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Keywords:

  • inflammatory bowel disease;
  • Crohn's disease;
  • ulcerative colitis;
  • bacterial translocation;
  • endotoxemia;
  • lipopolysaccharide-binding protein

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

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.

(Inflamm Bowel Dis 2007)

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients

Blood samples were taken from 104 patients with CD (54 men; mean age, 37 yr [range 15–84 yr]) and 52 patients with UC (29 men; mean age, 42 yr [range 20–87 yr]). Diagnosis was based on conventional clinical, radiological, and endoscopic criteria, supported by histopathologic findings for all UC patients and when possible for CD patients.16 UC disease activity was scored according to the Truelove & Witts Index (TW) and the Lichtiger Colitis Activity Index (LCAI).17, 18 We excluded patients with parasitic disease, active infection (discarded routinely by blood and feces cultures as clinically indicated), immunosuppressive treatment in the previous 3 months, neoplasia, pregnancy, antibiotics in the previous week, or congenital immunodeficiency or who had undergone surgery in the previous 3 months. Crohn's disease activity was graded according to the Crohn's Disease Activity Index (CDAI) and the Harvey-Bradshaw Index (HBI).19, 20 Active disease was defined as an HBI score of more than 4 for CD or an LCAI of more than 3 for UC.

The location (L) and behavior (B) of CD were classified according to Vienna criteria.21 L1 corresponds to disease in the terminal ileum, L2 to disease in the colon, L3 to disease in the ileocolon, and L4 to disease in the upper gastrointestinal tract. B1 corresponds to nonstricturing, nonpenetrating disease, B2 to stricturing disease, and B3 to penetrating disease. Extension of UC was assessed using classic criteria.16 Fifteen healthy volunteers matched with the patients in sex and age served as the control group. On the basis of endotoxin levels determined in the healthy controls, systemic endotoxemia and high endotoxin level were defined as an endotoxin concentration greater than 0.44 EU/mL, corresponding to the mean endotoxin concentration of the controls plus twice the standard deviation. Table I shows the main characteristics of the study population.

Table I. Characteristics of Patients with Crohn's Disease and Ulcerative Colitis
  Crohn's disease Ulcerative colitis
  • *

    Disease activity assessed using the Harvey-Bradshaw Index (HBI) for Crohn's disease and the Lichtiger Colitis Activity Index (LCAI) for ulcerative colitis. Active disease was defined as an HBI score > 4 or an LCAI > 3.

  • Location (L) and behavior (B) were defined according to the Vienna classification criteria for CD patients and to the Lennard-Jones classification criteria for UC patients.

  • Before inclusion in the study.

  • §

    Azathioprine, 6-mercaptopurine, or mycophenolic acid.

Number of patients 104 52
Sex (female/male) 50/54 23/29
Age (years), mean (range) 37 (15–84) 42 (20–87)
Progression (m), mean (range) 21 (1–400) 44 (2–144)
Disease activity*    
 Active 41 32
 Inactive 63 20
Localization    
 Distal colitis   18
 Left-sided colitis   14
 Total colitis   20
 L1 (ileum) 30  
 L2 (colon) 19  
 L3 (ileocolon) 53  
 L4 (upper gastrointestinal 2  
Behavior    
 B1 (inflammatory) 33  
 B2 (stricturing) 15  
 B3 (penetrating) 53  
 Inactive CDActive CDInactive UCActive UC
Maintenance treatment    
No medication18638
Steroids10954
5-ASA or sulfasalazine1612912
Steroids + 5-ASA9413
Immunomodulators§9825
Infliximab + immuno.12  

Thirty-one patients with CD (17 with active disease and 14 with inactive disease) and 15 patients with UC (10 with active disease and 5 with inactive disease) were followed for 6 weeks to establish correlations between serum levels of LBP, sCD14, and endotoxin with course of disease activity. Therapy was as undertaken in current medical practice (Table II).22

Table II. Active Treatment Strategies in Prospective Groups
MedicationInactive CD (n = 14)Active CD (n = 17)Inactive UC (n = 5)Active UC (n = 10)
  • *

    Azathioprine, 6-mercaptopurine or mycophenolic acid.

No medication4 1 
Steroids1713
5-ASA or sulfasalazine1433
Immunomodulators*52 1
Steroids + 5-ASA34 3

Assays

Blood samples were collected into endotoxin-free tubes (Endo Tube ET; Chromogenix AB, Stockholm, Sweden) and centrifuged, and the plasma was stored at −80°C until analysis. Plasma LBP was determined by the immunometric sandwich assay with a chemiluminescent substrate using an automated analyzing system (Immulite LBP; DPC, Los Angeles, CA). The lower assay sensitivity limit was 0.2 μg/mL. Plasma endotoxin was measured by a quantitative, chromogenic Limulus amebocyte lysate assay (QCL-1000; BioWhittaker Inc., Walkerswille, MD). Endotoxin inhibitors were removed from plasma by dilution with sterile endotoxin-free water and by treating the mixture at 75°C for 5 minutes. The lower limit of detection of this assay is 0.05 EU/mL. Intra-assay and interassay coefficients were 9.2% and 10.3%, respectively, for endotoxin and 3.0% and 4.1%, respectively, for LBP. Serum concentrations of TNF-α, IL-6, IL-8, and soluble receptor for interleukin-2 (sIL-2r) were measured by chemiluminescence (Immulite; DPC, Los Angeles, CA), and soluble CD14 (sCD14) was determined with an enzyme-linked immunosorbent assay (Quantikine; R&D Systems, Minneapolis, MN). The lower limits of detection of these assays were 1 pg/mL for TNF-α, IL-6, and IL-8; 5 IU/mL for sIL-2r; and 125 pg/mL for sCD14. Serum concentrations of alpha-1-glycoprotein/orosomucoid (A1G-ORO; Dade-Bering BN2; Eschborn, Germany) were quantified by nephelometry (Dade-Behring; BN2), and C-reactive protein (CRP) was determined in an AEROSET system (Abbott Laboratories, Abbott Park, IL) adapting the method provided by Sigma (Barcelona, Spain). The erythrocyte sedimentation rate (ESR) was measured in a VESMATIC-60 (Menarini diagnostics, Barcelona, Spain) analyzer.

Statistical Analysis

Data among groups were compared using the Kruskal-Wallis H nonparametric test, followed by a post hoc Mann-Whitney U test between distinct categories. We used the paired Wilcoxon's test to analyze changes in disease activity and serum markers after 6 weeks. Associations between continuous variables were assessed through linear regression analysis, and correlations between serum markers and disease activity were analyzed using Pearson's test. Data are expressed as means ± SD. The level of significance was set at P < 0.05. All statistical tests were performed using SPSS 12.0 software.

Ethical Considerations

The study protocol was approved by the ethics committee of the Hospital Ramon y Cajal, and informed consent was obtained from each participant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Endotoxin, LBP, and sCD14 Levels in Patients with Inflammatory Bowel Disease

Elevated endotoxin levels (>0.44 EU/mL) were observed in both CD (48%) and UC (28%) patients. Mean endotoxin levels were higher in CD patients (active and inactive) than in controls (0.46 ± 0.16 versus 0.32 ± 0.06 EU/mL; P < 0.0001). The percentage of patients with active CD who had a high concentration of endotoxin was double that of patients with inactive CD (63% versus 36%, P < 0.05). Patients with active disease also had higher plasma endotoxin levels (0.52 ± 0.18 EU/mL) than those with inactive disease (0.43 ± 0.12 EU/mL) and controls (0.32 ± 0.06 EU/mL; P < 0.01; see Fig. 1 and Table III). LBP values behaved similarly, with higher levels in patients with active CD than in patients with inactive CD or in controls. Note that even patients with inactive CD had serum endotoxin and LBP levels that differed significantly from those in healthy controls.

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Figure 1. Serum levels of endotoxin (LPS) and LBP in patients with IBD and in healthy controls. Levels were determined in 104 patients with CD (41 with active disease and 63 with inactive disease), in 52 patients with UC (32 with active disease and 20 with inactive disease), and in 15 healthy controls. Symbols show individual serum levels and means with 95% confidence intervals, shown by error bars (*P < 0.05, **P < 0.01, mean differences with healthy controls).

