is peptides elaborated by bacteria [119, 120]. The best studied of these oligopeptides is N-formyl-methionyl- leucyl-phenylalanine (FMLP), a neutrophil chemotactic factor produced by E. coli [121]. All chemotactic peptides appear to produce their effects by binding to cell-surface receptors, and FMLP receptors on human neutrophils have been characterized [119]. Hence both high- and low-affinity forms of the oligopeptide receptor have been identified by direct binding studies.

The chemotactic peptides induce directed migration of inflammatory cells toward their targets, and cause degranulation with lysosomal release, superoxide production, phagocytosis, and eicosanoid formation [119, 122]. Leucotriene B 4 poten- -tiates the response elicited. In perfused rabbit colon FMLP stimulates release of eicosanoids [123] and intrarectal administration of high concentrations of FMLP has been used as a model of UC [124]. Thus, it may be speculated that bacterial oligopeptides, originating in the colonic lumen, breach mucosal defences and initiate colonic inflammation.

Neuropeptidcs

The involvement of the nervous system in the generation of inflammatory responses is well established [125-127]. Polymodal nociceptor activation generates axon-reflexes in the terminal arborisa- tions of primary afferent neurones. These reflexes, in turn, cause the C fibres to release neuropeptides, for example, the undecapeptide, substance P [128, 129]. Neurokinin A (substance K), neurokinin B (neuromedin K), vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) are other possible candidates [125].

The neuropeptides are believed to exert their effects on vascular smooth muscles and endothelium to increase flow and permeability, in addition to altering the motility of the muscularis mucosae. It has been suggested that the effects of substance P at least in part are mediated by IL-1 [130].

So far, in contrast to other tissues, little is known about the role and mechanisms of neuropeptides in intestinal inflammation. Increased mucosal/submucosal concentrations of substance P have been observed in patients with UC, but not CD [131], whereas others have reported high concentrations of substance P receptor binding sites in colonic tissue obtained from both groups of patients [132].

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Free radicals - reactive oxygen species (ROS)

Free radicals are chemical species (atoms, ions or molecules) with one or more unpaired electrons in their outer orbital, rendering the compound chemi- cally reactive. The odd electron is often represented in the formula as a dot. The univalent reduction of oxygen by certain oxidases produces the superoxide anion radical (O2). Other reactive oxygen species (ROS) are hydrogen peroxide (H202) and the hydroxyl radical (OH). HzO 2 is formed by the diva- lent reduction of 02 (or the univalent reduction of O2 ) to yield the non-radical species 022 which is protonated at physiological pH to HzO 2. In the presence of iron II or copper I it is reduced and the hydroxide ion ( O H ) and the hydroxyl radical (OH) are formed. Several enzymes, including xanthine oxidase, aldehyde oxidase, and NADPH oxidase, are known to produce superoxide directly or indirectly as a reaction product [133]. Phagocytic leucocytes, when stimulated, produce large amounts of ROS via the NADPH oxidase system [134, 135] and intestinal microvascular and mucosal epi- thelium also contains various oxidases, for example, xanthine oxidase, a cytosolic enzyme normally present in its dehydrogenase form, but converted to an oxidase form during ischaemia [136].

The cellular toxicity associated with superoxide and hydrogen peroxide is generally ascribed to their role as a precursor of hydroxyl radicals and other secondary ROS [133]. The hydroxyl radicals oxidize sulphhydryl compounds that may result in inactivation of essential enzymes or membrane transport proteins. Furthermore, the radicals will react with DNA to cause strand scission [137] or other abnormalities, and attack of membrane associated polyunsaturated fatty acids results in the peroxidation of lipids [138]. Lipid peroxidation often precedes lysis of target cells and is also associated with increased rates of protein fragmentation and changes in composition [139] theoretically associated with changes in antigenicity. Finally, free radical production may have a potentiating effect on PG synthesis, initiating lipid peroxidation and providing the hydrogenperoxide required for the early stages of the PG cascade.

Since radical reactions are self-perpetuating unless two radicals interact, the net effect of generat- ing ROS in vivo will be widespread and un- predictable. The production of ROS is essential to normal metabolism, but they are theoretically de- structive unless tightly controlled. The endogenous defence is provided by catalytic free radical removal, for example, by various enzymes, such as superoxide dismutase, catalase, and gluthathione

peroxidase, by physiological non-enzymatic "free radical scavengers", such as alpha-tocopherol and ascorbate, and by removal of free transition iron and copper ions [133 135] through binding to transferrin or coeruloplasmin.

Although all cells possess numerous antioxidant enzymes and scavengers to protect themselves from ROS, the rate of production of ROS may exceed the capacity of the antioxidant defences, thereby resulting in substantial tissue damage. In animal experiments ROS mediate much of the microvascular injury associated with reperfusion of ischaemic intestine [140, 141]. It has also been suggested that transient ischaemia in patients with UC initiates a cascade of self-perpetuating cycles or ROS formation, inflammation and, ultimately, mucosal injury [133].

Implication for therapy in IBD

It is hoped that a better understanding of the intermediaries and mechanisms of inflammation will lead toward a better therapeutic control of undesirable elements of inflammation. It should be borne in mind, however, that the more potent the therapeutic intervention the more likely there is to be undesirable side effects. The current drug therapy of IBD established through controlled trials mainly relies on the use of corticosteroids and sulphasalazine [142].

Corticosteroids

Despite the widespread use of the corticosteroids it had for long been difficult to conceptualize their anti-inflammatory effects in any single model [143]. In recent years, however, a considerable amount of evidence points to a modulation of eicosanoid formation. Corticosteroids have also been shown to inhibit the release of non-arachidonic acid mediators from many inflammatory cell types (for example, histamine release from basophils). Because activation of these cell types may require phospholipase activity, it is not yet clear whether the corticosteroids inhibit these cellular processes via a similar mechanism [144]. Unlike aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), which block the cyclo-oxygenase pathway [145], the corticosteroids do not inhibit arachidonic acid metabolism in cell-free systems [145, 146]. In contrast, these drugs are considered to act by preventing formation of free arachidonic acid for eicosanoid formation [147 149]. This effect is believed to be

84

mediated by promotion of a natural phospholipase A2 inhibitor, commonly denoted as lipocortin [150]. Recently, the principal biological species in the lipocortin family in both rat and human, a 40 kiloDalton protein, has been isolated, purified to homogeneity, sequenced, and cloned [151, 152]. The described effect of corticosteroids is fairly re- producible in various in vitro systems [153] and in most experimental models of inflammation in animals, although in some experimental conditions exceptions to this concept have been found [154, 155]. Such failure of corticosteroids to prevent eicosanoid formation suggests that multiple functional pools of phospholipase are operative in relation to the arachidonic acid cascade [154]. Also basal PG production in vivo has been reported to be unaffected by pharmacological doses of corticosteroids [156, 157]. However, studies on the profile of eicosanoids in the inflamed rectum [49] in patients with severely active UC have demonstrated that prednisolone (1.5mg/kg/day orally) promptly (i. e., within 72 h) decreases luminal concentrations of both PGE 2 and LTB4, whereas indomethacin (150 mg/day orally) reduces PGE 2 concentrations in spite of unchanged, or even increased, LTB 4 levels. These prompt reductions are likely to be caused by a direct drug action, rather than being secondary to decreased tissue damage, and accord with the lipocortin theory [150].

Sulphasalazine and 5-aminosalicylic acid (5-ASA)

Interpretation of results obtained in vitro on the mode of action of sulphasalazine and 5-ASA has been hampered by the lack of knowledge of the therapeutically relevant concentrations of the drugs. Such results include inhibition of PG synthesis [24, 25, 28], increased intestinal "cytoprotection" [158] due to inhibition of PG degradation [159-162], and stimulation of PG synthesis [163], inhibition of TX synthesis [164, 165], reduction of LT formation [30, 165-169], modulation of polymorphonuclear leucocyte function [170-173], im- paired receptor-binding of chemotactic peptides [174], and impairment of folate absorption and metabolism [175]. A common feature of such in vitro experiments is the use of concentrations derived from in vivo measurements of so-called "therapeutic serum concentrations" (20-50 pg/ml) of sulphapyridine [176]. This may explain at least some of the conflicting data in the papers referred to. Although the exact concentrations of 5-ASA in the mucosa are unknown, the therapeutically relevant

concentrations seem to be those achieved intralu- minally. This notion is supported by the observations that 5-ASA acts topically after rectal instilla- tion [177-180] and that the concentration of 5-ASA in serum is negligible [181,182] in the presence of high luminal concentrations after oral administration [182].

That sulphasalazine and 5-ASA exert their therapeutic benefit through modulation of eicosanoid formation is a suggestion underlying a substantial amount of experimental work. Hence there has been considerable discussion whether the therapeutic effect relies on inhibition of the cyclooxygenase activity, suppression of the activity of PG-degrad- ing enzymes, or reduction of the lipoxygenase activity.

The hypothesis, that sulphasalazine acts through promotion of endogenous cytoprotective PGs [183] is based on in vitro observations of increased PGE 2 and PGI 2 production by normal colonic mucosa in the presence of 5-ASA, in addition to decreased enzymatic degradation of PGs in the presence of sulphasalazine (but not 5-ASA) [159-163], Nevertheless, the effect of sulphasalazine on PG formation may vary depending on substrate arachidonic acid availability [184], in addition to drug concentration [163]. Evidence against the proposed mechanisms of drug action is provided by experiments in humans, which demon- strate that therapeutic doses of the drug (2 g/day) result in concentrations of 5-ASA, which are 100 to 1,000 times higher [182] than those stimulating PG synthesis in vitro [163]. Furthermore, both sulphasalazine (2 g/day) and olsalazine (2 g/day) normal- ize significantly raised intraluminal PGE 2 levels, even in patients with inactive UC. These findings suggest that the named drugs modify colonic PG production in the absence of clinical and endoscopic disease activity [55].

A number of in vitro experiments [25, 28, 30, 185, 186], in addition to observations in vivo [55], have suggested that 5-ASA is a weak cyclooxygenase inhibitor. The latter trait is most likely un- related to its therapeutic efficacy, because more potent cyclo-oxygenase inhibitors, such as NSAIDs, are ineffective in this respect [43, 45, 185, 187]. Both sulphasalazine and 5-ASA block the soybean lipoxygenase [167, 168] (although this effect is disputed as regards sulphasalazine [188]) and inhibit the formation of 5-HETE and LTB 4 by human leucocytes [166, 169], in addition to inhibiting the colonic lipoxygenase [26, 30]. The inhibitory effect (for example, the drug concentration neces- sary for half-maximal reduction in the formation of lipoxygenase products), the target level in the path-

85

way, and the differential effect of sulphasalazine and 5-ASA appear, however, to depend on the system used and the experimental conditions.

Finally, there is some evidence to indicate that interventions directed toward oxidant scavenging and xanthine oxydase inhibition may provide a new approach to the management of IBD. 5-ASA is an extremely efficient oxygen radical scavenger, suggesting that the beneficial effect of salicylates in IBD may be due, in part at least, to scavenging of enterocyte- or phagocyte-derived oxidants [189 192].

New drug development

As mentioned previously, the rationale for seeking pharmacotherapeutic agents to limit LT biosynthesis and/or end-organ effects is based upon the increased generation of these compounds in IBD, their potent proinflammatory actions, and the lack of clinical efficacy by blocking one of the two major routes of arachidonic acid metabolism (i. e., the cyclooxygenase pathway), in addition to the con- trastingly remarkable efficacy of corticosteroids that inhibit the formation of oxygenation products via both routes. It is possible that such compounds possess corticosteroid-like therapeutic efficacy but are free from steroid-related toxicity. Even if this is over optimistic, the inhibition of LT synthesis must be seen as a promising concept and if one or more of these compounds ultimately prove to benefit patients with IBD, it would concomitantly substan- tiate the belief that LTs are mediators of primary importance for the inflammatory process.

Prospects

Progress in therapy of IBD is most likely to come from a better understanding of the role of the soluble intermediaries of inflammation. In particular, it appears likely that lipoxygenase products of arachidonic acid are potent intermediaries that may prove to be important in the pathogenesis of IBD. To confirm or refute this belief requires the demon- stration of clinical efficacy by selective lipoxygenase inhibitors or LT receptor antagonists through controlled clinical trials.

Improved knowledge of the role of inflammatory intermediaries has allowed reinterpretation of the rationale behind current therapy. Cortico- steroids and sulphasalazine, the drugs with established efficacy in IBD, affect arachidonic acid re-

lease and metabolism, which may prove to be the scientific basis of IBD therapeutics.

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Dr. J. Rask-Madsen Section of Gastroenterology Department of Medicine G Bispebjerg Hospital DK-2400 Copenhagen NV Denmark

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Inflammatory intermediaries in inflammatory bowel disease