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Keywords:

  • immune activation;
  • irritable bowel syndrome;
  • lipopolysaccharide;
  • lymphocyte;
  • probiotics

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Abstract  Patients with irritable bowel syndrome (IBS) may have a low grade immune activation. However, little is known about the properties of B cells of IBS patients. We therefore investigated activation level and antigen presenting phenotype of blood B cells of IBS patients. We also examined B-cell responses to lipopolysaccharide (LPS) and probiotic bacteria. Blood samples were obtained from 74 IBS patients and 30 healthy subjects. Peripheral blood mononuclear cells were isolated and stimulated with LPS or an UV-light inactivated bacterial cocktail consisting of the probiotic Gram-positive strains; Lactobacillus paracasei ssp. paracasei 19, Lactobacillus acidophilus La5, Bifidobacterium lactis B612. The phenotype of CD19+ B cells was investigated by flow cytometry before and after 72 h cell culture. Furthermore, IBS symptom severity was assessed. B cells isolated from blood of IBS patients displayed an amplified activation level as demonstrated by increased cell surface expression of IgG, and also the costimulatory molecules CD80 and CD86. Expression of antigen presenting HLA-DR and costimulatory molecule CD40 on B cells was, however comparable in IBS patients and controls. B cells of IBS patients displayed an impaired ability to increase expression of CD80, but not CD86, in response to both LPS as well as probiotic bacteria stimulations. To conclude, blood B cells of IBS patients have an increased activation level. Bacterial component induced expression of the costimulatory molecule CD80, regarded as important for tolerance induction, is impaired. These data suggest that B-cell antigen presentation in IBS patients is associated with altered capacity of providing costimulation to T cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Irritable bowel syndrome (IBS) may be the result of previous transient or chronic inflammation of the gut.1–3 Increased activity of the innate and the adaptive immune response has during the last decade been suggested to be associated with IBS, but the explanation for the increased immune activity observed in IBS patients remains unclear.

Recent studies have described the characteristics of the immune activity of IBS patients. Serum levels, and the degree of LPS triggered cell culture secretions of proinflammatory cytokines, seem to be enhanced in association with the development of IBS symptoms.4,5 Additionally, increased presence of intestinal T cells has also been a frequently reported feature of IBS, although the mechanism for the expanded T-cell pool and/or the increased T-cell recruitment to the mucosa remains unknown.6–11 So far, the properties of B cells of IBS patients have been relatively little studied. Several previous reports have however demonstrated increased serum levels of food specific IgG antibodies in IBS patients.12–15

Antibody producing B cells play a central role in adaptive immune responses, as antibodies are important in defence against and clearance of infections. However, B cells also function as professional antigen presenting cells, expressing HLA-DR (MHC class II), and thus have the ability to present antigens to CD4+ T-helper cells.16 In general, the ability of antigen presenting cells to induce T-cell activation is tightly linked to the maturation status of the presenting cell. B-cell activation and maturation largely depends on antigen recognition by the B-cell receptor (IgR). The maturation process of B cells results in increased surface expression of the costimulatory ligands CD80 and CD86. In addition, engagement of CD40 on B cells to CD40 ligand on T cells results in increased maturation level of the B-cell.

Antigen presenting properties of B cells of IBS patients have not previously been explored. We hypothesised that IBS patients have an increased immune activity, and that this would be reflected by augmented B-cell activation and antigen presenting capacity. Therefore, the antigen presenting phenotype of B cells in freshly isolated blood of IBS patients was examined. Furthermore, we investigated B-cell phenotype responses after stimulation with bacterial components.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Study subjects

The study was performed after receiving written informed consent from all subjects and approval from the ethics committee of the University of Gothenburg. Irritable bowel syndrome patients were defined according to the Rome II Criteria,17 and were recruited from our outpatient clinic, and from the general population by advertising in the local newspaper. We included 74 IBS patients (mean age 34 ± 16 years, 52 females) of which 26 had diarrhoea predominant IBS (IBS-d), 11 had constipation predominant IBS (IBS-c) and 37 had alternating bowel habits (IBS-a). Control subjects (n = 30, mean age 39 ± 10 years, 20 females) were healthy volunteers with no history of gastrointestinal disorders. None of the subjects included in the study were taking any medications known to affect the gastrointestinal tract or the immune system.

Bacterial strains

Isolates of the probiotic Gram-positive bacterial strains Lactobacillus paracasei ssp. paracasei 19, Lactobacillus acidophilus La5, Bifidobacterium lactis B612 were obtained from Arla Foods (Stockholm, Sweden). The strains were cultured anaerobically on Rogosa agar for 3 days. The bacteria were then harvested, washed and suspended in a dilution of 109 cells mL−1. The strains were inactivated by exposure to UV-light for 15 min to inhibit uncontrolled bacterial growth, and were then stored at −70 °C until further use.

Isolation of blood lymphocytes and cell cultures

Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by density-gradient centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Peripheral blood mononuclear cells were cultured for 3 days in Iscove’s medium supplemented with 5% AB+ serum, 1% Gentamycin (Sigma, St Louis, MO, USA) and 1%l-glutamine (Sigma). The cell cultures were either unstimulated or stimulated with 1 ug mL−1 LPS or a bacterial cocktail consisting of L. casei, L. acidophilus, B. infantis (2.5 bacteria of each bacterial strain/cell) in flat-bottomed microwell plates (Nunc, Roskilde, Denmark).

Flow cytometric analysis

Freshly isolated or cultured PBMCs, 1 × 105 cells per sample, were stained for flow cytometry analysis of various surface markers using combinations of the following antibodies: anti-CD19-PerCp was stained together with anti-CD80-FITC and anti-CD86-PE and anti-integrinβ7-APC, or anti-HLA-DR-FITC and anti-CD40-PE and anti-IgG-APC. All cells were fixed in cellfix (BD Pharmingen, San Diego, CA, USA) before flow cytometry analysis, which was performed using a LSR II flow cytometer (BD Pharmingen). At least 10 000 live lymphocytes per sample were analysed, as defined by forward and side scatter. The data were analysed using Flow Jo software (Treestar Inc, Ashland, OR, USA).

IBS severity scoring system (IBS-SSS)

The validated IBS severity scoring system (IBS-SSS) was used to evaluate the severity of IBS symptoms.18,19 It uses visual analogues scales (VAS), and an overall IBS score can be calculated as the sum of five items; pain severity, pain frequency, distension, bowel habit dissatisfaction and life interference. The maximum score is 500, and the higher the score, the more severe the symptoms.

Statistical analysis

All statistical evaluations were performed with the StatView Software (SAS Institute, Cary, NC, USA). Nonparametric, rather than parametric, analysis was performed due to extreme values in both the patient and the control groups. Mann–Whitney test was used to evaluate differences between two groups, whereas Kruskall–Wallis test was performed to evaluate differences between three or more groups. Spearman correlation was used to evaluate correlations between two sets of data within a group. A P-value of <0.05 was regarded as significant. Box plots indicate median values, 25% and 75% percentiles respectively, and error bars indicate 10% and 90% percentiles. Individual values are shown as separate dots, and horizontal lines indicate median values. Data given in the text are demonstrated as median ± interquartile range.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

IgG expression on B cells in IBS patients

Irritable bowel syndrome patients and controls had similar frequencies of lymphocytes (36.1 ± 13.3%vs 37.9 ± 9.2%) among mononuclear cells, and B cells (12.6 ± 4.5%vs 11.1 ± 4.2%) among lymphocytes in peripheral blood. The surface expression of IgG on B cells was investigated in IBS patients and controls. Freshly isolated blood B cells from IBS patients demonstrated an increased cell surface expression of IgG relative to controls (Fig. 1A). This was also true after LPS and bacterial cocktail stimulations (Fig. 1A). In accordance with the increased cell surface intensity of IgG on B cells of IBS patients, the frequency of IgG+ B cells was higher in IBS patients than controls. Additionally, the frequencies of IgG expressing B cells were augmented in both LPS, and bacterial cocktail stimulated cell cultures of IBS patients relative to controls (Fig. 1B). However, the IgG expression on B cells did not correlate to total score of IBS symptom severity (r = 0.187, P = 0.12) (not shown).

image

Figure 1.  IgG expression on B cells. The median fluorescence intensity (MFI) of IgG on B cells (A) and the frequency of IgG+ B cells (B) in freshly isolated blood, LPS and bacterial cocktail stimulated cell cultures (72 h) of IBS patients (IBS) and control subjects (CTRL).

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Expression of CD80 and CD86 on B cells in IBS patients

The cell surface expression of the costimulatory molecules CD80 and CD86 on B cells was investigated. Freshly isolated blood B cells displayed low expression of C80 and CD86 in both study groups. Nevertheless, IBS patients had higher expression of CD80 (Fig. 2A) and CD86 (Fig. 2B) relative to controls. In contrast, the expression of CD80 on B cells of IBS patients was impaired in cell cultures stimulated with LPS or bacterial cocktail (Fig. 2A). The expression of CD86 was however comparable in LPS and bacterial cocktail stimulated B cells of IBS patients and controls (Fig. 2B). Additionally, the frequencies of CD80+ B cells and CD86+ B cells in blood of IBS patients were higher than in controls (Figs 2C and D). The frequencies of CD80+ B cells, but not CD86+ B cells, were lower in cell cultures stimulated with LPS and with bacterial cocktail (Figs 2C and D). Neither the intensity of CD80 expression nor CD86 expression on B cells correlated to total score of IBS symptoms severity (r = 0.06, P = 0.6 and r = 0.2, P = 0.08 respectively) (not shown).

image

Figure 2.  CD80 and CD86 expression on B cells. The median fluorescence intensity (MFI) of CD80 and CD86, respectively, on B cells (A, B) and the frequency of CD80+ B cells (C) and CD86+ B cells (D) in freshly isolated blood, LPS and bacterial cocktail stimulated cell cultures (72 h) of IBS patients (IBS) and control subjects (CTRL).

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Integrin β7 expression on B cells of IBS patients

The intensity of the gut homing receptor integrin β7 expression on B cells in freshly isolated blood and in cell cultures stimulated with LPS was comparable in IBS patients and controls (Fig. 3A). Cell culture stimulation with bacterial cocktail resulted in more integrin β7 expression on B cells of IBS patients than controls (Fig. 3A). In addition, frequencies of integrin β7 expressing B cells were comparable in IBS patients and controls (Fig. 3B). However, the frequencies of coexpressing integrin β7+ CD80+ B cells and integrin β7+CD86+ B cells were increased in blood of IBS patients relative to controls (Fig. 3C). The frequencies of integrin β7 B cells expressing CD86 were higher in IBS patients than controls, whereas the frequency of CD80 expressing integrin β7 B cells was comparable in the two groups (Fig. 3D).

image

Figure 3.  Integrin β7 expression on B cells. The median fluorescence intensity (MFI) of integrin β7 on B cells (A) and the frequency of integrin β7+ B cells (B) in freshly isolated blood, LPS and bacterial cocktail stimulated cell cultures (72 h) of IBS patients (IBS) and control subjects (CTRL). The frequency of CD80+ and CD86+ on integrin β7+ B cells (C) and integrin β7 B cells (D), respectively, in freshly isolated blood.

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Expression of CD40 on B cells of IBS patients

No group differences were observed regarding the expression intensity of the costimulatory molecule CD40 on both freshly isolated B cells from blood as well as on LPS and bacterial cocktail stimulated B cells (Fig. 4A). However, the frequency of CD40 expressing B cells was lower in freshly isolated blood from IBS patients than controls (Fig. 4B). Furthermore, the frequency of CD40+ B cells was lower in LPS stimulated cell cultures from IBS patients than controls (Fig. 4B). The frequency of CD40+ B cells after bacterial cocktail stimulation was however comparable in the two groups.

image

Figure 4.  CD40 expression on B cells. The median fluorescence intensity (MFI) of CD40 (A) on B-cells, and the frequency of CD40+ B-cell (B) in freshly isolated blood, LPS and bacterial cocktail stimulated cell cultures (72 h) of IBS patients (IBS) and control subjects (CTRL).

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Expression of HLA-DR on B cells of IBS patients

The expression intensity of HLA-DR (MHC class II) on blood cells was equal in IBS patients and controls (Fig. 5A). The expression of HLA-DR on B cells was equally intense in IBS patients and controls after coculture with LPS and bacterial cocktail (Fig. 5A). However, the frequency of HLA-DR expressing B cells was lower in blood of IBS patients than controls (Fig. 5B), whereas the frequency of HLA-DR+ B cells after LPS and bacterial cocktail stimulations was comparable in the two groups (Fig 5B).

image

Figure 5.  HLA-DR expression on B cells. The median fluorescence intensity (MFI) of HLA-DR (A) on B-cells and the frequency of HLA-DR+ B-cell (B) in freshly isolated blood, LPS and bacterial cocktail stimulated cell cultures (72 h) of IBS patients (IBS) and control subjects (CTRL).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The evidence suggesting that IBS patients may have an increased immune activity is growing.20 There are examples from reports of increased levels of proinflammatory cytokines in serum and PBMC cultures,4,5 and increased presence of intestinal T cells.6–11 However, the understanding of the underlying mechanisms for an increased immune activation in IBS patients is incomplete. This study therefore focused on the activation level and antigen presenting phenotype of freshly isolated and stimulated blood B cells of IBS patients.

Irritable bowel syndrome patients displayed an increased cell surface expression intensity of the costimulatory molecules CD80 and CD86 on freshly isolated blood B cells, along with increased frequencies of CD80+ B cells and CD86+ B cells in blood, compared with controls. The relative expression of costimulatory molecules is determined by the activation status of the B-cell, which is influenced by recognition of bacterial components. The increased CD80 and CD86 expression recorded in this study therefore indicate that IBS patients have a somewhat augmented activation level of B cells in peripheral blood. Our demonstration of increased expression of IgG receptors on B cells further supports the hypothesis of an increased activation of B cells in IBS patients, as activated B cells switch from expressing IgM to IgG. Hence, our data imply that the activation level of B cells, and consequently the ability of B cells to activate T cells following cognate antigen presentation, is augmented in IBS patients relative to controls.

The mechanisms behind the increased expression intensity of costimulatory molecules on B cells and the increased frequency of B cells expressing costimulatory molecules in the blood of IBS patients are unclear. A plausible explanation is that IBS patients experience an increased antigen load, preferably in the gut as this is the symptom generating site. Therefore, it is interesting to note that several reports indicate that an altered intestinal flora is associated with IBS.21–24 Thus, it is possible that the increased immune activity recorded in IBS patients is caused by an increased intestinal reactivity to a transformed gut microbiota. Interestingly, the expression of the costimulatory molecules CD80 and CD86 on integrin β7+ B cells, preferentially activated in gut associated tissue, was higher in IBS patients than controls. Nevertheless, also integrin β7 B cells had increased expression of CD86, suggesting that IBS patients also have an increased B-cell activation in extra intestinal lymph nodes.

While the expression of CD86 on B cells after bacterial stimulation was comparable in IBS patients and controls, the expression of CD80 on B cells after stimulation with either LPS or bacterial cocktail was lower in IBS patients than controls. Although still a matter of debate, CD80 is thought to preferentially bind to the inhibitory receptor CTLA-4,25 a ligation that leads to restricted T-cell activation.26 On the other hand, CD86 ligation to its receptor CD28 results in T-cell stimulation.27 We therefore suggest that the reduced capacity of B cells of IBS patients to increase CD80 expression in response to bacterial component recognition demonstrated in this study may result in an increased T-cell activation. Thus, an impaired CD80 expression on B cells after stimulation with bacterial components might partly explain the increased T-cell activity of IBS patients previously reported.6,28

Despite increased intensity and frequency of both CD80 and CD86 on B cells in peripheral blood of IBS patients, the frequency of B cells expressing the costimulatory molecule CD40 was reduced. The intensity of CD40 on B cells was, however comparable in the two groups, and this was also true for antigen presenting HLA-DR. These data imply that although a slightly lower frequency of HLA-DR+ B cells, the B cells’ capacity to present antigen is not altered, whereas the costimulatory signals given by B cells to antigen specific T cells may be modulated in IBS patients.

Neither the increased expression of CD80 and CD86 on B cells in blood, nor the dysregulated expression of CD80 after bacterial stimulation, correlated to IBS symptoms. Furthermore, the exclusive expression pattern of the above mentioned immunological parameters was not related to gender, duration of IBS symptoms or subgroups of IBS patients according to predominant bowel habit.

Many IBS patients report food related symptoms.29 Previous studies have demonstrated increased levels of food specific IgG antibodies12,14,15 in the serum of IBS patients. It is therefore interesting to note that we demonstrated an increased frequency of blood IgG+ B cells in the IBS group in this study. Thus, it should not be excluded that IBS symptoms may be associated with an increased production of antigen specific IgG antibodies.

In summary, we have demonstrated that freshly isolated blood B cells of IBS patients displayed an augmented activation level with increased expression of IgG and the costimulatory molecules CD80 and CD86. Furthermore, the expression of CD80, suggested to be of importance for induction of T-cell tolerance, was impaired on B cells of IBS patients after stimulation with bacterial components. Hence, our data suggest the increased immune activity of IBS patients might partly be the result of a modified costimulatory capacity of B cells.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was supported by the Swedish Medical Research Council (Grant no. 13409), The Sahlgrenska Academy, (LUA/ALF), Swedish Society of Medicine, Magnus Bergwall Foundation, O.E. and Edla Johansson’s Foundation, Sigurd and Elsa Golje Foundation, Ollie and Elof Ericsson Foundation, Sahlgrenska University Hospital Foundation, The Royal Science and Literature Society of Gothenburg, Tore Nilsson Foundation, Wilhelm & Martina Lundgren Foundation and Arla Foods.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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