Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carol, M.
Right arrow Articles by Malagelada, J.-R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carol, M.
Right arrow Articles by Malagelada, J.-R.
(Journal of Leukocyte Biology. 2006;79:917-922.)
© 2006 by Society for Leukocyte Biology

Modulation of apoptosis in intestinal lymphocytes by a probiotic bacteria in Crohn’s disease

Monica Carol1, Natalia Borruel, Maria Antolin, Marta Llopis, Francesc Casellas, Francisco Guarner and Juan-R. Malagelada

Digestive System Research Unit, University Hospital Vall d’Hebron, Autonomous University of Barcelona, Spain

1Correspondence: Digestive System Research Unit, University Hospital Vall d’Hebron, Passeig Vall d’Hebron 129, E-08035 Barcelona, Spain. E-mail: mcarol{at}vhebron.net

ABSTRACT

Apoptosis of active T lymphocytes constitutes a major control mechanism of immune homeostasis and tolerance. In Crohn’s disease, abnormal activation of mucosal T lymphocytes against enteric bacteria is the key event triggering intestinal inflammation. Resistance of lymphocytes to apoptosis has been proposed as the pathogenetic defect. We examined the influence of bacteria-mucosa interactions on apoptosis of mucosal T lymphocytes. Ileal specimens were obtained at surgery from 12 patients with Crohn’s disease. Mucosal explants from each specimen were cultured with nonpathogenic Escherichia coli ATCC 35345, Lactobacillus casei DN-114 001, or no bacteria. Cytokine release was measured in supernatant, and mononuclear cells were isolated for phenotypic characterization and Bcl-2 family protein expression. Coculture of inflamed tissue with L. casei significantly reduced the release of interleukin (IL)-6 and tumor necrosis factor {alpha} (P<0.05). In addition, coculture with L. casei significantly reduced the number of T cells displaying the IL-2 receptor in the lamina propria. Expression of the antiapoptotic protein Bcl-2 in lamina propria lymphocytes was also reduced after coculture with L. casei, and the percentage of deoxyuridine triphosphate nick-end labeling positive lymphocytes increased. The nonpathogenic E. coli strain had no significant effect. In conclusion, L. casei reduces the number of activated T lymphocytes in the lamina propria of Crohn’s disease mucosa. A balanced, local microecology may restore immune homeostasis.

Key Words: human • T lymphocytes • inflammatory disorders • intestinal mucosa

INTRODUCTION

The number and diversity of mature T lymphocytes are controlled tightly by programmed cell death or apoptosis [1 ], which can occur throughout the life of a T cell in its resting and activated state, but it plays a particularly important role after antigen-specific lymphocyte activation and proliferation. After antigen clearance, only a minority of the T cells generated survives to become memory-type T cells, which protect from recurrent infection, whereas the vast majority of activated T cells undergoes apoptosis as part of the normal immune response [2 ]. Failure of this mechanism of lymphocyte control can lead to the development of autoimmunity or lymphoma [2 ]. Thus, lymphocyte apoptosis is a fundamental mechanism of immune homeostasis and tolerance [1 , 2 ].

In Crohn’s disease, abnormal activation of intestinal T lymphocytes as an exaggerated response to bacterial antigens in the gut lumen plays an important role in the development of intestinal mucosal lesions [3 , 4 ]. Several studies have suggested that in patients with Crohn’s disease, mucosal T cells escape normal apoptotic regulatory mechanisms. Consequently, the lifespan of antigen-primed T cells is extended, and an abnormal population of activated cells is retained within the mucosal compartment [5 6 7 ]. In vitro studies showed that such mucosal T cells are resistant to multiple apoptotic stimuli and have a reduced expression of the proapoptotic Bax protein [7 ]. In addition, studies by Atreya and co-workers [8 ] showed that interleukin (IL)-6 and the complex formed with its soluble receptor sIL-6R can stimulate intestinal T cells to express antiapoptotic genes such as Bcl-2 and Bcl-xl. Indeed, an enhanced expression of Bcl-2 and Bcl-xl was shown in lamina propria T cells of patients with Crohn’s disease when compared with controls. These investigators also reported that treatment with a neutralizing antibody against human IL-6R led to apoptosis in lamina propria T cells obtained from patients with Crohn’s disease but not in those from controls [8 ]. Enhanced production of IL-6 and sIL-6R would account for the observed resistance of T cells to apoptotic signals in Crohn’s disease.

