The mechanisms of prednisone inhibition of inflammation in Crohn's disease involve changes in intestinal permeability, mucosal TNFα production and nuclear factor kappa B expression

Authors


Dr G. E. Wild, Montreal General Hospital, 1650 Cedar Avenue, Room D7-120, Montreal, Quebec, Canada, H3G A4.
E-mail: gary.wild@muhc.mcgill.ca

SUMMARY

Background : The clinical course of Crohn's disease after the induction of remission with medical therapy is characterized by unpredictable relapse.

Aim : To evaluate three surrogate markers, intestinal permeability, mucosal TNFα and nuclear factor (NF)-κB/IκBα expression, in order to determine the relationship of these parameters to clinical relapse.

Methods : Thirty patients with active Crohn's disease were treated with a 10 week course of prednisone using a tapering dosing regimen. Intestinal permeability (lactulose/mannitol [L/M ratio]) was determined at baseline and at the end of prednisone tapering. TNFα production and the levels of expression of NF-κB/IκBα were measured in colonic mucosal biopsies obtained after the induction of remission.

Results : Twenty-two patients (73%) achieved remission and 50% of patients experienced a clinical relapse during the ensuing 12 months. Treatment with prednisone resulted in a significant decrease in the L/M ratio. Of the patients that relapsed, 75% had a raised L/M ratio at the time of remission compared with 20% of patients with a normal L/M ratio (P < 0.008; hazard ratio = 6.094; CI 1.55, 17.43). Mucosal TNFα production was greater in relapsers compared with those who remained in remission. The levels of NF-κB in relapsers were significantly greater and levels of cytosolic IκBα were significantly lower compared with those measured in patients who remained in remission.

Conclusions : These findings underscore the importance of incorporating biological parameters of inflammation in determining the clinical course of Crohn's disease.

Introduction

The intestinal epithelium constitutes a dynamic cellular interface that orchestrates the regulated absorption of water, electrolytes, and a diverse array of macro and micronutrients, while at the same time serving as a critical barrier preventing the migration of pathogens from the intestinal lumen to the submucosa.1 Abnormalities in this barrier function, manifested biochemically as increased intestinal permeability to sugars and macromolecules, has been documented in Crohn's disease and appears to be a useful surrogate biological marker of inflammation.2–7

Wyatt and coworkers8 reported that a single measure of intestinal permeability, as determined by measuring the urinary clearance of lactulose and mannitol (L/M ratio), was predictive of clinical relapse in Crohn's disease in remission. This observation has been confirmed in a series of subsequent studies using single or serial estimates of intestinal permeability.9–12 By contrast, there is a relative paucity of data pertaining to the effects of medical treatment on intestinal permeability in active Crohn's disease.7

Several studies have shown that proinflammatory cytokines such as tumour necrosis factor (TNF)α and interferon (IFN)γ, both of which are key determinants of the pathophysiology of the inflammatory response in Crohn's disease,13 diminish epithelial barrier integrity both in cultured intestinal epithelial cell and in vivo.14 The alterations in intestinal epithelial barrier integrity elicited by proinflammatory cytokines occur even in the absence of overt cellular damage or ulcers, which ultimately leads to an increase in intestinal permeability.

One major downstream target of TNFα is the inducible transcription factor, nuclear factor kappa B (NF-κB), which up-regulates the transcriptional activity of a diverse array of promoters of proinflammatory cytokine genes.15, 16 NF-κB is sequestered in the cytoplasm where it is associated with inhibitory proteins of the IκB family.15, 16 NF-κB is activated by a broad range of stimuli, including TNFα, IL-1 and lipopolysaccharide. In response to stimuli, IκB is phosphorylated, thus allowing the NF-κB p65 subunit to translocate to the nucleus where it initiates the transcription of a host of target genes involved in the inflammatory response.15, 16

NF-κB p65 activation and increased expression has been demonstrated in the intestinal mucosa in inflammatory bowel disease,17, 18 in isolated epithelial cells19 and in models of colitis.20 In inflammatory bowel disease, the level of activation of NF-κB correlated with the degree of intestinal mucosal inflammation.17, 18 The use of corticosteroids is central to the clinical management of patients with inflammatory bowel disease.21, 22 Corticosteroids inhibit inflammation by a diverse array of mechanisms, including decreased transcription of cytokine genes, decreased eicosanoid production, inhibition of adhesion molecule synthesis and decreased chemotaxis of monocytes and neutrophils.23 Importantly, the immunosuppressive and anti-inflammatory properties of corticosteroids are mediated, at least in part, by steroid-dependent inhibition of NF-κB mediated gene expression through blockade of NF-κBp65 and up-regulation of IκBα gene expression.24, 25

In the present study, we sought to determine the effects of prednisone on intestinal permeability, and whether the anti-inflammatory effects of prednisone are mediated through an inhibitory effect on mucosal TNFα and NF-κB expression.

Materials and methods

Patients

Thirty consecutive patients with Crohn's disease, who were attending a gastroenterology clinic, participated in this study. The study protocol was approved by the institutional review board and all patients gave informed consent. The diagnosis of Crohn's disease was based on established radiologic, endoscopic and histological criteria.26 Patients included in the study had active symptoms as indicated by a Crohn's disease activity index27 (CDAI) > 200. Patients were started on 40 mg of prednisone per day for 3 weeks and tapered completely over the following 7 weeks. At each clinic visit, the CDAI was calculated according to the patient's diary card. Patients who entered into remission were maintained on the same dose of mesalazine throughout the 12 month follow-up period, during which time they were seen at 6 week intervals. The clinical end point was defined as a clinical relapse (CDAI > 150 and a 100 point increase above baseline, and/or need for surgery)27 during the 1 year follow-up after prednisone tapering. Remission was defined by a CDAI < 150.27

Measurement of intestinal permeability

Intestinal permeability was measured as described by Wyatt and coworkers.8 At bedtime, subjects ingested 150 mL of a solution containing 5 g mannitol, 10 g lactulose and 22 g glucose (1300 mOsmol/L). Subjects were instructed to void prior to ingesting the test solution and all urine, including the first morning void, was subsequently collected in a preweighed container. The concentrations of urinary lactulose and mannitol were measured by high performance liquid chromatography according to methodology described elsewhere.28 Intestinal permeability was determined by the urinary excretion ratio of lactulose/mannitol (L/M ratio) for ingested dose. The L/M ratio was measured at baseline and at the end of prednisone tapering. In addition, intestinal permeability was determined in 23 healthy volunteers.

Measurement of TNFα in colonic mucosal biopsies

Colonic mucosal biopsies were obtained from endoscopically normal appearing noninflamed mucosa, from patients who were in clinical remission at the end of prednisone tapering. Mucosal biopsy samples were placed on the wire grids of Falcon organ culture dishes (Fisher Scientific, Montreal, Canada), the mucosal surface being uppermost, and cultured in 1.5 mL of RPMI medium containing 2 mmol/L glutamine, 25 mmol/L HEPES buffer (pH 7.4), 10% fetal bovine serum and antibiotics (penicillin, gentamycin, fungizone) according to the method described by Allan and Jewell.29 The colonic mucosal biopsies were explants placed for 8 h in an incubator at 37 °C in a humidified atmosphere containing 5% CO2. Duplicate aliquots of the media were snap frozen and stored at −70 °C until determination of TNFα concentration using a standardized ELISA protocol from R & D Systems (Minneapolis, MN, USA). The limit of detection of the ELISA for TNFα was 0.5–32 pg/mL. Results were expressed as pg/mg of tissue.

Western blot analysis of NF-κB and IκB

Colonic mucosal biopsies from noninflamed mucosa were obtained from patients who were in clinical remission at the of prednisone tapering. Biopsies were snap frozen in liquid nitrogen and stored at −70 °C. Nuclear extracts and cytosolic fractions were prepared as described in detail elsewhere.30 Briefly, biopsy samples were pulverized on liquid nitrogen, and cytosolic extracts were collected in a buffer containing 10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, and 10 mmol/L KCl. Nuclear extracts were prepared by solubilizing the remaining nuclei in a buffer containing 20 mmol/L HEPES (pH 7.9), 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA and 25% glycerol. Both buffers were supplemented with a cocktail of protease inhibitors which included the following: 1 mmol/L dithithreitol, 0.5 mmol/L phenylmethylsulphonyl fluoride, along with aprotinin, leupeptin and pepstatin (each 1 µg/mL) and sodium fluoride, benzamidine and sodium vanadate (each 1 mmol/L). Total protein was measured using the Bio-Rad reagent (Bio-Rad Laboratories, Mississauga, Ontario, Canada), and all samples were adjusted to an equal protein content prior to fractionation on polyacrylamide gels.

Aliquots (15 µL) of cytosolic (20 µg protein) and nuclear (10 µg of protein) fractions were fractionated on 12% denaturing polyacrylamide gels followed by electroblotting to nitrocellulose membranes. Equal loading was confirmed by Ponceau S staining of the membranes. The membranes were incubated in Tris-saline buffer (pH 7.5) containing 0.5% Tween-20 (TTBS), and 5% nonfat milk, for 1 h at 22 °C. The membranes were subsequently probed for 1 h at 22 °C with antibody to NF-κB p65 (1 : 100) or IκBα (1 : 100) (Santa Cruz Biotechnology, Santa-Cruz, CA, USA) diluted in the same buffer. The membranes were washed in TTBS and incubated with alkaline phosphatase conjugated goat antirabbit IgG (1/1500 dilution) for 1 h at 22 °C. After further washes with TTBS, the immune complexes were visualized using the CDP-Star detection system (Boehringer Mannheim, Laval, Quebec, Canada). The fluorograms were digitized with a Hewlett Packard Scanner and analysed using Gel-Cypher Analyser software (Lightools Research, Encinitas, CA, USA). Determination of the signal area was determined using duplicate samples.

Data analysis

Results are expressed as means (s.d.). The Mann–Whitney test was used to compare intestinal permeability, TNFα, NF-κBp65 and IκBα in the relapse and nonrelapse groups. The Wilcoxon matched pairs test was used to assess dependent samples. Time-to-relapse curves were derived using the Kaplan–Meir method31 and statistical significance was determined using the log rank test.

Results

Patient characteristics

The baseline clinical features of the patient group are reported in Table 1. All patients had been in remission for > 6 months. None of the patients was taking immunosuppressive therapy for Crohn's disease, nor had any patients taken corticosteroids during the past 6 months. Isolated ileal disease was present in 13 patients and the remainder had ileocolonic disease.

Table 1.  Clinical characteristics
CharacteristicDistribution
  1. Values are expressed as the mean.

Demographics
 Gender (M/F)14/16
 Age (years)33.9 (19–52)
Clinical data
 Duration of disease (years)5 (1.5–12)
 Time elapsed since last relapse (months)10 (6.5–16)
 Baseline CDAI (points)285 (205–409)
 Ileal involvement alone (number of patients)13
 Ileocolonic involvement (number of patients)17
 Previous resection (number of patients)5

Treatment with prednisone induced remission in 22 of 30 (73%) patients, who were subsequently followed for a 12 month period or until relapse. None of the patients withdrew from the study. All of the patients were taking mesalazine at baseline, and the doses were held constant during the time course of treatment with prednisone and during the subsequent follow-up period.

All of the patients underwent assessment of intestinal permeability at baseline and at the time of completion of prednisone tapering. Only those patients who were in clinical remission (CDAI < 150) underwent flexible sigmoidoscopy, at which time mucosal biopsies were taken for the measurement of TNFα production and the determination of NF-κB expression. None of the eight patients who failed to enter into remission after prednisone therapy required surgery.

Effects of prednisone on intestinal permeability

We examined intestinal permeability in 23 healthy volunteers; the L/M ratio in this group was 0.021 ± 0.004 and the variation within the group was low. We defined the upper limit of normal for the L/M ratio as 0.029 (mean of the controls + 2 s.d.). Patients with active Crohn's disease had a higher mean L/M ratio compared with controls (0.088 ± 0.026, P < 0.001) and the L/M ratio varied considerably within this group (0.052–0.153) (Figure 1). Overall, the L/M ratio was significantly decreased after treatment with prednisone (0.046 ± 0.024, P < 0.001). In the group of patients that did not enter into remission after treatment with prednisone (n = 8) we observed a significant difference between the pre- (0.095 ± 0.062) and post (0.062 ± 0.018)-treatment levels of the L/M ratios (P < 0.001). The change in L/M ratio was 33 ± 5% in the patients who did not enter remission compared with 55 ± 4% in patients who were in remission after prednisone treatment (P < 0.008). Finally, we compared the L/M ratios in those patients who entered into clinical remission after treatment with prednisone and observed a significant difference in the L/M ratio between relapsers and nonrelapsers (0.055 ± 0.018 vs. 0.026 ± 0.017; P < 0.003) (Figure 2).

Figure 1.

Changes in intestinal permeability in Crohn's disease patients (n = 30) after treatment with prednisone. Patients with active Crohn's disease (CDAI > 200) were treated with 40 mg prednisone per day for 3 weeks and tapered completely over the following 7 weeks. Intestinal permeability was determined by measuring the urinary clearance of lactulose and mannitol (L/M ratio).

Figure 2.

Intestinal permeability as measured by the L/M ratio in relapsers (n = 11) and nonrelapsers (n = 11) who initially entered into clinical remission after treatment with prednisone.

Predictive value of intestinal permeability

To determine the predictive value of the L/M ratio, the data from Crohn's disease patients in remission after treatment with prednisone were divided into groups with normal (L/M < 0.029) (n = 10) and raised (L/M > 0.029) (n = 12) intestinal permeability. Overall, 11 out of 22 (50%) patients experienced a clinical relapse during the ensuing 12 months. Of the patients that relapsed, nine of the 12 (75%) had a raised L/M ratio at the time of remission compared with two out of 10 (20%) with a normal L/M ratio (Figure 3) (P < 0.008; hazard ratio = 6.094; CI 1.55, 17.43). The sensitivity of the L/M ratio was 82% and the specificity was 73%. The positive predictive value of the L/M ratio was 75%.

Figure 3.

Percentage of patients who remained in remission after treatment with prednisone. Patients with active Crohn's disease (n = 30) were treated with 40 mg prednisone per day for 3 weeks and tapered completely over the following 7 weeks. All patients underwent assessment of intestinal permeability at baseline and at the time of completion of prednisone tapering. Treatment with prednisone induced remission in 22 of 30 patients who were subsequently followed for a 12 month period or until relapse.

Effects of prednisone on TNFα production and the expression of NF-κB and IκBα in colonic mucosal biopsies

Colonic mucosal biopsies were obtained from the 22 patients who attained a clinical remission with prednisone treatment, and TNFα production was measured after culturing the biopsies in vitro(Figure 4). The levels of production of TNFα in the colonic mucosa of patients who subsequently relapsed were significantly greater than those in the patients who remained in remission (43.5 ± 8.7 vs. 23.9 ± 5.1; P < 0.001).

Figure 4.

Colonic mucosal TNFα production in relapsers (n = 11) and nonrelapsers (n = 11) who initially entered into clinical remission after treatment with prednisone.

The levels of expression of NF-κB and IκBα were measured by western blotting of nuclear and cytosolic extracts prepared from colonic mucosal biopsies of patients after induction of remission with prednisone. The levels of NF-κB in the colonic mucosa from patients who relapsed during the follow-up period were significantly greater than those in patients who remained in remission (8.7 ± 2.3 vs. 2.4 ± 1.1; P < 0.001) (Figure 5a). By contrast, the levels of IκBα, the endogenous inactivator of NF-κB,15, 16 were significantly lower in the colonic mucosa at remission in those patients that subsequently relapsed compared with those who remained in remission (3.6 ± 1.6 vs. 11.1 ± 2.4; P < 0.001) (Figure 5b).

Figure 5.

Colonic mucosal NF-κB p65 (a) and IκBα (b) levels of immunodetectable protein in relapsers (n = 11) and nonrelapsers (n = 11) who initially entered into clinical remission after treatment with prednisone.

Discussion

Corticosteroids comprise the current mainstay for the induction of remission in moderate to severe Crohn's disease,21, 22 and previous studies have reported clinical response rates ranging from 60 to 90%, depending upon the treatment protocol used and the definition of remission.26, 32–34, 42 Additionally, oral corticosteroids have been shown to be superior to mesalazine in the treatment of moderate to severe Crohn's disease.21, 22

The clinical course of Crohn's disease is characterized by periods of remission punctuated by episodes of clinical relapse associated with an increase in symptoms due to increased inflammatory activity.26, 32 Symptomatic recurrences occur at a rate of 40–60% over a period of 12 months in patients with Crohn's disease who enter a state of medically induced remission.26, 32 Corticosteroids are effective in the induction of remission in Crohn's disease, although there is no evidence to support the use of corticosteroids, including budesonide, for the maintenance of remission in Crohn's disease.21, 22 While mesalazine is modestly effective in the treatment of mild to moderate Crohn's disease, it has not been shown to be effective in the maintenance of remission or in the prevention of relapse after the induction of remission with corticosteroids.34–38

In the present study, clinical remission was induced with a 10 week course of prednisone in 73% of patients with moderate Crohn's disease who were relapsing while taking mesalazine. This observation is congruent with previously reported corticosteroid-induced remission rates in this clinical setting.34 Overall, 50% of patients experienced a clinical relapse during the ensuing 12 months. These findings are in agreement with previously reported rates of relapse following medically induced remission.26, 32, 34

At present, the management of patients with Crohn's disease is focused on periodic clinical assessment. The identification of prognostic factors reflective of the inflammatory process, and which allow for the identification of patients at high risk of clinical relapse, represents an unmet need in the management of these patients. In this regard, an extensive array of laboratory parameters (e.g. C-reactive protein, erythrocyte sedimentation rate) including direct measurement of serum cytokine levels (e.g. IL-6), along with a variety of clinical indices, have been proposed as useful measures to assess the risk of relapse.39–41 Unfortunately, these indices lack sensitivity and the predictive value of the markers examined thus far has been disappointing. This may be explained, at least in part, by the fact that these markers are not reflective of the intestinal inflammatory response.

Intestinal permeability has been proposed as a surrogate marker of intestinal inflammation.1, 2 Support for this contention is based on evidence that the proinflammatory cytokines (e.g. IFNγ and TNFα),13, 42–44 which are overly expressed in the intestinal mucosa in Crohn's disease, can elicit increased epithelial permeability.14 An increase in intestinal permeability has been suggested by Wyatt and coworkers8 as a predictor of recurrence of clinical symptoms in quiescent Crohn's disease, with a sensitivity of 81% and specificity of 73%, and represents a substantial improvement over nonspecific markers of inflammation.39–41 The observations of Wyatt et al. pertaining to the utility of the L/M ratio as a predictor of relapse in inactive Crohn's disease have been recently corroborated by other investigators.9–12 In the present work, our findings of increased intestinal permeability in active Crohn's disease are in agreement with those reported elsewhere.3–7 In addition, the value for the upper limit of the L/M ratio, which we defined in a normal population, is in agreement with data pertaining to the normal population reported by others.8, 10 Our findings constitute the first report of the effects of prednisone on intestinal permeability in active Crohn's disease (Figure 1). Treatment with prednisone resulted in an approximately 50% reduction in intestinal permeability as measured by the L/M ratio. The proportion of patients who entered into clinical remission after prednisone therapy, and who had a normal L/M ratio, was similar to the proportion observed in the group of patients with inactive Crohn's disease and a normal L/M ratio reported elsewhere.8 In the present study, 75% of the patients who experienced a clinical relapse during the ensuing 12 months had a raised L/M ratio at the time of remission, whereas 20% of patients with a normal L/M ratio relapsed over a similar time frame (Figure 3).

Our observations pertaining to increased intestinal permeability as a predictor of relapse confirm and extend those findings reported by other groups.8–12 Here, we report that intestinal permeability has a sensitivity of 82% and specificity of 73% for the prediction of relapse at 1 year. D'Inca and coworkers9 observed that in a group of 130 patients with inactive Crohn's disease, the L/M ratio had a sensitivity of 53.3% and a specificity of 84.6% for the prediction of relapse in the ensuing 4 months. Arnott et al.12 identified the lactulose to rhamnose ratio as an independent variable predictive of relapse over a 1 year follow-up interval in 50 patients with inactive Crohn's disease, and defined a sensitivity of 89% and specificity of 76%. Using the same probes, Tibble et al. found that intestinal permeability had a sensitivity of 84% and specificity of 64% for the identification of relapse in a cohort of 50 patients with inactive Crohn's disease.11 These investigators determined the value of faecal calprotectin levels as a predictor of relapse in this cohort, and defined a sensitivity of 90% and specificity of 83% for this neutrophil associated protein. While faecal calprotectin appears to be a simple noninvasive marker of subclinical inflammation, the utility of this method in the assessment of clinical prognosis and response to treatment in Crohn's disease awaits confirmation. In contrast to the findings pertaining to the potential utility of intestinal permeability as a predictor of relapse, previous studies have shown that levels of erythrocyte sedimentation rate and C-reactive protein did not differ between patients who relapsed and those who remained in remission.9, 11, 12

Corticosteroids possess a diverse array of anti-inflammatory properties, including the ability to inhibit the expression of proinflammatory cytokines such as TNFα.23 While recent work has shown that TNFα is an important therapeutic target in Crohn's disease,45 there is a paucity of data examining the expression of this pivotal cytokine in response to therapeutic interventions. Schreiber and coworkers have recently examined the secretion of TNFα by cultured lamina propria mononuclear cells of colonic mucosal biopsies obtained from patients with Crohn's disease in whom remission was induced by corticosteroids.46 In this study, increased secretion of TNFα (> 70 pg/mL) was predictive for acute relapses in the following year. These findings are supported by the present work, where we examined the production of TNFαin vitro by intact colonic mucosal biopsies and observed a nearly twofold increase in TNFα production in mucosal biopsies obtained from patients at the time of remission who subsequently relapsed during the follow-up period (Figure 4). Finally, the secretion capacity for TNFα, measured by whole blood cytokine assays, was down-regulated after treatment of refractory Crohn's disease patients with anti-TNFα, and relapsers were characterized by a rise in TNFα secretion prior to relapse of symptoms.47

Glucocorticoids have been shown to inhibit NF-κB activation and stimulate IκB expression in a variety of cell types.24, 25 Thus, in the present study, we examined the expression of NF-κB p65 and IκBα expression in mucosal biopsies obtained from patients at the time of remission, and observed significantly increased mucosal levels of NF-κB p65 and decreased levels of IκBα in those patients who subsequently relapsed during the follow-up period (Figure 5). Thiele et al. reported a similar pattern of changes in NF-κB p65 and IκBα expression in resection specimens from five Crohn's disease patients with active disease who had received corticosteroids for varying intervals prior to surgery.51 While sulfasalazine and mesalazine have previously been shown to modestly inhibit NF-κB activation and inhibit the degradation of IκB,48–50 the most compelling evidence for the importance of NF-κB as a therapeutic target comes from a recent report in which down-regulation of colonic mucosal NF-κB p65 was observed, after treatment of patients with refractory Crohn's disease with a single infusion of anti-TNFα; reaccumulation of nuclear NF-κB p65 was seen prior to clinical relapse.47

In summary, our findings demonstrate that the induction of remission in active Crohn's disease with corticosteroids is associated with an improvement in intestinal permeability as measured by the L/M ratio. These observations add to the body of evidence which suggests that intestinal permeability may be a clinically useful surrogate marker of intestinal inflammation. The extent to which other markers of intestinal inflammation are modulated by corticosteroid treatment, including TNFα and signalling through the NF-κB p65/IκBα pathway, support the rationale for measuring inflammatory markers in order to predict relapse in Crohn's disease. The ability to predict relapse based on continuing subclinical mucosal inflammation, as determined by the L/M ratio, may allow for the optimization of maintenance therapy in high risk Crohn's disease patients.

Acknowledgements

This work was supported by an operating grant from the Crohn's and Colitis Foundation of Canada and from the Canadian Institute for Health Research. Gary E. Wild is a senior clinician scientist and Alain Bitton is a junior clinician scientist supported by Les Fonds de la Recherche en Sante du Quebec.

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