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- MATERIALS AND METHODS
Background: In inflammatory bowel disease (IBD), enhanced inflammatory activity in the gut is thought to increase the risk of bacterial translocation and endotoxemia. By searching for signs of endotoxin-signaling cascade activation, including augmented levels of endotoxin, lipopolysaccharide-binding protein (LBP), and soluble CD14 receptor (sCD14), this prospective study sought to establish whether endotoxemia could contribute to greater clinical activity of disease.
Methods: Concentrations of plasma endotoxin, LBP, sCD14, several cytokines, acute phase proteins and clinical activity indices were determined in 104 patients with Crohn's disease (CD) and 52 patients with ulcerative colitis (UC).
Results: Endotoxemia was present in 48% of the patients with CD and in 28% of the patients with UC. The mean LBP was higher in patients with active CD (23.1 ± 13.7 μg/mL) and UC (21.4 ± 10.9 μg/mL) than in healthy controls (7.2 ± 1.8 μg/mL; P < 0.01). Elevated serum concentrations of endotoxin and LBP were even detected in patients with inactive CD. Among the patients with active IBD, those with higher endotoxin levels had the worst clinical activity scores and the highest LBP levels. Treatment normalized LBP concentrations, from 29.1 ± 13.0 to 15.2 ± 7.3 μg/mL; (P < 0.05) in active CD and from 21.7 ± 9.8 to 13.6 ± 5.7 μg/mL; (P < 0.01) in active UC, along with normalizing endotoxin and sCD14 plasma concentrations.
Conclusions: Patients with IBD show increased serum levels of endotoxin, LBP and sCD14. This alteration correlates with disease activity, with normal levels recovered after treatment, although less completely in Crohn's disease, and parallels a rise in proinflammatory cytokines, suggesting a contribution of bacterial products to the inflammatory cascade in these patients.
T he exaggerated intestinal inflammatory response observed in ulcerative colitis (UC) and Crohn's disease (CD), collectively termed inflammatory bowel disease (IBD), is thought to result from a combination of genetic, immunological, and bacterial factors.1 In turn, intestinal inflammation is suspected to lead to enhanced intestinal permeability with an increased risk of bacterial translocation and endotoxemia.2, 3 Indeed, the administration of endotoxin [Gram-negative bacterial lipopolysaccharide (LPS)] to human volunteers induces many of the systemic signs (fever, tachycardia, proinflammatory cytokine release, hypercoagulable state, etc.4) frequently observed in IBD. Several studies have provided evidence that circulating endotoxins are detectable in IBD,5–8 but it is not yet known whether endotoxemia contributes to disease activity in these patients. This is partly because there is no marker that reliably identifies individuals who suffer the frequent passage of bacteria or their products into circulation because measurement of endotoxin in biological fluids is notoriously difficult9 and because the endotoxemia that follows bacterial translocation is episodic and short-lived.4
Once in the bloodstream, endotoxin or endotoxin-containing particles (including intact Gram-negative bacteria) form complexes with lipopolysaccharide-binding protein (LBP) and activate monocytes and macrophages through toll-like receptors, probably involving the nucleotide-binding oligomerization domain 2 protein (NOD2) pathway.10 This leads to cytokine production (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and interleukin-8 [IL-8]), shedding of the extracellular domain of the soluble CD14 receptor (sCD14), and further LBP production. Studies of other disease states commonly associated with endotoxemia, such as sepsis or cirrhosis, have detected elevated levels of sCD14 and LBP.11, 12 Indeed, given the long half-lives of sCD14 and LBP (24-48 hours) compared to endotoxin (1–3 hours), sCD14 and plasma LBP seem to reflect long-term exposure to endotoxin rather than to the endotoxin itself.13, 14 To our knowledge, plasma LBP levels in IBD have not yet been investigated, and only a single published report has described high plasma sCD14 levels in a subgroup of IBD patients who had a promoter polymorphism in this gene.15 The aim of the present study was to examine whether systemic endotoxemia contributes to clinical activity in IBD patients by searching for elevated levels of endotoxin, LBP, sCD14, and proinflammatory mediators as an indication of activation of the endotoxin-signaling cascade.
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- MATERIALS AND METHODS
Our present findings confirm and extend earlier observations of circulating endotoxin in IBD5 in a large group of patients. The rates of endotoxin challenge we found are intermediate relative to those observed in other studies, which ranged from 17% to 88% in UC and from 31% to 98% in CD.5–8 These wide ranges may be explained by the different methods of assessing clinical activity and quantifying endotoxin and different inclusion criteria used. In addition, we found that higher plasma endotoxin and LBP levels were associated with more severe disease both in patients with UC and in patients with CD, which is in line with the increased concentrations of endotoxin, plasma IgG antiendotoxin, and anti-OmpC (Escherichia coli porin protein C antibodies) correlating with severity of IBD.7, 24 Nevertheless, other studies have not established a link between clinical endotoxemia and active disease.6
The endotoxemia in these patients has been attributed to several factors. Patients suffering from IBD show enhanced permeability of mucosa, leading to an increased absorption of macromolecules, which has been correlated with disease severity.25 In addition, bacterial overgrowth, abnormal microflora within the gut,26 and reduced bacterial clearance by immune cells6 are thought to increase the risk of bacterial translocation in these patients. The concentrations of both endotoxin and LBP reported here for active IBD were lower than those previously observed in septic patients12 and similar to those seen in patients with cirrhosis and chronic heart failure.11, 27 Our patients had no signs of active infection, and thus the behavior shown by endotoxin-related markers was more in line with the hypothesis of translocation of bacteria, rather than a focal infection, as the source of endotoxemia.
Endotoxin binds to LBP and activates monocytes through surface CD14/toll-like receptor complexes. This promotes the release of proinflammatory cytokines, including TNFα, IL-8, and IL-6, which activate or suppress the expression of acute-phase response genes in hepatocytes, the vascular endothelium, and other target cells.28 Consistent with this, our patients with active IBD who had high concentrations of endotoxin, showed raised LBP and sCD14 levels and an inflammatory immune and systemic response characterized by greater levels of cytokines and acute phase proteins, contributing to clinical worsening of their disease.
The picture changes in inactive IBD disease and also differs between UC and CD. Endotoxemia seems to be rare in patients with inactive UC; in effect, mean plasma concentrations of endotoxin, LBP, sCD14, and inflammatory markers were similar to those observed in healthy controls. In contrast, up to 36% of patients with inactive CD showed high amounts of endotoxin. This different behavior of endotoxemia could reflect differences in the inflammatory pattern of the two diseases after treatment. Thus, in UC, therapy achieves complete endoscopic and histological recovery, and the presence of a residual inflammatory infiltrate correlates with the relapse rate.29 A smaller percentage of patients with CD, however, show complete mucosal healing.30 Hence, although therapy can control symptoms, many CD patients still suffer significant ongoing inflammation, which is consistent with our finding of increased serum levels of endotoxin and LBP, as well as of A1G-ORO and TNF-α in inactive CD. These findings indicate a need for better tools to define disease remission and activity in CD because CDAI and HBI indices apparently misclassify some CD patients as inactive despite significantly elevated serum levels of some inflammatory markers.
We also observed a moderate increase in LBP in patients with inactive CD, with no greater stimulatory effects on the proinflammatory cascade. Previous studies have suggested that the stimulatory and inhibitory effect of LBP on the inflammatory cascade depends on the LBP/endotoxin ratio. Thus, in some experiments, high concentrations of LBP were inhibitory only when the endotoxin concentration was low, whereas at higher endotoxin concentrations, much higher concentrations of LBP were required to inhibit responses to the endotoxin.31 Of note is that inactive CD and UC are related to a similar LBP/endotoxin ratio (30.0 versus 28.2), both superior to controls (22.5). Thus, although it is difficult to ascribe a clear-cut physiological function to LBP, we could speculate that the increase in LBP may be sufficient to neutralize the stimulatory effects of moderate endotoxemia in inactive CD.
Only in active disease was the LBP release moderately correlated with the endotoxin challenge, and plasma LBP associations were strongest with CRP and A1G-ORO, proteins that share the same coordinated mechanism of hepatic expression.32 Interestingly, A1G-ORO, which has many similarities to LBP in its binding capacity for endotoxin and sCD14,33 was also elevated in patients with inactive CD, supporting a possible protective role for both proteins in moderate endotoxemia.
A link among endotoxemia, LBP and sCD14 release, cytokine production, and clinical activity is indicated by the normalization of these variables after medical treatment. Therapies including an elemental diet,34 prednisolone,35 and anti-TNF antibodies36 have been found to induce clinical remission and decreased gut permeability in patients with active IBD. Our results did not detect an association between different therapies and endotoxemia. Thus, reduced endotoxemia may be a consequence of the ameliorated intestinal permeability after treatment, which does not seem to be related with differences in medication. However, as recent studies have demonstrated, an endotoxin challenge is able to promote neutrophil recruitment from circulation into the gut mucosa, causing increased intestinal permeability.37 Under this scenario, a definition of causality is difficult to establish because both factors, endotoxin and intestinal permeability, at the same time also are secondary, perpetuating and worsening the processes in the intestinal mucosa. Further studies exploring the mucosa of IBD patients may help to define the contribution of endotoxemia to systemic and intestinal inflammation.
We hypothesize that if increased intestinal permeability and subsequent endotoxemia were a consequence of mucosal inflammation, the percentage of patients affected by systemic endotoxemia would be higher if the affected site was at greater risk of allowing the passage of macromolecules, such as the small bowel.38 Effectively, in some series, increased permeability has been shown to be more common in small bowel CD than in colonic CD.39 Nevertheless, our results indicated no relationship between plasma endotoxin level and the affected site in CD, whether inactive or active. In UC, we found differences in endotoxin and LBP levels between distal and fully extended disease, suggesting that the complete spread of the inflammatory process itself, rather than the location of the lesions, could condition the magnitude of the endotoxin challenge.
Taken together, our results support the hypothesis that passage of enteric bacterial products into circulation contributes to the inflammatory response in IBD patients. Endotoxemia defines a subset of active IBD patients with increased LBP and sCD14 levels and excess proinflammatory cytokine production, rendering worse clinical activity scores. We hypothesize that it is the disturbed permeability of the gut mucosa that is responsible for the high endotoxin levels in these patients. The link between plasma LBP and endotoxin levels confirms our previous finding that this protein is a useful marker of bacterial challenge.40 Further studies could possibly confirm the usefulness of this determination in IBD.