In a previous study by our group, we reported that certain bacteria down-regulate inflammatory activity in the inflamed intestinal mucosa of patients with Crohn’s disease [9 ]. Using an organ-culture model, we found that co-culture of ileal explants with viable Lactobacillus casei DN-114 001 significantly reduced the release of tumor necrosis factor {alpha} (TNF-{alpha}) by the inflamed mucosa and the expression of TNF-{alpha} protein by intraepithelial lymphocytes (IEL). In addition, co-culture with L. casei reduced the number of T cells displaying the {alpha} chain of IL-2R (CD25), an activation marker highly expressed by lamina propria lymphocytes (LPL) in Crohn’s disease [10 , 11 ].

In the present study, our specific aim was to investigate the role of L. casei DN-114 001 in the modulation of lymphocyte apoptosis in ileal mucosa obtained from patients with active Crohn’s disease.

MATERIALS AND METHODS

Patients
Samples of intestinal mucosa were obtained at surgery from 12 patients with Crohn’s disease (seven women and five men; mean age 37 years; range 25–67) who underwent ileal resection for ileal stricture unresponsive to conventional medical treatment. At the time of surgical tissue sampling, all patients were receiving treatment with oral corticosteroids (prednisolone 0.5–1.0 mg/Kg/day) and azathioprine (2–2.5 mg/Kg/day). The diagnosis of Crohn’s disease had been established previously by routine clinical, radiological, and endoscopic criteria and was confirmed by histological evaluation of the surgical specimen. Ileal stricture was diagnosed on the basis of clinical symptoms and imaging studies (small bowel barium studies and/or computed tomography scan). Macroscopic examination confirmed ileal stricture in all patients, and histological examination demonstrated transmural inflammation, intense fibrosis, lymphoid aggregates in the submucosa and granulomas. Macroscopically normal ileal specimens were obtained from four patients undergoing right hemi-colectomy for proximal colonic cancer (two men and two women; median age 67 years; range 45–78). All patients received similar preparation for colonic surgery, including gut lavage with electrolyte-polyethylene glycol solution and broad-spectrum antibiotic therapy.

Organ culture
Full-thickness ileal wall specimens, including areas with macroscopic lesions, were collected at surgery. After intensive rinsing and washing with sterile saline, the specimens were transferred immediately to our laboratory in sterile saline at 4°C. Multiple mucosal samples weighing 20–30 mg each were separated from underlying tissue, placed on culture filter plates (15 mm diameter wells with 500 µm bottom mesh, Netwell culture systems, Costar, Cambridge, MA), and orientated so that the epithelial surface was upper-most. Filters were suspended over wells containing 1500 µL medium, consisting of RPMI 1640 (CanSera, Rexdale, Ontario, Canada), supplemented with 10% fetal calf serum (FCS; Gibco-BRL, Eggenstein, Germany), 16 mg/mL vancomycin (Eli Lilly & Co., Indianapolis, IN), 2500 U/mL colistin (Pharmax, Kent, UK), and 50 µg/mL gentamycin (Normon, Madrid, Spain). Samples were preincubated with antibiotics for 3 h at 37°C in a humidified 5% CO2 atmosphere to eradicate the indigenous flora. Thereafter, medium was replaced by fresh RPMI 1640 supplemented with 10% FCS and sodium bicarbonate at 24 mmol/L. Bacteria strains (Escherichia coli or L. casei) were added to the incubation at appropriated concentrations as described later. Each study included control wells with no bacteria in the organ culture. After 18 h at 37°C in a 5% CO2 chamber, supernatants were collected and stored at –80°C until analysis, and tissues were treated as described below.

Bacteria strains
L. casei DN-114 001 was provided by Danone Vitapole (Palaiseau, France). A nonpathogenic E. coli strain (ATCC 35345, Ecor-26) from the Ochman-Selander collection of standard reference strains of E. coli isolated from natural populations [12 ] was provided by Professor Juan Aguilar (Biochemistry, Faculty of Pharmacy, University of Barcelona, Spain). The lactobacillus strain was grown in Man-Rogosa-Sharpe liquid medium (MRS broth; Difco, Detroit, MI) and the E. coli strain, in Luria Bertani broth (Pharmacy, Hospital Vall d’Hebron, Autonomous University of Barcelona, Spain) at 37°C under aerobic conditions for 24 h. Bacteria were harvested in the stationary phase, cell counts in the bacterial suspension were estimated by optical density at 600 nm absorbance, and bacteria were added to the tissue-culture wells at the appropriate dilution to reach a final concentration of 106 colony-forming units per mL incubation medium. Aliquots of the supernatants after organ culture were plated in MRS broth or blood agar to confirm bacterial growth and exclude the presence of contaminant bacteria.

Cell isolation
Cell populations were isolated after organ culture of ileal mucosa by the procedure described above. Tissues from four organ-culture experiments performed under the same conditions were pooled to obtain a single data point per patient in each condition (control, E. coli and L. casei). IEL and LPL were isolated as described previously [13 ]. Briefly, the biopsies were washed and incubated in Iscove medium (Gibco-BRL) supplemented with 40 mg/mL gentamycin, 10% FCS, and 1 mM EDTA (Sigma-Aldrich, Madrid, Spain) for 1 h at 37°C under continuous stirring. Thereafter, IEL and epithelial cells were collected in the supernatant. Histological examination of the remaining fragment revealed that the villous and lamina propria structures were still preserved, whereas all the cells within the epithelium had disappeared during the procedure. The remaining fragments were then cut into small pieces and incubated under stirring for 1 h at 37°C in Iscove medium containing 1 mg/mL collagenase-dispase (Sigma-Aldrich), and lamina propria mononuclear cells were collected in the supernatant. For both suspensions, the numbers of mononuclear cells were counted using a Bürker chamber, and the percentages of CD3+ lymphocytes were estimated by flow cytometry.

Phenotypic analysis of intestinal T lymphocytes
Phenotypic characterization of the intestinal lymphocytes was performed by flow cytometry on isolated LPL. Cell suspensions were resuspended directly in complete Iscove medium at 1 x 106 cells/mL, and 100 µL of the cell suspension was incubated with anti-CD3 fluorescein isothiocyanate (FITC), anti-CD4 allophycocyanin, anti-CD8 peridinin chlorophyll protein, anti-CD25 phycoerythrin (PE) monoclonal antibodies (Becton Dickinson, San Jose, CA) at 4°C for 30 min in the dark. After staining, cells were washed, and at least 5000 cells were analyzed by flow cytometry (FACScan, Becton Dickinson). Lymphocytes were gated by forward/side-scatter light and by gating for CD3+ cells. Results are given as percentages of positive cells per CD3+ lymphocytes.

Analysis of Bax and Bcl-2 proteins on lamina propria T lymphocytes
The phenotype of Bax- and Bcl-2-positive cells was analyzed by flow cytometry in isolated LPL by labeling intracytoplasmic proteins and lymphocyte membrane markers (FACSCalibur instrument and CellQuest sofware, Becton Dickinson). Intestinal cells were analyzed according to the instructions of the manufacturer of the Cytofix/Cytoperm product (PharMingen, San Diego, CA), using monoclonals anti-CD3 FITC and anti-Bax and anti-mouse immunoglobulin G1 FITC or anti-Bcl-2 antibody (PE-conjugated antibody reagent set, all from Becton Dickinson). The cut-off point at which a Bax- or Bcl-2-specific signal was considered positive was determined using cells stained with control antibodies of the same isotype. Results were expressed as percentages of positive cells per CD3+ cell and also as mean fluorescence intensity (MFI).

Assessment of apoptosis
Apoptotic cells were detected by in situ DNA end-labeling [deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) method] using the Apo-DirectTM kit (PharMingen). Briefly, 1 x 106 lamina propria mononuclear cells were suspended in 0.5 mL Iscove medium (Gibco-BRL) supplemented with 40 mg/mL gentamycin and 10% FCS and fixed by paraformaldehyde (1% in phosphate-buffered saline; Sigma-Aldrich, St. Louis, MO) in ice for 15 min and permeabilized by ice-cold, 70% ethanol and stored at –80°C at least 1 week. The manufacturer provided positive and negative control cells.

Controls and test samples (500,000 cells/mL) were centrifuged, and ethanol was removed by aspiration. Pellets were washed twice with the wash buffer and resuspended in the staining solution (15.8% FITC-dUTP), as provided by the manufacturer. The cells were incubated in the staining solution for 60 min at 37°C. After incubation, cells were rinsed twice using the buffer provided and resuspended in propidium iodure (PI) solution. Cells were incubated in the dark for at least 30 min at room temperature and analyzed within 3 h of staining by a laser scanning cytometer (LSC instrument and WinCyte software, CompuCyte, Cambridge, MA). Lymphocytes were gated by forward/side-scatter light. Results were expressed as percentage of apoptotic lymphocyte (FITC-positive nucleus) from all lymphocytes (PI-positive nucleus).