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Table III. Plasma Concentrations (Mean ± SD) of LBP, sCD14, Endotoxin, and Inflammatory Markers in Patients and Controls in the Study
 Controls (n = 15)Crohn's diseaseUlcerative colitis
Inactive (n = 63)Active (n = 41)Inactive (n = 20)Active (n = 32)
  • *

    P < 0.01 active versus controls;

  • P < 0.05 active versus controls;

  • c

    P < 0.01 inactive versus controls;

  • §

    P < 0.05 inactive versus controls;

  • P < 0.01 active versus inactive;

  • P < 0.05 active versus inactive.

Endotoxin (EU/mL)0.32 ± 0.060.43 ± 0.120.52 ± 0.18*,0.33 ± 0.050.45 ± 0.19,
LBP (μg/mL)7.2 ± 1.812.9 ± 5.123.1 ± 13.7*,9.3 ± 3.521.4 ± 10.9*,
sCD14 (ng/mL)1336 ± 3081237 ± 3931537 ± 4801167 ± 2031439 ± 370
A1G-ORO (mg/dL)88.9 ± 13.9103.6 ± 32.7§148.1 ± 56.5*,89.8 ± 19.3152.3 ± 66.2*,
CRP (mg/L)2.9 ± 1.75.0 ± 7.840.2 ± 53.4*,1.9 ± 2.935.8 ± 45.7*,
ESR (mm/h)6.8 ± 6.115.2 ± 13.643.5 ± 26.2*,16.0 ± 10.634.6 ± 29.9*,
IL-6 (pg/mL)3.4 ± 4.18.6 ± 16.522.5 ± 52.9*,6.0 ± 7.012.0 ± 12.1
IL-8 (pg/mL)7.6 ± 2.97.8 ± 6.419.7 ± 36.1,1.8 ± 10.329.2 ± 32.3*,
sIL-2r (IU/mL)625 ± 396512 ± 196767 ± 509,689 ± 2681167 ± 704*,
TNF-α (pg/mL)6.2 ± 6.59.7 ± 38.39.0 ± 24.01.5 ± 1.24.2 ± 4.7
HBI/LCAI2 ± 18 ± 32 ± 110 ± 3
CDAI/TW84 ± 36235 ± 917 ± 111 ± 3

In patients with UC, disease activity was also associated with higher serum endotoxin (0.45 ± 0.19 EU/mL) and LBP (21.4 ± 3.7 μg/mL) compared to those of the controls (P < 0.05 and P < 0.01, respectively). When the disease was active, endotoxemia was less frequent in UC (41%, 13/32) than in CD, and, interestingly, could only be detected in 1 patient with inactive UC (5%, 1/20). sCD14 levels were significantly higher in patients with active IBD than in patients with inactive IBD regardless of pathology.

Relationship among Activity Markers with Endotoxin-Related Mediators

As expected, CRP, ESR, and A1G-ORO levels were higher in patients with active CD and UC than in those with inactive disease (see Table III). A1G-ORO was significantly increased in patients with inactive CD (103 ± 32.7 mg/dL; P < 0.01) compared to in controls (68.9 ± 13.9 mg/dL). Serum concentrations of IL-6, IL-8, and the soluble receptor sIL2-r were higher (P < 0.05) in active IBD patients (both CD and UC) than in healthy controls (see Table III), but no significant differences were found in mean IL-6, IL-8, and sIL-2r levels between patients with inactive IBD and controls. TNF-α levels were increased in patients with active and inactive CD relative to the controls (P < 0.05), but this was not so in UC patients.

As shown in Table IV, patients with active CD and high endotoxin levels exhibited increased serum levels of LBP, CRP, and IL-6 and poorer clinical status, reflected by higher HBI and CDAI scores. Similarly, in endotoxemic patients with active UC, disease activity was more pronounced, and activation of the inflammatory response was greater, indicated by higher levels of LBP, soluble receptors, and proinflammatory cytokines, than in patients with active UC disease whose endotoxin levels were normal. In contrast, among patients with inactive CD, we found no differences between those with high and normal endotoxin levels (data not shown).

Table IV. Plasma Concentrations (Mean ± SD) of LBP, sCD14, and Inflammatory Markers in Patients with Active IBD (CD or UC) and Normal or High Endotoxin (LPS) Levels*
 Active CD (n = 41)Active UC (n = 32)
Normal LPS (n = 16)High LPS (n = 25)Normal LPS (n = 19)High LPS (n = 13)
LBP (μg/mL)16.5 ± 2.626.7 ± 2.815.3 ± 1.531.5 ± 3.2
sCD14 (ng/mL)1388 ± 3801647 ± 5301247 ± 2971733 ± 265
A1G-ORO (mg/dL)125.2 ± 50.8156.9 ± 49.0114.4 ± 32.0216.4 ± 66.1
CRP (mg/L)19.5 ± 11.549.3 ± 10.813.1 ± 15.978.2 ± 16.4
ESR (mm/h)35.3 ± 27.147.3 ± 23.424.5 ± 24.051.7 ± 33.1
IL-6 (pg/mL)6.1 ± 5.533.1 ± 13.76.6 ± 9.418.9 ± 11.7
IL-8 (pg/mL)11.8 ± 14.017.4 ± 25.618.6 ± 5.361.1 ± 16.5
sIL-2r (IU/mL)637 ± 302804 ± 400932 ± 4131607 ± 265
TNF-α(pg/mL)3.5 ± 2.313.2 ± 7.23.2 ± 4.85.1 ± 4.8
HBI/LCAI7 ± 39 ± 38 ± 312 ± 3
CDAI/TW202 ± 85257 ± 9010 ± 214 ± 2

In patients with active CD, LBP (R2 = 0.40, P < 0.01) and endotoxin (R2 = 0.41, P < 0.05) correlated with activity (HBI), with those with high endotoxin levels having improved scores (R2 = 0.45, P < 0.01; and R2 = 0.59, P < 0.05, respectively). In patients with UC, LBP, endotoxin, and sCD14 also correlated with LCAI (R2 = 0.49, P < 0.01; R2 = 0.59, P < 0.01; and R2 = 50, P < 0.05, respectively) in active disease, but no better correlation was observed in those patients with high endotoxin levels. Concentration of LBP correlated significantly with endotoxin and sCD14 levels both in patients with active UC and in patients with active CD (see Fig. 2), but not in patients with inactive disease. In patients with active IBD (CD and UC), LBP also correlated with CRP (r = 0.76 and r = 0.78, respectively; P < 0.01), A1G-ORO (r = 0.71 and r = 0.74, respectively; P < 0.01), and ESR (r = 0.64 and r = 0.65, respectively; P < 0.01). Correlations were also detected in patients whose disease was inactive with CRP (r = 0.71 and r = 0.60, respectively; P < 0.01) and with A1G-ORO (r = 0.50 and r =, respectively; 0.57; P < 0.01). In contrast, no correlations were observed between LBP, endotoxin, or sCD14 and proinflammatory cytokine level.

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Figure 2. Correlation of LBP plasma level with endotoxin (LPS) and sCD14 in active IBD.

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Effect of Treatment on Endotoxin-Related Markers

Because an effect of the differences in treatment on serum parameters could not be excluded, we investigated this association. We found lower LBP levels in active CD patients pretreated with immunomodulators (13.8 ± 4.7 μg/mL, P < 0.05) than in those receiving steroids (25.4 ± 6.2 μg/mL) or 5-ASA (23.5 ± 5.0 μg/mL); however, we did not detect significant differences in the endotoxin concentrations. We also investigated the effects of therapy-induced disease remission on endotoxin, LBP, and sCD14 levels in a subset of successfully treated patients with active disease (see Table V). In patients with CD, concentrations significantly changed after therapy (LBP from 29.1 ± 13 to 15.2 ± 7.3 μg/mL, P < 0.01; endotoxin from 0.61 ± 0.20 to 0.46 ± 0.13 EU/mL, P < 0.05; and sCD14 from 1680 ± 460 to 1330 ± 406 ng/mL, P < 0.05), showing values similar to those recorded for patients with inactive CD. As expected, the levels of the acute-phase reactants IL-6 and sIL2-r and clinical indices also returned to normal. In UC patients, LBP and sCD14 levels also diminished following medical control of the disease. We would like to highlight that serum endotoxin, LBP, and sCD14 concentrations remained unchanged in both inactive CD and inactive UC patients, with no clinical signs of disease relapse after 6 weeks (see Table VI).

Table V. Plasma Concentrations (Mean ± SD) of LBP, sCD14, Endotoxin, and Inflammatory Markers in Patients with Active IBD (CD or UC) at Baseline and After 6 Weeks
 Active CD (n = 17)Active UC (n = 10)
Baseline6 WeeksBaseline6 Weeks
  • *

    P < 0.01;

  • P < 0.05.

Endotoxin (EU/mL)0.61 ± 0.200.46 ± 0.130.54 ± 0.200.35 ± 0.07
LBP (μg/mL)29.1 ± 1315.2 ± 7.321.7 ± 9.813.6 ± 5.7*
sCD14 (ng/mL)1680 ± 4601330 ± 4061417 ± 3311208 ± 236
A1G-ORO (mg/dL)162.5 ± 54.9121.4 ± 30.8153.6 ± 52.3113.1 ± 26.8
CRP (mg/L)59.2 ± 56.26.7 ± 8.714.9 ± 10.511.1 ± 16.7
ESR (mm/h)53.1 ± 28.726.2 ± 16.634.6 ± 25.125.1 ± 24.9
IL-6 (pg/mL)29.9 ± 655.9 ± 7.418.8 ± 15.97.2 ± 8.0
IL-8 (pg/mL)11.3 ± 15.912.5 ± 12.741.4 ± 35.815.4 ± 7.0
sIL-2r (IU/mL)833 ± 680597 ± 1911225 ± 476948 ± 453
TNF-α (pg/mL)4.6 ± 7.91.9 ± 1.53.6 ± 5.62.9 ± 2.9
HBI/LCAI9 ± 34 ± 212 ± 25 ± 4*
CDAI/TW270 ± 90135 ± 613 ± 59 ± 3*
Table VI. Plasma Concentrations (Mean ± SD) of LBP, sCD14, Endotoxin, and Inflammatory Markers in Patients with Inactive IBD (CD or UC) at Baseline and After 6 Weeks
 Inactive CD (n = 14)Inactive UC (n = 5)
Baseline6 WeeksBaseline6 Weeks
Endotoxin (EU/mL)0.42 ± 0.100.46 ± 0.110.36 ± 0.070.35 ± 0.07
LBP (μg/mL)11.1 ± 3.614.1 ± 6.49.1 ± 3.413.6 ± 5.7
sCD14 (ng/mL)1231 ± 4451302 ± 4501081 ± 3121208 ± 236
A1G-ORO (mg/dL)89.3 ± 31.599.9 ± 38.789.3 ± 13.183.7 ± 5.5
CRP (mg/L)4.4 ± 6.58.6 ± 12.54.1 ± 2.42.5 ± 1.7
ESR (mm/h)17.3 ± 15.421.8 ± 11.616.8 ± 3.310.7 ± 9.5
IL-6 (pg/mL)9.4 ± 12.36.5 ± 6.04.4 ± 3.36.9 ± 10.7
IL-8 (pg/mL)5.3 ± 3.710.6 ± 10.66.5 ± 3.46.8 ± 2.8
sIL-2r (IU/mL)450 ± 180524 ± 196595 ± 145583 ± 229
TNF-α(pg/mL)2.1 ± 5.322.1 ± 67.47.5 ± 1.96.3 ± 0.5
HBI/LCAI1 ± 11 ± 14 ± 21 ± 1
CDAI/TW63 ± 2770 ± 407 ± 26 ± 1

We could not confirm previous reports linking the extent of endotoxemia with the location and behavior of CD.7, 8 Nevertheless, as shown in Figure 3, we did find greater increases of endotoxin and LBP concentrations in UC patients with full extension of the disease as opposed to UC patients with distal colitis.

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Figure 3. Plasma endotoxin (LPS) and LBP concentrations in inactive and active IBD according to disease location. CD patients were classified as having ileal (L1), colonic (L2), or ileocolonic (L3) disease according to the Vienna criteria. Disease extension in UC was grouped into 3 categories: distal colitis, left-sided colitis, and total colitis. Bars represent mean endotoxin levels, and error bars indicate 95% confidence intervals (*P < 0.05, mean differences with patients with inactive disease at the same location; #P < 0.02, mean differences between distal colitis and total colitis).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Óscar Pastor Rojo is recipient of a grant from the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (CM03/00041). We are indebted to Sergio Ávila Padilla and Manuela Díaz Enríquez for excellent technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES