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Monitoring disease activity by stool analyses: from occult blood to molecular markers of intestinal inflammation and damage
  1. D Foell1,
  2. H Wittkowski1,
  3. J Roth2
  1. 1
    Department of Pediatrics, University of Muenster, Germany
  2. 2
    Institute of Immunology, University of Muenster, Germany
  1. Dr D Foell, Department of Pediatrics, University of Muenster, Albert-Schweitzer-Str 33, D-48149 Muenster, Germany; dfoell{at}uni-muenster.de

Abstract

It is a common experience that gastrointestinal symptoms urge us to differentiate inflammatory bowel disease (IBD) from functional disorders. Furthermore, in patients with proven IBD the disease activity has to be accurately monitored. Faecal markers of neutrophil influx into the mucosa are promising indicators of intestinal inflammation. Some neutrophil-derived proteins may be linked to the pathogenesis of IBD due to their functions as damage-associated molecular pattern molecules (DAMPs). Phagocyte-specific DAMPs of the S100 family are released from neutrophils or monocytes, followed by pro-inflammatory activation of pattern recognition receptors. The complex of S100A8/S100A9 was termed “calprotectin” and has been in use as a faecal marker for 10 years. More recently, faecal S100A12 has been reported to be an even more accurate faecal marker of inflammation. We review the biology of this novel group of molecules which can be used as surrogate markers directly linked to the molecular mechanisms of gut inflammation.

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Although the aetiology of chronic inflammatory bowel disease (IBD), consisting of ulcerative colitis and Crohn’s disease, is still incompletely understood, recent scientific achievements have led to significant improvements in patient care. Therapies targeting cytokines such as tumour necrosis factor α (TNFα) mainly produced by monocytes and macrophages attenuate the clinical course of IBD, thus pointing to phagocytes as key players in the pathophysiology of these disorders.1 2 In recent years the involvement of innate immunity in IBD has been emphasised even more; in this regard the association of Crohn’s disease with abnormalities in nuclear oligomerisation domain receptor 2 (NOD2) is notable, since this pattern recognition receptor (PRR) is involved in innate host defence.36 Our current understanding of IBD pathogenesis involves complex interactions among susceptibility genes, the environment and the immune system. The inflammatory processes involve both adaptive and innate immune mechanisms and, finally, mucosal damage.2

Since intestinal symptoms are a frequent cause for referrals to gastroenterologists, it is crucial to distinguish between non-inflammatory functional problems such as irritable bowel syndrome (IBS) and IBD.7 8 Furthermore, chronic IBD is characterised by unpredictable flare-ups of symptoms that can severely impair a patient’s quality of life. Most patients have recurrent periods of active disease or even continuous active inflammation. In those who do respond to treatment, the duration of the response varies considerably. Identifying biomarkers for this variation would enable the design of individualised treatment strategies for patients with refractory disease.9 In addition, improved markers of inflammation are important in the follow-up of patients especially during periods of low disease activity, when it is crucial to detect sub-clinical intestinal inflammation and to predict relapsing disease. This could aid in adjusting treatment to the actual needs of individual patients. The risk of relapses is influenced by intestinal inflammation which, at present, is only accurately detected by endoscopy and histology, if at all. These invasive procedures are neither feasible for frequent routine follow-up examinations nor for the screening of individuals at risk of chronic active IBD.

To date, no simple diagnostic tests for monitoring intestinal inflammation are available.7 10 11 Commonly used laboratory parameters, such as C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR) are acute phase reactants which correlate poorly with intestinal disease activity. These markers reflect a summation of systemic host responses rather than being specific for intestinal inflammation, and they have no predictive value for the further course of the disease. Therefore, more reliable biological markers are needed to confirm remission, to early detect relapses or local reactivation, and to monitor anti-inflammatory therapies.7 Ideal markers to be used in the clinical follow-up would be “non-invasive”, easy to perform, reproducible, inexpensive, and reflect intestinal disease activity with high sensitivity and specificity. Faecal biomarkers are attractive given the fact that the faecal stream is in close contact with the intestinal mucosa and can therefore take up molecules serving as markers of mucosal inflammation or damage. We provide an overview of the molecular basis of novel faecal markers directly linked to mucosal damage and intestinal inflammation.

Innate immunity plays a fundamental role in the pathogenesis of inflammatory bowel disease

FAECAL BIOMARKERS OF MUCOSAL DAMAGE

In table 1, molecules proposed as faecal biomarkers for IBD in at least three studies are summarised. Markers without independent investigation by at least two scientific groups have been excluded from this summary. Some of the markers are indicators of mucosal damage and loss of its normal barrier function. Since phagocyte influx into the mucosa is associated with IBD, phagocyte markers have been studied to some extent. Feasible stool assays have been introduced for these new biomarkers which are stable in faeces over an appropriate period of time.

Table 1 Faecal markers of inflammation

It has to be emphasised that none of the reported stool markers are entirely specific for chronic IBD but rather increased in all inflammatory disorders characterised by mucosal inflammation, irrespective of an infectious or non-infectious origin. As a general rule, markers expressed by phagocytes are more specific for inflammation, while markers also inducible in epithelial cells are more sensitive but can, on the other hand, increase in conditions of non-inflammatory stress, such as lactose intolerance.20 In addition, infants and small children can differ in their expression level of epithelial proteins, which appears at least partially related to feeding formula and the establishment of the normal gut flora.2123

It is a well-known phenomenon that the occurrence of blood in faeces indicates mucosal damage in the intestine, especially in the context of malignancies or inflammation. The oldest indicator for occult bleeding is gum guaiac, today used in an assay format modified from the original method.24 Tests screening for the presence of haemoglobulin in stool are of limited accuracy with reported sensitivites of 36–76%, and quantification of disease activity is not possible by these means. Furthermore, faecal blood is a late symptom of inflammatory tissue damage and does not reflect an underlying molecular pathomechanism (fig 1).

Figure 1

(A) The occurrence of different biomarkers in stool reflects distinct phases of intestinal inflammation. Initially, unidentified triggers lead to a cascade of events which involve epithelial alterations and the activation of innate immune mechanisms. Innate immunity is rapidly engaged to ensure host integrity and defence against pathogens. While the exact triggers are only poorly understood, our current understanding of the IBD pathogenesis involves complex interactions among the environment and the aberrations of immune responses in susceptible hosts. These responses involve the influx of granulocytes into the affected mucosa. Variations in innate immunity, eg, of PRRs, are a predisposing factor for inflammatory amplification. (B) Whenever counter-mechanisms fail, perpetuation of active IBD into chronic disease becomes possible. Mucosal damage leads to clinical and laboratory signs of IBD including blood in faeces, while the earlier phases are detectable by endoscopy and histology. Novel faecal biomarkers may have the potential to already detect early phases of intestinal inflammation. (C) In a more detailed view, early events of inflammation are illustrated. 1. Granulocytes are abundant as infiltrating cells in affected mucosa. These cells actively secret cytokines and chemokines. Degranulation may also cause release of granule proteins without pro-inflammatory but rather anti-inflammatory functions, such as lactoferrin. The cytosol is the source of the DAMP proteins S100A8/S100A9 (calprotectin) and S100A12. 2. These pro-inflammatory molecules activate microvascular intestinal endothelial cells and serve as chemoattractants resulting in further recruitment of leucocytes (especially monocytes, macrophages, T cells) into the mucosa. 3. There is some spill-over of cytokines, chemokines, and DAMPs from the mucosa into the gut lumen in this early phase of inflammation. (D) The amplification phase involves complex networks of molecules mainly released from phagocytes and receptors on different cell types including the epithelium. 4. There is a further recruitment of leucocytes by ongoing chemotaxis. Infiltrating cells secrete cytokines and DAMPs. 5. Transmigration of neutrophils occurs; there is both active secretion and passive release of DAMPs by necrotic cells in the gut lumen. 6. Activation of epithelial cells facilitates transepithelial migration of neutrophils and promotes release of host defence proteins. Eventually, mucosal damage is the consequence of the chronic intestinal inflammation. 7. There is an exudation of proteins in this phase. Breakdown of epithelial barrier function explains the loss of serum proteins, eg, α1-antitrypsin. 8. Finally, bleeding from damaged mucosa can be detected by occult blood assays. DAMPs, damage-associated molecular patterns; IBD, inflammatory bowel disease; PRR, pattern recognition receptor.

An attempt to more accurately quantify mucosal damage was introduced by analysing protein loss through stool. A measure of disturbed barrier function related to intestinal inflammation is faecal alpha 1-antitrypsin (α1-AT), which may be elevated in IBD.25 26 However, the reported accuracy does not suggest the use as a faecal surrogate marker in IBD.18 27 Over the last decades, more accurate faecal markers of inflammation have been introduced which are directly linked to the pathogenesis of IBD.

FAECAL BIOMARKERS OF INTESTINAL INFLAMMATION

Markers of phagocyte influx and activation are especially interesting considering the role of innate immunity in IBD, where phagocytes are both abundant at sites of inflammation and the major source of cytokines targeted by effective therapies. Indium-111-labelled granulocyte scintigraphy is a sensitive and specific method of analysing intestinal inflammation. Indium-111 excretion in faeces correlates with disease activity of IBD.28 However, this technique is reserved for specialised facilities, is expensive, involves long-term stool sampling, and implies exposure to radiation. In addition, the appearance of leucocytes in stool may be less sensitive for early changes than markers released from activated phagocytes within the mucosa, which spill over into the lumen and remain stable in single random stool samples.

In the group of neutrophil activation markers cytokines are certainly attractive, as they are involved in the network of events which link immune responses with the pathogenesis of IBD (fig 1). In particular, TNFα has a central role in inflammation, which is emphasised by the fact that therapies targeting this cytokine are undisputedly effective in IBD. Unfortunately, cytokines and chemokines like interleukin 6 (IL6), IL1, IL8, and TNFα are rather unstable small peptides.29 Their detection in stool varies with methodology and handling, making clinical application impractical.17 18 3033

Eosinophil cationic protein (ECP) and eosinophilic protein X (EPX) are markers of eosinophil degranulation that have been described as faecal markers of intestinal hypersensitivity and eosinophilic inflammation. Their accuracy in the context of Crohn’s disease and ulcerative colitis is inferior to that of other markers.19 34 35 Lactoferrin, polymorphonuclear (PMN) elastase, myeloperoxidase, and human neutrophil lipocalin (HNL) are markers of neutrophil degranulation detectable in stool.19 3639 Of the proteins stored in neutrophilic granules, lactoferrin is the most accurate marker.15 However, lactoferrin is a member of the transferring family of iron-binding glycoproteins not only expressed in neutrophils. Epithelial cells secrete lactoferrin into the mucus and may therefore also serve as a source of lactoferrin detected in stool.40 Extracellular lactoferrin has several biological functions, eg, in iron homeostasis and host defence. It also has anti-inflammatory properties which may be related to interactions with surface receptors,41 42 but the exact molecular mechanisms attenuating inflammation still need to be defined.43 44 A direct link of lactoferrin to the pathogenesis of chronic IBD has not been established.

More recently, proteins which originate from phagocytic cytosol have been shown to bear an excellent potential as serum and faecal markers of inflammation.45 46 These pro-inflammatory molecules also point to the central role of innate immunity and play a pivotal role in the pathogenesis of IBD.1 2 46 Proteins released from phagocytes within the mucosa may also spill over into the gut lumen, and the additional release by inflammatory cells in crypt abscesses results in significant amounts that mix with other secretions and gut contents which ultimately appear in the patients’ stool.47 They are released at sites of inflammation and their faecal concentrations correlate with disease activity.

PAMPS, DAMPS AND INFLAMMATORY BOWEL DISEASE

The innate immune system has a pivotal function in host defence as well as in promoting inflammatory processes.48 49 However, the critical role of phagocytes and epithelial cells in the pathogenesis of IBD has been emphasised in its full extent only in recent years. Activation of the local mucosa and especially intestinal epithelial cells is supposed to be an initial event of IBD probably triggered by infectious stimuli. Subsequently, infiltration of neutrophils and monocytes/macrophages is a hallmark of the developing inflammatory reaction. Furthermore, inflammatory products of phagocytes like cytokines, reactive oxygen species (ROS) and proteolytic enzymes promote inflammation and cause significant tissue damage.50 51 However, there is growing evidence that during the early phase additional factors of the innate immune response trigger disease activity of IBD (fig 1).

Innate immunity achieves our primary host defence by recognising invading microorganisms through so-called pathogen-associated molecular patterns (PAMPs). These highly conserved structures on pathogens are detected by PRRs, eg, toll-like receptors (TLR) or intracellular receptors of the NOD family.5254 Recently, a novel group of molecules has been described as important pro-inflammatory factors of innate immunity. Due to their release by activated or damaged cells under conditions of cell stress, these molecules are summarised under the term damage-associated molecular pattern proteins (DAMPs).5557 The emerging concept of DAMP recognition involves the multiligand receptor for advanced glycation end products (RAGE) and TLRs in sensing not only PAMPs but also endogenous DAMPs.55 58 59 The exact mechanism by which microorganisms contribute as triggers of IBD is only partially understood so far. Probably, multiple positive feedback loops between DAMPs and PAMPs and their overlapping receptors amplify these processes. This overlap between endogenous and exogenous triggers of innate immunity may represent the molecular basis for the clinically obvious experience that infections as well as non-specific stress factors can induce flares in IBD.

Since DAMPs are involved in the initial trigger of cell stress and inflammation and are released at sites of IBD these molecules are promising candidates as sensitive markers for monitoring local disease activity. Three novel members of this group of DAMPs expressed in cells of myeloid origin are phagocytic calcium-binding S100-proteins. S100A8, S100A9 and S100A12 are novel ligands of PRRs like TLR-4 and RAGE directly amplifying inflammatory processes.56 59 60 These S100 molecules are active extracellularly, which makes them distinct to most of the other members of this family. DAMP molecules accumulating in the intestinal mucosa represent excellent markers for mucosal inflammation. Therefore, assays which determine intestinal inflammation by detecting neutrophil-derived DAMPs in stool bear an excellent diagnostic potential.

Damage-associated molecular pattern proteins (DAMPs) are endogenous activators of pattern recognition receptors

S100 PROTEINS IN INFLAMMATORY BOWEL DISEASE

S100 proteins comprise a family of more than 20 calcium-binding proteins which are characterised by a tissue-specific expression pattern.61 Three S100 proteins are specifically linked to innate immune functions by their expression in phagocytes. S100A8 (also named calgranulin A; myeloid-related protein 8, MRP8) and S100A9 (calgranulin B; MRP14) are found in granulocytes, monocytes and early differentiation stages of macrophages. S100A8 and S100A9 represent the majority of the calcium-binding capacity in phagocytes. S100A9 exists in various isoforms which associate with S100A8 to different heterodimers and tetramers which are all summarised under the term “calprotectin”.62 Expression of these proteins can also be induced in epithelial cells under inflammatory conditions.63 64 In contrast, S100A12 (calgranulin C) is more restricted to granulocytes. In the presence of calcium S100A12 forms homodimers, but there is no complex formation with S100A8 or S100A9. Thus, S100A12 acts independently of S100A8/S100A9 during calcium-dependent signaling.65

Non-covalently associated complexes of S100A8/S100A9 are secreted by activated phagocytes and epithelial cells during inflammatory processes.66 67 Secretion of S100A8/S100A9 is induced during contact of phagocytes with inflamed endothelium. Both S100 proteins lack structural requirements for classical transport via the endoplasmic reticulum and the Golgi complex. However, release of S100A8 and S100A9 by human monocytes is a specific and energy-dependent process. Secretion involves activation of protein kinase C (PKC) and depends on an intact microtubule network.67 Release of S100A12 by granulocytes follows a similar intracellular pathway (fig 2).46 68

Figure 2

Initiation of secretion of S100A9/S100A9 and S100A12 is dependent on the activation of two intracellular signalling pathways. Inflammatory stimuli, such as cytokines (eg, interleukin 1 (IL1)) or bacterial products (eg, lipopolysaccharide (LPS)) activate protein kinase C (PKC). A parallel calcium signal, triggered by adherence to activated endothelial cells, is necessary to start release of these S100-proteins. The intracellular transport follows a so-called alternative secretory pathway which is dependent on an active transport via microtubules. Similar to other alternatively secreted proteins such as IL1 the transmembraneous transport mechanism of S100-proteins is presently not known. MT, microtubule.

S100A8/S100A9 induce a number of pro-inflammatory chemokines as well as adhesion molecules like vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and modulate transendothelial migration of leucocytes increasing binding activity of the integrin receptor CD11b/CD18.69 70 Furthermore, enzymes of superoxide metabolism were upregulated in endothelial cells and phagocytes by S100A8 and S100A9.59 71 S100A8 and S100A9 induce a specific inflammatory pattern in endothelial cells which is characterised by the induction of a prothrombotic and pro-inflammatory response and an increased vascular permeability due to a loss of endothelial cell–cell contacts and cell junction proteins (fig 3A,B).69 71

Figure 3

(A) Various inflammatory triggers like cytokines (eg, interleukin 1 (IL1)) or bacterial products (eg, lipopolysaccharide (LPS)) activate monocytes and intestinal epithelial cells (1, red) and stimulate specific secretion of S100A8/S100A9 (calprotectin) (2, black) during the initial phase of inflammatory bowel disease (IBD). In particular, S100A8/S100A9 expressed and secreted by epithelial cells may contribute to elevated stool concentrations in early stages of IBD. In parallel, S100A8/S100A9 triggers a positive feedback mechanism by activation of macrophages via interaction with toll-like receptor 4 (TLR4) (3, blue). (B) During established IBD S100A8/S100A9 activate endothelial cells via a so far unidentified receptor (1, black). It induces expression of endothelial adhesion molecules, stimulates leucocyte adherence, and promotes recruitment of leucocytes to inflamed intestinal tissue (1, black). Activation of TLR4 on neutrophils (2, blue) amplifies inflammation by release of cytokines (IL1, tumour necrosis factor (TNF)) and reactive oxygen species (ROS) (3, red). S100A8/S100A9 released by neutrophils and the additional secretion by the intestinal epithelium leads to further increase of concentrations of this protein complex in stool during IBD. Thus, S100A8/S100A9 reflects the orchestrated inflammatory response of the local innate immune system in IBD. (C) S100A12 is exclusively released after inflammatory activation of granulocytes. S100A12 interacts with the receptor of advanced glycation end-products (RAGE) and additional, so far unidentified receptors on endothelial cells and induces leucocyte adhesion and transmigration comparable to S100A8/S100A9 (1, black). S100A12 stimulates granulocytes in an autocrine mechanism (2, blue) resulting in release of cytokines like IL1 and TNF as well as ROS production which results in additional release of S100A12 by mucosal and intraluminal granulocytes in a positive feedback (3, red). S100A12 is thus a very sensitive and specific marker of granulocyte activation in IBD.

S100A12 has been implicated in a novel inflammatory axis binding RAGE, a multiligand receptor of the immunoglobulin superfamily expressed on endothelium and cells of the immune system.58 68 Recently, S100A8 and S100A9 have been identified as endogenous ligands of TLR4 (fig 3).59 S100A12 stimulation increases surface expression of VCAM-1 on endothelial cells and promotes binding of integrin VLA-4 bearing mononuclear cells to S100A12-stimulated endothelium.58 68 S100A12 upregulates expression of pro-inflammatory cytokines by macrophages such as TNF and IL1β (fig 3C). In addition, S100A12 increases expression of ICAM-1, thereby providing a mechanism by which polymorphonuclear leucocytes might be attracted to S100A12-stimulated endothelium. Induction of VCAM-1 and ICAM-1 expression by S100A12 is, at least in part, mediated by activation of nuclear factor kappa B (NF-κB).58 68 The interaction of S100A12 with RAGE has been initially studied in the murine system. This work is difficult to translate into the human setting since mice normally lack S100A12, but there is evidence for pro-inflammatory effects in humans.47 72 A direct pro-inflammatory response of human intestinal microvascular endothelial cells upon S100A12 stimulation has been demonstrated.47

Damage-associated molecular pattern (DAMP) molecules are markers of inflammation which are specific for cells critically involved in the underlying immunological disturbances of inflammatory bowel disease

Numerous S100A8, S100A9 and S100A12 expressing phagocytes have been detected as pro-inflammatory cells at sites of intestinal inflammation in patients with Crohn’s disease or ulcerative colitis. In addition, S100A8 and S100A9 are expressed by intestinal epithelial cells during IBD.73 74 S100A8/S100A9 as well as S100A12 serum levels are highly elevated under many inflammatory conditions and are useful in monitoring inflammation in individual patients with IBD.46 73 S100A8 and S100A9 expressing cells are the major source of ROS in IBD and are thus directly responsible for intestinal tissue damage.74 S100A12 also promotes intestinal inflammation.58 75

Taken together, secretion of S100A8/S100A9 and/or S100A12 at sites of IBD constitute a positive feedback mechanism in which interaction of primed phagocytes with endothelial cells promotes further recruitment of leucocytes and subsequent tissue damage. S100A12 is very specific for neutrophil activation, and its release from inflamed mucosa into the gut explains the occurrence in stool samples in the presence of gut inflammation.47 76

PERFORMANCE OF DAMPS AS FAECAL BIOMARKERS

Complexes of S100A8/S100A9, referred to as calprotectin in most of the literature, are certainly among the best characterised faecal biomarkers in IBD since its introduction into the diagnostic scenery.12 High stability at room temperature, resistance to degradation and a homogenous distribution in stool have been initially described as prerequisites for biomarkers as well a strong correlation between S100A8/S100A9 (calprotectin) and indium-111-labelled granulocyte scintigraphy as the gold standard method for detecting inflammatory activity in IBD.12 28 Numerous publications have emphasised different clinical applications for this biomarker.

In the initial diagnostic work-up clinicians have to differentiate organic diseases and functional disorders. Sensitivity of S100A8/S100A9 (calprotectin) for the diagnosis of IBD ranges from 63% to 100%, while specificity was reported to be between 79% and 93% (table 2).36 7784 One meta-analysis has been published in 2007, which states an overall sensitivity and specificity of 95% and 91%, respectively.85 Direct comparison between S100A8/S100A9 (calprotectin) and lactoferrin revealed comparable levels of diagnostic accuracy.36 77 86 However, it has to be emphasised that these studies were designed to compare IBD patients with healthy controls or IBS, respectively, or alternatively to reflect endoscopic activity in Crohn’s disease.87 Due to the non-specific character of these inflammatory markers, other inflammatory conditions cannot be differentiated from IBD with such accuracy, including infectious enteritis, polyps, tumours and NSAID-related or ischaemic colitis. Two studies proposed that faecal calprotectin may be a specific marker of IBD, given the fact that it was also found elevated in non-affected first degree relatives of IBD patients, but this observation warrants further confirmation.88 89

Table 2 Reported diagnostic accuracy of faecal calprotectin, faecal lactoferrin, and faecal S100A12 in the differentiation of inflammatory bowel disease versus inflammatory bowel syndrome36 7784

Reflecting severity of disease and monitoring disease activity is an urgent issue in chronic remitting and relapsing conditions like IBD. Patients with known IBD, especially ulcerative colitis, have normal faecal S100A8/S100A9 (calprotectin) levels in clinical remission, being thereby predictors of a further stability of remission in adults and children. Contradictory results have been reported for the prediction of Crohn’s disease relapses by means of biomarkers.9092 However, the actual severity of mucosal inflammation is well reflected by faecal S100A8/S100A9 (calprotectin) concentrations, as reported by a number of studies, which additionally indicate response to anti-TNF therapy in Crohn’s disease.36 87 93 94 On the other hand, several studies showed that the concentrations of faecal and serum biomarkers of inflammation may be elevated despite clinical remission, which may reflect ongoing subclinical inflammation.46 80 95 With this regard it is remarkable that faecal biomarkers correlate better with endoscopic findings than with clinical activity scores. It is therefore conceivable that these markers can be used as sensitive indicators of residual or relapsing disease even in presumably stable patients.

Clinical studies showing that neutrophil specific protein S100A12 is a useful surrogate marker of inflammation when measured in the faeces of IBD patients have been initiated more recently. De Jong et al showed for the first time increased faecal concentrations of S100A12 in children with proven IBD, with a sensitivity and specificity of 96% and 92% versus healthy controls, and a significant correlation to disease activity.13 In another study, sensitivity and specificity of faecal S100A12 in the differentiation from IBD versus IBS were 86% and 96%, respectively. Direct comparison to faecal S100A8/S100A9 (calprotectin) in this study revealed a better diagnostic accuracy and a higher correlation to mucosal inflammation for S100A12.80 In the most recent study, in contrast to faecal S100A8/S100A9 (calprotectin), faecal S100A12 was highly specific (97% vs 67%) in distinguishing paediatric patients with IBD from children without IBD in a prospective setting.96 Release of S100 proteins from mucosal tissue is significantly higher in patients with active Crohn’s disease and ulcerative colitis compared to non-inflamed tissue. The correlation of release to inflammatory activity was higher for S100A12 compared to S100A8/S100A9 (calprotectin).47 76

The results from the first clinical studies utilising faecal S100A12 reveal a diagnostic performance that may be superior to that of faecal S100A8/S100A9 (calprotectin). This difference could result from the different expression patterns of these proteins. While S100A12 is a protein very specifically expressed in neutrophilic granulocytes, S100A8/S100A9 are, like lactoferrin, also inducible in epithelial cells.46 64 65 97 Differences in the release of these proteins from affected mucosa may thus be explained.47 As debris of both leucocytes and intestinal epithelial cells may be a hallmark of mucosal damage, the release from these cells can add to the faecal concentrations of S100A8/S100A9 (calprotectin), making them excellent markers for mucosal damage. On the other hand, however, elevated faecal S100A8/S100A9 (calprotectin) has been shown also in colorectal carcinoma, due to lactose intolerance, or related to concomitant therapy with non-steroidal anti-inflammatory drugs.81 98 In addition, S100A8 and S100A9 exist in different, less defined complex forms in vivo which are all summarised under the term “calprotectin”. This fact may impede with direct comparison of data obtained with different immunoassays using antibodies against different epitopes. The most widely used test for detecting faecal S100A8/S100A9, for example, is an assay using a mixture of polyclonal coating antibodies and a variety of capture antibodies against six different epitopes.12 28 In contrast, the antigen in S100A12-immunoassay is more accurately defined.

As biomarkers that can be quantified in stool, damage-associated molecular patterns (DAMPs) give cell-specific information about local disease activity and tissue damage

PRACTICAL ASPECTS RELATED TO FAECAL BIOMARKERS

Today there are sufficient data available to underline the usefulness of faecal biomarkers in the clinical setting. Whenever gastrointestinal symptoms urge us to differentiate organic from functional disorders, proteins derived from neutrophils, monocytes and epithelium can serve as markers of intestinal inflammation and mucosal damage. This ability makes them feasible tools for the identification of those patients who warrant further invasive check-up, once infections have been ruled out. Up to date, other causes of non-infectious mucosal diseases involving inflammation and damage, such as NSAID-related ischaemic enteritis, polyps or tumours cannot be reliably differentiated by the available stool markers but may be identified by endoscopy. The differentiation of IBD from functional disorders by faecal biomarkers may be especially attractive in childhood, because (1) they can be applied as non-invasive screening tests, and (2) the differential diagnoses mentioned above for organic intestinal disease are less likely than IBD during childhood.

Another very useful application of faecal biomarkers is certainly the application in monitoring disease activity, as they correlate closer to endoscopic and histological evidence of inflammation than clinical activity scores. They can be used for detecting subclinical disease activity before it becomes clinically evident. With this regard, the ability of faecal S100A8/S100A9 in predicting disease relapses especially in ulcerative colitis is remarkable both in adults and in children.90 92 99 On the other hand, quantifying mucosal damage is important, as mucosal healing both in Crohn’s disease and in ulcerative colitis is associated with decreased risk of hospitalisation and surgery. Therefore, aiming for complete remission including normalisation of stool markers of inflammation seems reasonable, keeping their correlation to endoscopy and histology in mind.

Markers of leucocyte degranulation are available commercially. From the DAMP group, S100A8/S100A9 (calprotectin) assays are available, while an assay for S100A12 is under development and will be launched in the future. For faecal S100A8/S100A9 (calprotectin), first results of rapid tests are promising.100 101 Rapid, qualitative or semi-quantitative tests are suitable for discrimination of IBD from IBS. The cut-off of these tests is so low that their value in monitoring IBD is limited. A rapid quantitative test would be useful in monitoring IBD. Future studies will have to confirm whether phagocyte-related proteins offer advantages over molecules that are also derived from epithelial cells. It is conceivable that the latter are sensitive indicators of mucosal damage while markers specific for neutrophils are excellent indicators of early inflammatory events, which is a benefit for depicting relapsing disease.

Faecal biomarkers are especially useful in monitoring disease activity, detecting unapparent intestinal inflammation and predicting relapses

CONCLUSIONS

Although the pathogenesis of IBD is still only partially understood, increases in our knowledge about the role of local danger signals and their receptors have shed new light on the inflammatory processes underlying Crohn’s disease and ulcerative colitis. DAMP molecules may link mechanisms of inflammation with molecular targets which can be used as markers of disease activity. The reason for the diagnostic accuracy of these novel biomarkers rests upon the fact that DAMPs give cell-specific information about local disease activity and tissue damage.

Faecal S100A8/S100A9 and lactoferrin are reliable markers of inflammatory activity in the gut, able to distinguish organic from functional disease with high diagnostic accuracy and helpful in a number of clinical conditions related to IBD. Neutrophil-specific S100A12 is a “newcomer” in this field but has already yielded promising results in first studies. The diagnostic performance may be even better than that of S100A8/S100A9 (calprotectin) and lactoferrin, which, however, has to be confirmed by further studies. The clinical application of S100A8/S100A9 and lactoferrin measurements has been further facilitated by the implementation of faecal rapid tests, an easy method, which should be developed also for S100A12.101

In the case of DAMPs and innate immunity, the translation of research into clinical practice is a bidirectional process. Having feasible markers of inflammation and tissue damage will foster research in multiple ways. Bearing in mind the pro-inflammatory mechanisms engaged by DAMP molecules, the diagnostic capacities of these proteins due to their dramatic over-expression at sites of inflammation also point to a key function in the pathogenesis of IBD. It is conceivable that DAMP molecules represent potential targets for novel therapies. The overlapping effector functions of PAMPs and DAMPs may also help to further emphasise the link between the gut flora, pathogens and disturbed innate immune functions in IBD, which is even more significant taking into account the reported association of PRR alterations (eg, NOD proteins) with IBD.

Since the introduction of occult blood tests, there has been a constant improvement of molecular markers for intestinal inflammation. DAMP molecules are excellent examples for contemporary biomarkers that are specific for cells critically involved in the underlying pathophysiology, secreted once these cells are activated, and appear in faeces during early inflammatory events of IBD. Future improvements of assay formats and further progress in our knowledge about the biology of these proteins will help translating IBD research into patient care.

REFERENCES

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Footnotes

  • Competing interests: None.

  • Funding: This work was supported by grants from the Broad Medical Research Program (IBD-0201R) and the Crohn’s and Colitis Foundation of America (Ref# 1911).

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