Analytical methods
Concentration of TNF-{alpha} and IL-6 in the supernatants was measured using a commercially available assay system for human TNF-{alpha} and IL-6 (DuoSet, R&D Systems, Minneapolis, MN). All samples were analyzed in duplicate. Cytokine concentration is expressed as pg (TNF-{alpha}) or ng (IL-6) per mg of tissue.

Supernatants were analyzed for lactate dehydrogenase (LDH) using the spectrophotometric method of Henry and co-workers [14 ]. Some tissue samples were homogenized in Tris/HCl (100 mmol/L, pH 7.4), and protein concentration was determined using the bicinchoninic acid reagent for protein assay (Pierce, Rockford, IL). Tissue viability was assessed as the release of LDH per mg of tissue.

Ethical considerations
Prior informed consent was obtained from every patient, and the study had been approved by the Ethical Committee of our institution (Comitè d’Ètica i Investigació Clínica, Hospital Universitari Vall d’Hebron).

Statistical analysis
Results are expressed by the mean and SEM, or by individual data in plots. Statistical differences were determined using the repeated measures ANOVA test and Tukey-Kramer multiple comparisons post-test (GraphPad Instat, San Diego, CA).

RESULTS

Cytokine release
As previously observed [9 ], coculture of the tissue with bacteria did not modify LDH release. Coculture of inflamed tissue from Crohn’s disease patients with the L. casei strain significantly reduced the release of IL-6 and TNF-{alpha} (P<0.05; Fig. 1 ). The nonpathogenic E. coli strain had no significant effect on the release of IL-6 or TNF-{alpha}. Organ culture experiments with normal mucosa showed no difference in the release of cytokines between blank, L. casei, or E. coli coculture (data not shown).


Figure 1
View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Levels of TNF-{alpha} and IL-6 in the incubation medium after coculture of inflamed intestinal mucosa from Crohn’s disease patients with or without bacteria. The release of TNF-{alpha} and IL-6 was reduced significantly in the presence of L. casei. Data are mean ± SEM (*, P<0.05, vs. control; n=12).

 
Phenotype of lymphocytes
Table 1 shows the phenotype of lamina propria T lymphocytes (CD3+ cells) in normal and Crohn’s disease mucosa. As observed in control experiments (with no bacteria), significantly higher numbers of CD4+ and CD25+ T cells were found in the lamina propria of Crohn’s disease mucosa when compared with normal ileal mucosa. Conversely, the inflamed mucosa showed a significantly lower proportion of CD8+ T cells. In normal mucosa, no changes were observed after coculture with bacteria in the proportion of CD4+, CD8+, or CD25+ T cells. In Crohn’s disease mucosa, however, coculture with L. casei significantly reduced the rate of CD25+ T cells within the lamina propria. Figure 2 shows individual values from the 12 Crohn’s disease patients studied. In contrast, coculture with E. coli had no effect (Table 1) .


View this table:
[in this window]
[in a new window]
  		Table 1. Phenotype of Lamina Propria T Lymphocytes after Organ Culture

 

Figure 2
View larger version (13K):
[in this window]
[in a new window]
  		Figure 2. Percentage of activated T cells in the lamina propria of inflamed mucosa from Crohn’s disease patients. Coculture with L. casei significantly reduced the proportion of T cells expressing the IL-2R (CD25+). Individual data from 12 Crohn’s disease patients are shown (*, P<0.05).

 
Apoptosis of lymphocytes
The percentage of apoptotic lymphocytes in the lamina propria after organ culture with or without bacteria was assessed in mucosal samples from 10 patients with Crohn’s disease. (In two of the 12 patients, the number of isolated lymphocytes was insufficient.) As shown in Figure 3 , an increase in TUNEL-positive lymphocytes was observed in nine out of 10 patients after coculture with L. casei as compared with control cultures. The percentage of TUNEL-positive lymphocytes was significantly higher in mucosal samples cocultured with L. casei as compared with control culture, but no effect was observed in tissue cocultured with E. coli (control, 12±1.3%; L. casei, 19±2.5%, P<0.05 vs. control; E. coli, 13±1.5%, not significant).


Figure 3
View larger version (11K):
[in this window]
[in a new window]
  		Figure 3. Apoptosis of LPL isolated from Crohn’s disease mucosa was assessed by in situ DNA end-labeling (TUNEL). The percentage of TUNEL+ lymphocytes was higher in the mucosa cocultured with L. casei as compared with control culture. Individual data from 10 Crohn’s disease patients are shown (*, P<0.05).

 
The expression of Bcl-2 and Bax proteins was assessed in T lymphocytes from the lamina propria of mucosal samples from Crohn’s disease ileum (appropriate sample size was obtained from eight patients only). Data are shown in Table 2 . It is interesting that the ratio of Bcl-2+ over Bax+ T cells in the lamina propria was reduced significantly by coculture with L. casei as compared with control culture (Fig. 4 ). There were no differences between groups in MFI for Bax (data not shown), but Bcl-2 MFI was lower in T cells from tissues cocultured with L. casei than in controls (control, 12±1.6 fluorescence units; L. casei, 9±2.1, P<0.05 vs. control; E. coli, 11±2.2). Individual data are shown in Figure 5 .


View this table:
[in this window]
[in a new window]
  		Table 2. Expression of Anti- and Proapoptotic Proteins in Isolated Lamina Propria T Cells of Crohn’s Disease Mucosa

 

Figure 4
View larger version (9K):
[in this window]
[in a new window]
  		Figure 4. Lamina propria T lymphocytes expressing the antiapoptotic Bcl-2 or proapoptotic Bax proteins were identified by flow cytometry in mucosal samples from eight Crohn’s disease patients. In all patients, the ratio of Bcl-2+ to Bax+ T cells decreased in mucosal samples cocultured with L. casei as compared with control culture (*, P<0.05).

 

Figure 5
View larger version (9K):
[in this window]
[in a new window]
  		Figure 5. Intracellular staining of isolated lamina propria T lymphocytes for the antiapoptotic Bcl-2 protein expressed as fluorescence units per T cell. Coculture of inflamed tissue with L. casei as compared with control culture reduced MFI in six out of seven patients tested (*, P<0.05).

 
DISCUSSION

Using an organ culture model with intestinal mucosa explants and selected bacteria strains, this study shows that a L. casei strain is capable of decreasing the number of activated T lymphocytes in the lamina propria of inflamed mucosa from patients with Crohn’s disease. The effect is not observed in noninflamed, intestinal tissue from controls without Crohn’s disease. Our data also show that coculture of the inflamed mucosa with the probiotic bacteria decreases the expression of the antiapoptotic protein Bcl-2 in lamina propria T cells and increases the number of lymphocytes that undergo apoptosis. By favoring apoptosis of T lymphocytes, L. casei may restore immune homeostasis in the inflamed ileal mucosa of patients with Crohn’s disease.

Although the etiology of Crohn’s disease is unknown, substantial experimental and clinical evidence suggests that uncontrolled T lymphocyte activation is a central pathogenetic mechanism of chronic intestinal inflammation [3 , 15 ]. Cell-mediated immunity against the luminal flora appears to be the key event in driving the inflammatory process that leads to intestinal lesions [4 , 16 ]. Several factors may contribute to the pathogenesis of the aberrant immune response toward the autologous flora, including genetic susceptibility, a microbial imbalance of the enteric flora, and a defect in mucosal barrier function [3 , 16 , 17 ]. However, few data exist about the effects of nonpathogenic bacteria on intestinal lymphocyte populations. Several studies have shown that colonizing bacteria play a major role in the growth and differentiation of gut-associated lymphoid tissue [18 19 20 ]. Nonpathogenic bacteria may modify immune responses of the intestinal mucosa by interaction and signaling at mucosal surfaces [21 ], and different bacteria elicit different responses [22 ]. When exposed to peripheral blood lymphocytes and other mononuclear cells, lactobacilli and streptococci induce proinflammatory cytokines [23 ]. However, our group demonstrated that some lactobacillus strains may down-regulate inflammatory responses when exposed to inflamed intestinal mucosa in organ culture [9 ]. This effect is manifested by a reduced mucosal release of TNF-{alpha} and also by a decrease in the number of intraepithelial CD3+ cells. Our current data confirm our previous observation in that L. casei down-regulates the release of TNF-{alpha} by Crohn’s disease-inflamed tissue and reduces the number of lymphocytes expressing the IL-2R in the lamina propria. In addition, this manuscript shows that interaction of L. casei with the inflamed intestinal tissue down-regulates the production of IL-6. The cellular origin of these cytokines is not clear at this stage. Epithelial cells, macrophages, and dendritic cells can produce IL-6, but our study design does not allow us to obtain adequate tissue sections for immunohistochemical studies to investigate the cellular origin of the cytokines.

In Crohn’s disease, IL-6 and sIL-6R are present at high levels [24 , 25 ]. The complex of IL-6 with sIL-6R is capable of binding to the membrane of T cells, which do not express IL-6R. By this way, IL-6 triggers intracellular signaling cascades involving the activation of antiapoptotic genes for expression of Bcl-2 and Bcl-xl proteins [26 ]. It is interesting that IL-6 has been shown to play a crucial role in the pathogenesis of intestinal inflammation in a murine model of colitis induced by transfer of T helper cell CD45RBhigh to severe combined immunodeficiency mice [27 ]. In this colitis model, the transferred T helper cells expand and react against luminal bacteria inducing chronic colonic inflammatory lesions. However, treatment with a monoclonal anti-IL-6R antibody reduced T helper cell expansion and prevented the development of macroscopic and histological lesions in the colon. As mentioned in the introduction, purified lamina propria T cells from patients with Crohn’s disease show an enhanced production of IL-6 and an increased expression of the antiapoptotic protein Bcl-2 [8 ]. Blockade of the IL-6 trans-signaling pathway with a neutralizing antibody against human IL-6R induced apoptosis of lamina propria T cells from Crohn’s disease patients. These studies suggest that IL-6 plays an important role in chronic intestinal inflammation by mediating the resistance of lamina propria T cells against apoptosis. In Crohn’s disease, IL-6 rescues T cells from apoptosis, and this observation is consistent with previous in vitro investigations about fundamental mechanisms of lymphocyte apoptosis [28 ].

In our study, coculture of inflamed mucosa with L. casei significantly reduced the release of IL-6 in the supernatant as well as the expression on Bcl-2 in lamina propria T cells. These findings were associated with an increase of TUNEL+ lymphocytes in the lamina propria of tissue cocultured with L. casei. According to the studies commented above, these observations are likely to be related, although our in vitro studies do not prove a causative link. Our model does not allow us to investigate signals between cells, as mucosa explants include a great variety of cells in their natural disposition for cell-to-cell communication. However, our model is useful to study whole tissue responses in Crohn’s disease. We may speculate that a reduction in cytokine release (TNF-{alpha}, IL-6) produced by the interaction between L. casei (but not E. coli) and the inflamed mucosa would reduce the level of signals generated at the epithelial level. Changes would be transduced to the lamina propria and would result in reduced T cell activation and an increased number of activated lymphocytes undergoing apoptosis.

The therapeutic efficacy of infliximab or azathioprine in Crohn’s disease seems to be related with the ability of these drugs to induce apoptosis of activated T cells [29 30 31 32 ]. It is interesting that infliximab or azathioprine stimulates apoptosis of CD3/CD28-activated T cells but not of resting T lymphocytes. Our current findings suggest that certain bacteria, such as the L. casei strain, are able to interact with immunocompetent cells using the mucosal interface and modulate locally the production of cytokines. Furthermore, signals generated at the interface can be transduced to the lamina propria and induce apoptosis of activated T cells in the lamina propria of inflamed intestinal mucosa. It is conceivable that bacteria possessing such properties might be of therapeutic value in vivo, provided that the appropriate concentrations of living bacteria were achieved at the mucosal surface. Clinical evaluation of this putative therapeutic effect of bacteria is needed. Our findings also suggest that a balanced, local microecology within the gut lumen and particularly at the mucosal niches constitutes a promising target for future strategies directed to prevent the spread of inflammatory bowel diseases among developed societies [33 ].

ACKNOWLEDGEMENTS

This work was supported in part by Ministerio de Ciencia y Tecnología (SAF 2003-05262), Generalitat de Catalunya (RE: 2001SGR 00389), Instituto de Salud Carlos III (C03/02), and Danone Vitapole (Palaiseau, France). The authors thank Mrs. Montserrat Casellas and Mrs. Milagros Gallard for technical assistance in the analytical procedures. We are also grateful to Dr. Eloy Espin and Dr. Javier Naval (Department of Surgery, Hospital Vall d’Hebron) for their collaboration in obtaining surgical specimens and to Dr. Raphaëlle Bourdet-Sicard (Danone Vitapole) for her helpful discussion and critical reading of the manuscript.

Received April 8, 2005; revised November 3, 2005; accepted December 15, 2005.

REFERENCES

  1. Lenardo, M., Chan, K. M., Hornung, F., McFarland, H., Siegel, R., Wang, J., Zheng, L. (1999) Mature T lymphocyte apoptosis immune regulation in a dynamic and unpredictable antigenic environment Annu. Rev. Immunol. 17,221-253[CrossRef][Medline]
  2. Rathmell, J. C., Thompson, C. B. (2002) Pathways of apoptosis in lymphocyte development, homeostasis, and disease Cell 109,S97-S107[CrossRef][Medline]
  3. Shanahan, F. (2002) Crohn’s disease Lancet 359,62-69[CrossRef][Medline]
  4. D’Haens, G. R., Geboes, K., Peeters, M., Baert, F., Penninckx, F., Rutgeerts, P. (1998) Early lesions of recurrent Crohn’s disease caused by infusion of intestinal contents in excluded ileum Gastroenterology 114,262-267[CrossRef][Medline]
  5. Boirivant, M., Marini, M., Di Felice, G., Pronio, A. M., Montesani, C., Tersigni, R., Strober, W. (1999) Lamina propria T cells in Crohn’s disease and other gastrointestinal inflammation show defective CD2 pathway-induced apoptosis Gastroenterology 116,557-565[CrossRef][Medline]
  6. Ina, K., Itoh, J., Fukushima, K., Kusugami, K., Yamaguchi, T., Kyokane, K., Imada, A., Binion, D. G., Musso, A., West, G. A., Dobrea, G. M., McCormick, T. S., Lapetina, E. G., Levine, A. D., Ottaway, C. A., Fiocchi, C. (1999) Resistance of Crohn’s disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance J. Immunol. 163,1081-1090[Abstract/Free Full Text]
  7. Itoh, J., Motte, C., Strong, S. A., Levine, A. D., Fiocchi, C. (2001) Decreased Bax expression by mucosal T cells favors resistance to apoptosis in Crohn’s disease Gut 49,35-41[Abstract/Free Full Text]
  8. Atreya, R., Mudter, J., Finotto, S., Mullberg, J., Jostock, T., Wirtz, S., Schutz, M., Bartsch, B., Holtmann, M., Becker, C., Strand, D., Czaja, J., Schlaak, J. F., Lehr, H. A., Autschbach, F., Schurmann, G., Nishimoto, N., Yoshizaki, K., Ito, H., Kishimoto, T., Galle, P. R., Rose-John, S., Neurath, M. F. (2000) Bockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in Crohn disease and experimental colitis in vivo Nat. Med. 6,583-588[CrossRef][Medline]
  9. Borruel, N., Carol, M., Casellas, F., Antolín, M., de Lara, F., Espín, E., Naval, J., Guarner, F., Malagelada, J. R. (2002) Increased mucosal TNF{alpha} production in Crohn’s disease can be modulated in vitro by probiotic bacteria Gut 51,659-664[Abstract/Free Full Text]
  10. Pallone, F., Fais, S., Squarcia, O., Biancone, L., Pozzilli, P., Boirivant, M. (1987) Activation of peripheral blood and intestinal lamina propria lymphocytes in Crohn’s disease. In vivo state of activation and in vitro response to stimulation as defined by the expression of early activation antigens Gut 28,745-753[Abstract/Free Full Text]
  11. Choy, M. Y., Walker-Smith, J. A., Williams, C. B., MacDonald, T. T. (1990) Differential expression of CD25 (interleukin-2 receptor) on lamina propria T cells and macrophages in the intestinal lesions in Crohn’s disease and ulcerative colitis Gut 31,1365-1370[Abstract/Free Full Text]
  12. Ochman, H., Selander, R. K. (1984) Standard reference strains of Escherichia coli from natural populations J. Bacteriol. 157,690-693[Abstract/Free Full Text]
  13. Carol, M., Lambrechts, A., Van Gossum, A., Libin, M., Goldman, M., Mascart-Lemone, F. (1998) Spontaneous secretion of interferon {gamma} and interleukin 4 by human intraepithelial and lamina propria gut lymphocytes Gut 42,643-649[Abstract/Free Full Text]
  14. Krieg, A. F., Gorton, S., Henry, J. B. (1967) Effect of temperature on activity and lactate optima of LDH isoenzymes Clin. Chim. Acta 17,363-366[CrossRef][Medline]
  15. Fiocchi, C. (1998) Inflammatory bowel disease: etiology and pathogenesis Gastroenterology 115,182-205[CrossRef][Medline]
  16. Shanahan, F. (2001) Inflammatory bowel disease: immunodiagnostics, immunotherapeutics, and ecotherapeutics Gastroenterology 120,622-635[CrossRef][Medline]
  17. Guarner, F., Malagelada, J. R. (2003) Gut flora in health and disease Lancet 361,512-519[CrossRef][Medline]
  18. Umesaki, Y., Setoyama, H., Matsumoto, S., Okada, Y. (1993) Expansion of {alpha} ß T-cell receptor-bearing intestinal intraepithelial lymphocytes after microbial colonization in germ-free mice and its independence from thymus Immunology 79,32-37[Medline]
  19. Helgeland, L., Vaage, J. T., Rolstad, B., Midtvedt, T., Brandtzaeg, P. (1996) Microbial colonization influences composition and T-cell receptor V ß repertoire of intraepithelial lymphocytes in rat intestine Immunology 89,494-501[CrossRef][Medline]
  20. Falk, P. G., Hooper, L. V., Midtvedt, T., Gordon, J. I. (1998) Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology Microbiol. Mol. Biol. Rev. 62,1157-1170[Abstract/Free Full Text]
  21. Haller, D., Bode, C., Hammes, W. P., Pfeifer, A. M., Schiffrin, E. J., Blum, S. (2000) Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures Gut 47,79-87[Abstract/Free Full Text]
  22. Borruel, N., Casellas, F., Antolín, M., Carol, M., Llopis, M., Espín, E., Naval, J., Guarner, F., Malagelada, J. R. (2003) Effects of nonpathogenic bacteria on cytokine secretion by human intestinal mucosa Am. J. Gastroenterol. 98,865-870[CrossRef][Medline]
  23. Miettinen, M., Matikainen, S., Vuopio-Varkila, J., Pirhonen, J., Varkila, K., Kurimoto, M., Julkunen, I. (1998) Lactobacilli and streptococci induce interleukin-12, IL-18, and {gamma} interferon production in human peripheral blood mononuclear cells Infect. Immun. 66,6058-6062[Abstract/Free Full Text]
  24. Gross, V., Andus, T., Caesar, I., Roth, M., Scholmerich, J. (1992) Evidence for continuous stimulation of interleukin-6 production in Crohn’s disease Gastroenterology 102,514-519[Medline]
  25. Mitsuyama, K., Toyonaga, A., Sasaki, E., Ishida, O., Ikeda, H., Tsuruta, O., Harada, K., Tateishi, H., Nishiyama, T., Tanikawa, K. (1995) Soluble interleukin-6 receptors in inflammatory bowel disease: relation to circulating interleukin-6 Gut 36,45-49[Abstract/Free Full Text]
  26. Desreumaux, P. (2000) Specific targeting of IL-6 signaling pathway: a new way to treat IBD? Gut 47,465-466[Free Full Text]
  27. Yamamoto, M., Yoshizaki, K., Kishimoto, T., Ito, H. (2000) IL-6 is required for the development of Th1 cell-mediated murine colitis J. Immunol. 164,4878-4882[Abstract/Free Full Text]
  28. Teague, T. K., Marrack, P., Kappler, J. W., Vella, A. T. (1997) IL-6 rescues resting mouse T cells from apoptosis J. Immunol. 158,5791-5796[Abstract]
  29. Lügering, A., Schmidt, M., Lügering, N., Pauels, H. G., Domschke, W., Kucharzik, T. (2001) Infliximab induces apoptosis in monocytes from patients with chronic active Crohn’s disease by using a caspase-dependent pathway Gastroenterology 121,1145-1157[CrossRef][Medline]
  30. Ten Hove, T., Van Montfrans, C., Peppelenbosch, M. P., van Deventer, S. J. H. (2002) Infliximab treatment induces apoptosis of lamina propria T lymphocytes in Crohn’s disease Gut 50,206-211[Abstract/Free Full Text]
  31. Tiede, I., Fritz, G., Strand, S., Poppe, D., Dvorsky, R., Strand, D., Lehr, H. A., Wirtz, S., Becker, C., Atreya, R., Mudter, J., Hildner, K., Bartsch, B., Holtmann, M., Blumberg, R., Walczak, H., Iven, H., Galle, P. R., Ahmadian, M. R., Neurath, M. F. (2003) CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes J. Clin. Invest. 111,1133-1145[CrossRef][Medline]
  32. Van den Brande, J. M. H., Braat, H., Van den Brink, G. R., Versteeg, H. H., Bauer, C. A., Hoedemaeker, I., Van Montfrans, C., Hommes, D. W., Peppelenbosch, M. P., Van Deventer, S. J. H. (2003) Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn’s disease Gastroenterology 124,1774-1785[CrossRef]
  33. Bach, J. F. (2002) The effect of infections on susceptibility to autoimmune and allergic diseases N. Engl. J. Med. 347,911-920[Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carol, M.
Right arrow Articles by Malagelada, J.-R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carol, M.
Right arrow Articles by Malagelada, J.-R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS