Abstract
T lymphocytes and their cytokines have an important role in the regulation of immune responses in the gut and in the pathogenesis of intestinal inflammation such as in Crohn's disease. The aim of this study was to analyse the Th1/Th2 cytokine profile (IFN-γ, IL-2, IL-4 and IL-10) in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) in Crohn's disease (CD) and ulcerative colitis (UC) in relation to healthy controls (C). Colonic and ileal biopsy specimens were obtained from controls (n = 13) and patients with CD (n = 32). Colonic biopsies were obtained from patients with UC (n = 11). Intracytoplasmic IFN-γ, IL-2, IL-4 and IL-10 were determined by flow cytometry after PMA–ionomycin stimulation in IEL and LPL. In colonic LPL, a significant proportional decrease of IFN-γ and IL-2 producing CD3+ cells was observed in patients with CD and UC compared to controls. In ileal LPL, a similar tendency was found although differences were not significant. In IEL no differences in cytokine profiles could be observed. Flow cytometric analysis of intracytoplasmic cytokines at single cell level showed a proportional decrease of IFN-γ and IL-2 producing T cells in colonic lamina propria in patients with inflammatory bowel disease.
Keywords: Th1, Th2, mucosal lymphocytes, Crohn's disease, ulcerative colitis
INTRODUCTION
Both in vitro [1] and in vivo investigations [2] indicate that T lymphocytes and their cytokines play an important role in the regulation of gut immune responses and in the pathogenesis of intestinal inflammation. Human CD4+ T cells can be divided into two major subsets [3]. Th1 cells produce IFN-γ and IL-2 and mediate cellular immune responses; whereas IL-4 and IL-10 producing Th2 cells are more implicated in humoral responses and allergy.
T cells are the major source of IFN-γ which activates several important inflammatory cells [4], thereby controlling the local immune responses [5]. IL-2 is produced by lymphocytes. It functions as an obligatory signal for T and B cell growth by interacting with the IL-2 receptor complex on T and B cells [6,7]. IL-2 is the prototypical T cell growth factor and functions in an autocrine and paracrine manner to stimulate clonal expansion of antigen-stimulated lymphocytes. IL-2 is also involved in activation induced cell death and lymphocyte homeostasis [8]. Thus, disruption of the IL-2 gene leads to uncontrolled accumulation of activated lymphocytes and manifestations of autoimmunity, apparently due to failure of self-tolerance and lymphocyte homeostasis [8]. IL-4 is a pleiotropic cytokine and has a wide variety of effects, including T cell growth and regulation, B cell growth and differentiation, haematopoiesis and immunoglobulin class regulation [9]. IL-4 has the ability to influence the balance between Th1 and Th2 cells [10] and has a potent anti-inflammatory activity [11,12]. IL-10 is produced by T cells, B cells and macrophages [13–15] and exerts inhibitory effects on various cell types. It suppresses lipopolysaccharide-induced production of IL-1, GM-CSF, TFN-α, IL-6 and IL-8 by macrophages and diminishes IL-2 and IFN-γ production, together with the allogeneic proliferative and cytotoxic T cell response [16,17].
The aim of the present study is to analyse the T cell cytokine profile (IFN-γ, IL-2, IL-4 and IL-10) in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) from patients with Crohn's disease (CD) and ulcerative colitis (UC) in order to identify abnormal patterns. Flow cytometry was used to measure the T cell cytokine profile in mucosal lymphocytes.
Methods
Patients and samples
Fifty-six patients entered the study, including 32 patients with CD, 11 patients with UC and 13 controls. The diagnosis of CD and UC was based on the presence of characteristic endoscopical findings and the results of the pathological examination. The clinical and histological data on CD and UC patients are summarized in Table 1 and Table 2, respectively. Colonic and ileal biopses were obtained during ileocolonoscopy from patients with CD, and colonic biopsies were obtained during colonoscopy from patients with UC. Control samples were obtained from 13 persons (6 females, 7 males, median age 47 years, range 18–74 years), who underwent an ileocolonoscopy for irritable bowel syndrome or for follow-up of polyps. All ileocolonoscopies were normal in these patients. This study was approved by the Ethical Committee of the local Faculty of Medicine.
Table 1.
Clinical and histological characteristics of Crohn's disease patients included in this study
| Patient | Sex | Age | Ileum | Colon | Medication |
|---|---|---|---|---|---|
| GJ | M | 63 | Active | Normal, NB | Azathioprine |
| NE | M | 39 | Normal | Normal* | Sulphasalazine |
| BB | M | 20 | Normal | Active | Mesalazine/Methylprednisolon |
| LR | M | 57 | Active, NB | Normal | Mesalazine |
| VDBP | M | 49 | Normal | Normal* | Mesalazine |
| VM | F | 32 | Active | Normal* | None |
| CP | M | 19 | Normal | Active | Mesalazine/Methylprednisolon/Azathioprine |
| PS | F | 24 | Active | Active | None |
| BM | F | 14 | NB | Active | Azathioprine |
| DJ | M | 33 | Normal | Normal* | Mesalazine |
| LM | F | 25 | Normal | Active | Mesalazine |
| VDW | M | 25 | Active, NB | Active | Mesalazine |
| DRK | M | 29 | Normal | Active | Azathioprine |
| CJ | M | 18 | Active, NB | Active | Mesalazine |
| BM | M | 45 | Active, NB | Active | Sulphasalazine/Methylprednisolon/Azathioprine |
| DBE | F | 42 | Normal, NB | Active | Mesalazine/Methylprednisolon/Azathioprine |
| DCN | F | 31 | Normal, NB | Active | Azathioprine |
| SC | F | 31 | Normal, NB | Active | Mesalazine/Azathioprine/Budesonide |
| VDBA | F | 26 | Normal, NB | Active | Azathioprine |
| VDDV | F | 35 | Normal, NB | Active | Azathioprine |
| VDS | F | 29 | Normal, NB | Active | Mesalazine/Methylprednisolon/Azathioprine |
| HN | F | 21 | Normal, NB | Active | Mesalazine |
| BJ | M | 47 | Active, NB | Active | Mesalazine |
| VM | M | 42 | Normal | Active | Methotrexate |
| DS | F | 28 | Normal, NB | Active | Azathioprine |
| DL | M | 45 | Normal, NB | Active | Sulphasalazine |
| DBA | F | 32 | Normal, NB | Active | Methylprednisolon |
| AH | M | 39 | Normal, NB | Active | None |
| GD | M | 32 | Normal, NB | Active | Sulphasalazine/Azathioprine |
| MK | M | 27 | Active, NB | Active | Sulphasalazine/Azathioprine |
| MR | M | 17 | Normal, NB | Active | Azathioprine |
| SDK | F | 26 | Normal, NB | Active | Methylprednisolon/Azathioprine |
NB: no biopsies available for lymphocyte extraction.
Active colitis was documented by earlier colonoscopies.
Table 2.
Clinical and histological characteristics of ulcerative colitis patients included in this study
| Patient | Sex | Age | Localization | Histology | Medication |
|---|---|---|---|---|---|
| DCA | M | 80 | RS | Active | Mesalazine |
| KM | M | 44 | LC | Active | Mesalazine |
| LG | F | 68 | RS | Active | Corticosteroids |
| NM | M | 23 | TC | Active | Mesalazine |
| RN | M | 16 | TC | Normal | Mesalazine/Corticosteroids |
| GS | F | 24 | TC | Active | Corticosteroids |
| DL | F | 40 | RS | Normal | Mesalazine |
| VDBM | M | 70 | TS | Normal | Mesalazine |
| NT | F | 51 | TC | Active | Mesalazine |
| VA | M | 64 | RS | Active | Corticosteroids |
| LH | F | 68 | RS | Active | Corticosteroids |
LC: left colon; RS: recto-sigmoid; TC: total colon.
Isolation of intraepithelial and lamina propria lymphocytes
Eight to 10 colonic biopsies and eight to 10 ileal biopsies were collected in 10 ml phosphate buffered saline (PBS) (Gibco BRL, Grand Ysland, NY, USA). The isolation protocol was done as previously described [18]. Briefly, the biopsies were transferred into fresh PBS and stirred during 20 min at 37°C in PBS to remove blood, mucus and debris. Next, the biopsies were transferred to fresh PBS and stirred for another 60 min at 37°C to isolate IEL. Subsequently, lamina propria cells were obtained by cutting the remaining biopsy fragments into approximately 1–5 mm3 fragments, which were then incubated at 37°C for 3 h in collagenase type IV (50 U/ml) (Sigma Chemical Co., St. Louis, Missouri, USA) in RPMI 1640 medium (Gibco). The IEL and LPL were resuspended in 2 ml RPMI 1640 medium with 10% autologous serum and put overnight in a humidified chamber at 37°C.
Cell viability
The viability of isolated IEL and LPL from two healthy controls and two patients with Crohn's disease was compared between immediate analysis and analysis after overnight interval. Therefore, cells were incubated with anti-CD3 (Leu4, clone SK7) monoclonal antibody (MoAb) from Becton Dickinson (BD) (San Jose, CA, USA) for 30 min. After washing with PBS, cells were resuspended in PBS and analysed by flow cytometry by adding propidium iodide (0·5 µg/ml; Sigma), which discriminates dead cells from viable cells.
Detection of intracellular cytokines
Cells were stimulated as described by Morita et al. [19]. IEL and LPL, after overnight standing in the humidified chamber at 37°C, were stimulated with a combination of 25 ng/ml phorbol 12–myristate 13–acetate (PMA; Sigma) and 1 µg/ml calcium ionophore ionomycin (Sigma) for 5 h at 37°C in a humidified atmosphere containing 5% CO2. After 1 h 10 µg/ml brefeldin A (Sigma) was added. Brefeldin A was used to enhance flow cytometric analysis of intracellular cytokine staining, through its inhibitory effect on protein secretion by interfering with the function of the Golgi apparatus.
Human cytokine specific MoAb and isotype-matched control MoAb conjugated with FITC and PE were obtained from BD: anti-Hu IFN-γ (25723.11), anti-Hu IL-2 (5344.11) and anti-Hu IL-4 (3010.211). Anti-Hu IL-10 (JES3-9D7) was purchased from Biosource (Biosource Europe, Nivelles, Belgium). MoAb against human surface antigens labelled with PerCP or APC were also purchased from BD: anti-CD8 (Leu-2a, clone SK1) and anti-CD3 (Leu4, clone SK7).
After stimulation, the cells were washed and incubated with 10 µl of anti-CD8 and 5 µl of anti-CD3 for 30 min. Two ml of lyse buffer (BD) was added for 10 min. After centrifugation, 500 µl of permeabilization buffer (BD) was added for 10 min. After washing with PBS, the cells were incubated with the MoAb anti-Hu IFN-γ, anti-Hu IL-4, anti-Hu IL-2 and anti-Hu IL-10 for 30 min. After washing with PBS, the cells were resuspended in 300 µl CellFIX (BD) and kept at 4°C until analysis by flow cytometry. All incubations were carried out at room temperature and in the dark.
Flow cytometric analysis
Flow cytometric analysis was performed using FACSort type (BD) by four colour flow cytometry. A biparametric gate in the forward/side scatter dot plot was drawn around the lymphocyte population and T lymphocytes were subsequently gated by their CD3 expression. Four colour flow cytometry allowed to analyse the percentages of cytokine-producing cells within the CD3+CD8+ and CD3+CD8− populations. Since CD4 expression is known to be down-regulated after stimulation with phorbol esters [20], the CD4+ population could only be analysed indirectly by gating on the CD3+CD8− population. Acquired data were analysed using Cellquest® software (BD).
Immunohistochemistry
Immunohistochemistry was performed in a subset of controls and IBD patients, from whom also gut lymphocytes extraction were available. Five-µm thick sections of formalin-fixed, paraffin-embedded bowel mucosa from four controls (DHI, DHR, VP and BD), five Crohn's disease (patients DJ, LM, PS, BM and DS from Table 1) and six ulcerative colitis (patients RN, VDBM, NT, KM, NM and GS from Table 2) were immunohistochemically stained with a monoclonal rabbit-antihuman CD3 antibody (A0452, DAKO, Glostrup, Denmark). Before applying the antibody, proteolytic predigestion with trypsin was performed for unmasking of antigens. Immunostaining was visualized using 3-amino-9-ethylcarbazole (DAKO). An isotype-specific irrelevant antibody was used to control for nonspecific binding of the primary antibody. In the lamina propria, the CD3 positive cells were counted in 5 high power fields (magnification × 400) and in the epithelium, CD3 positive cells were counted among 100 epithelial cells.
Statistical analysis
Values were expressed as percentage positive cells with median and range. To avoid statistical biases created by multiple comparisons, the Kruskal–Wallis test was used comparing the different groups; only when this test showed significant differences, the exact P-values were calculated with the Mann–Whitney U-test and corrected for the number of comparisons.
RESULTS
Cell viability
The viability of IEL and LPL from both controls and Crohn's disease patients after overnight interval was more than 95% as determined with propidium iodide by flow cytometric analysis. No significant differences in the fraction of dead cells was observed between the immediate analysis and the analysis following overnight interval.
T cell cytokine pattern
In a first analysis, the relative numbers of cytokine-producing cells were analysed between controls, patients with CD and UC. In colonic LPL, from actively inflamed tissue, we observed a significant proportional decrease of IFN-γ and IL-2 producing CD3+ cells in patients with CD compared with controls (P = 0·003; P = 0·003, respectively) (Table 3) and in patients with UC compared with controls (P = 0·018; P = 0·024, respectively) (Table 3). No differences were observed in colonic IEL between the groups.
Table 3.
Percentages of cytokine-producing CD3+, CD3+CD8+ and CD3+CD8− T cells in colon epithelium and lamina propria from normal controls (C), patients with Crohn's disease (CD) and ulcerative colitis (UC) after PMA–ionomycin stimulation
| CD3+ | CD3+CD8+ | CD3+CD8− | |||||||
|---|---|---|---|---|---|---|---|---|---|
| C n = 10 | CD n = 27 | UC n = 11 | C | CD | UC | C | CD | UC | |
| IEL | |||||||||
| IFN-γ | 29 (15–65) | 28 (3–45) | 28 (16–40) | 42 (18–77) | 44 (4–69) | 49 (38–72) | 19 (9–38) | 20 (5–43) | 15 (8–30) |
| IL-2 | 43 (16–72) | 49 (2–62) | 34 (10–59) | 37 (12–85) | 48 (2–68) | 33 (18–64) | 47 (20–58) | 50 (13–65) | 41 (25–56) |
| IL-4 | 1·1 (0·2–3·6) | 0·9 (0·2–4) | 0·7 (0–1·5) | 0·9 (0·5–2·7) | 1·1 (0–4·3) | 0·5 (0·3–2·7) | 1·1 (0–4·4) | 0·8 (0·1–2·8) | 0·5 (0·4–1·7) |
| IL-10 | 2·9 (0·1–4·8) | 3·4 (1·2–13) | 3·5 (1·4–7) | 2·2 (0·1–4·2) | 3·3 (1–11) | 3·3 (1–6·2) | 4·2 (0·1–5·9) | 3·2 (0·6–10) | 3·3 (1·4–8·8) |
| LPL | |||||||||
| IFN-γ | 64 (42–72)*† | 41 (11–71)* | 49 (24–63)† | 78 53–88) | 61 (15–81) | 73 (15–84) | 57 (37–67)*† | 34 (9–71)* | 35 (15–56)† |
| IL-2 | 71 (49–85)*† | 50 (10–79)* | 51 (22–71)† | 60 (38–87) | 44 (8–74) | 48 (30–74) | 74 (47–84)*† | 54 (10–82)* | 53 (17–72)† |
| IL-4 | 1·8 (0·9–4·2) | 1·1 (0·2–3·3) | 1·5 (0·7–6) | 3·4 (0·7–6·5) | 1·5 (0·3–5·9) | 2·2 (1–4·6) | 1·4 (0·5–3·3) | 1 (0·2–3·3) | 1 (0·4–5·3) |
| IL-10 | 3 (1·4–7·3) | 3·4 (1·5–15) | 3·4 (1·8–7) | 3 (1–8) | 3 (1·8–7·5) | 3·6 (1·9–8·8) | 2·6 (1·5–6·9) | 3·3 (1·3–9·5) | 3 (1·4–6) |
Results are expressed as the median with range percentages of positive cells. Statistical significant differences (P < 0·05) are noted.
C ⇔ CD.
C ⇔ UC.
IFN: interferon; IL: interleukin; IEL: intraepithelial lymphocytes; LPL: lamina propria lymphocytes; PMA: phorbol 12-myristate13-acetate.
In ileal LPL, a tendency towards a lower proportion of IFN-γ and IL-2 producing CD3+ cells was noted in CD patients compared with controls, but no significance was reached (Table 4). No differences were observed in ileal IEL between the groups.
Table 4.
Percentages of cytokine-producing CD3+, CD3+CD8+ and CD3+CD8− T cells in ileum epithelium and lamina propria from normal controls (C) and patients with Crohn's disease (CD) after PMA–ionomycin stimulation
| CD3+ | CD3+CD8+ | CD3+CD8− | ||||
|---|---|---|---|---|---|---|
| C n = 10 | CD n = 11 | C | CD | C | CD | |
| IEL | ||||||
| IFN-γ | 33 (20–67) | 28 (3·5–67) | 52 (31–74) | 42 (8–73) | 15 (4–53) | 18 (3–61) |
| IL-2 | 50 (6–79) | 51 (5–73) | 64 (7–84) | 58 (8–81) | 42 (4–69) | 41 (6–66) |
| IL-4 | 1·1 (0·2–2) | 0·9 (0·4–2·5) | 0·7 (0·4–1·3) | 0·8 (0·3–2·6) | 1·2 (0·5–1·9) | 0·8 (0·6–3·9) |
| IL-10 | 3·9 (2·9–9·7) | 4·6 (1·5–13) | 3·8 (2·9–9·3) | 3·6 (1–10·9) | 3·1 (2·6–9·7) | 5·5 (1·7–14) |
| LPL | ||||||
| IFN-γ | 65 (13–82) | 51 (14–73) | 77 (35–89) | 62 (30–78) | 58 (5·4–74) | 39 (10–72) |
| IL-2 | 83 (26–89) | 78 (17–86) | 88 (30–88) | 75 (12–90) | 81 (8·8–89) | 76 (18–89) |
| IL-4 | 1·6 (0·6–3·8) | 1·5 (0·4–3·9) | 1·1 (0·4–3·8) | 1·8 (0·3–6·1) | 2 (0·4–3·8) | 1·1 (0·3–3·3) |
| IL-10 | 2·5 (1–4) | 2·5 (0·8–4·1) | 2·1 (0·6–4·8) | 3·9 (0·7–5·5) | 2·4 (1·1–4·5) | 2 (0·8–3·9) |
Results are expressed as the median with range percentages of positive cells. IFN: interferon; IL: interleukin; IEL: intraepithelial lymphocytes; LPL: lamina propria lymphocytes; PMA: phorbol 12-myristate13-acetate.
Four colour flow cytometry allowed to analyse the percentages of cytokine-producing cells within the CD3+CD8+ and CD3+CD8− populations. Since CD4 expression is known to be down-regulated after stimulation with phorbol esters [20], the CD4+ population could only be analysed indirectly by gating on the CD3+CD8− population. In all tested situations (IEL and LPL) both CD3+CD8+ as well as CD3+CD8− subsets were found to have the potential to produce the cytokines under investigation. The proportional decrease of IFN-γ and IL-2 producing cells observed in CD3+ LPL cells was also noted in CD3+CD8− cells (Table 3).
In a further analysis, we wanted to correct the relative changes in T cell subsets for the changes in absolute numbers of CD3 cells in lamina propria and epithelium in CD and UC. Therefore, we made a quantitative analysis of CD3+ cells in situ, using immunohistochemistry, in a subgroup of patients in whom lymphocyte extractions were performed. Immunohistochemical CD3 staining was performed on biopsies from four controls, five Crohn's disease patients and six ulcerative colitis patients. In the lamina propria, the CD3+ cells were counted in 5 high power fields and in the epithelium CD3+ cells were counted among 100 epithelial cells.
A calculated estimation of the absolute number of IFN-γ and IL-2 producing LPL CD3+ cells in situ, was obtained by multiplying (per patient) the number of CD3+ cells per 5 high power fields with the percentage of IFN-γ and IL-2 positive CD3 cells in the lymphocyte populations extracted from lamina propria. The results are summarized in Table 5. No differences were observed in the calculated absolute number of IFN-γ and IL-2 producing CD3+ colon LPL between controls, CD and UC. Thus, the absolute number of CD3+ cells was increased in IBD, but the absolute number of Th1 cells was the same in controls and IBD patients.
Table 5.
Calculated absolute numbers of IFN-γ and IL-2 producing LPL CD3+ cells in colon from controls, Crohn's disease and ulcerative colitis
| CD3+/5 HPF | % IFN-γ positive cells among extracted LPL | Calculated absolute number of IFN-γ positive cells/5 HPF | % IL-2 positive cells among extracted LPL | Calculated absolute number of IL-2 positive cells/5 HPF | |
|---|---|---|---|---|---|
| Controls | |||||
| DHI | 323 | 62·47 | 202 | 74·31 | 240 |
| DHR | 359 | 65·21 | 234 | 66·79 | 240 |
| VP | 514 | 65·73 | 338 | 73·59 | 378 |
| BD | 424 | 61·48 | 261 | 71·88 | 305 |
| Mean 405 | Mean 259 | Mean 291 | |||
| s.d. 84 | s.d. 58 | s.d. 66 | |||
| Crohn's disease | |||||
| DJ | 525 | 35·58 | 187 | 36·37 | 191 |
| LM | 1252 | 44·65 | 559 | 44·04 | 551 |
| PS | 870 | 49·36 | 429 | 56·2 | 489 |
| BM | 394 | 10·86 | 43 | 9·69 | 38 |
| DS | 339 | 30·19 | 102 | 42·46 | 144 |
| Mean 676 | Mean 264 | Mean 283 | |||
| s.d. 382 | s.d. 221 | s.d. 225 | |||
| Ulcerative colitis | |||||
| RN | 476 | 49·18 | 234 | 66·51 | 317 |
| VDBM | 751 | 40·85 | 307 | 47·47 | 357 |
| NT | 330 | 62·88 | 208 | 69·96 | 231 |
| KM | 564 | 60·49 | 341 | 40·7 | 230 |
| NM | 552 | 42·16 | 233 | 54·79 | 302 |
| GS | 718 | 63·05 | 453 | 59·78 | 429 |
| Mean 565 | Mean 296 | Mean 311 | |||
| s.d. 156 | s.d. 92 | s.d. 77 | |||
The CD3+ cells were expressed as the number of CD3+ cells per 5 high power fields (HPF). The absolute number of IFN-γ and IL-2 producing CD3+ cells was calculated on the basis of the percentage IFN-γ or IL-2 positive CD3 cells among the extracted lymphocyte population and was expressed per 5 HPF.
IFN, interferon; IL, interleukin; s.d., standard deviation; LPL, lamina propria lymphocytes
No differences were observed in Th1 cells in the epithelium from controls and IBD patients (neither in relative number, nor in absolute number).
Influence of medical treatment
In colonic LPL, IFN-γ and IL-2 producing CD3+ T cells from patients with Crohn's disease were analysed in relation to medical treatment (mesalazine, prednisolone and azathioprine). The results are summarized in Table 6. There were no significant intergroup differences observed according to the medication intake.
Table 6.
Percentage of IFN-γ and IL-2 producing CD3+ T cells in colon lamina propria from controls and Crohn's disease (CD) patients in relation with medical treatment
| Controls (n = 10) | CD with mesalazine (A) (n = 11) | CD without mesalazine (B) (n = 15) | A vs. B | |
|---|---|---|---|---|
| IFN-γ | 64 (42–72) | 49 (20–71) | 35 (11–67) | P = 0·108 |
| IL-2 | 71 (49–85) | 48 (19–79) | 51 (10–68) | P = 0·659 |
| Controls (n = 10) | CD with prednisolon (A) (n = 5) | CD without prednisolon (B) (n = 21) | A vs. B | |
|---|---|---|---|---|
| IFN-γ | 64 (42–72) | 49 (27–69) | 39 (11–71) | P = 0·205 |
| IL-2 | 71 (49–85) | 51 (19–78) | 49 (10–79) | P = 0·770 |
| Controls (n = 10) | CD with azathioprine (A) (n = 14) | CD without azathioprine (B) (n = 12) | A vs. B | |
|---|---|---|---|---|
| IFN-γ | 64 (42–72) | 35 (11–69) | 43 (28–71) | P = 0·543 |
| IL-2 | 71 (49–85) | 49 (10–78) | 54 (30–79) | P = 0·369 |
Results are expressed as the median with range percentages of positive cells.
IFN, interferon; IL, interleukin.
DISCUSSION
Many mucosal cells produce immunoregulatory and pro-inflammatory cytokines: epithelial cells, endothelial cells, muscle cells, lymphocytes or monocytes. Different techniques have been used to measure these cytokines. First, cytokines can be measured at the mRNA level using quantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR) technique. However, the presence of cytokine message in cells does not necessarily correlate with the presence of the expressed protein. Second, bioassays detect the biologically active secreted cytokine in serum or cell culture supernatant. Third, cytokine immunoassays like ELISA measure secreted cytokines. One of the major limitations is the lack of standardization between cytokine immunoassay detection methods. As the production of most cytokines is not confined to a single cell type, a drawback of these techniques is that they do not allow to identify the cellular source of the produced cytokines. In the early 1990s, two important techniques were developed for determination of cytokine production at the single cell level. First, Sander et al. [21] described a microscopic method to detect intracellular cytokines in single cells by paraformaldehyde fixation, saponin permeabilization and indirect immunofluorescent staining. Second, by the method of Jung et al. [22] cells can be stimulated in the presence of monensin, which disrupts intracellular protein transport and leads to cytokines accumulating in the Golgi apparatus; this yields an enhanced cytokine signal that can be analysed by flow cytometry. This approach allows for the precise identification of the cellular source of the cytokine using phenotypic surface markers and the easy quantification of the number of cytokine-producing cells. Therefore, four colour flow cytometry was used to study the expression of T cell cytokines in intraepithelial and lamina propria lymphocytes in a normal population and in patients with Crohn's disease and ulcerative colitis.
A first observation is that in all groups studied there was a strong predominance of gut mucosal T cells producing Th1 cytokines, with IFN-γ predominantly produced by CD3+CD8+ cells and IL-2 by both CD3+CD8+ as CD3+CD8− cells. In contrast, the percentage of Th2 cytokine (IL-4 and IL-10) producing T lymphocytes was rather low. These findings correlate well with previous reports on IFN-γ and IL-2 producing T cells in peripheral blood [19,23] and studies showing less than 5% Th2 peripheral blood T cells [22,24].
The second observation is that the proportion of Th1 cells, IFN-γ and IL-2, is decreased in patients with Crohn's disease and ulcerative colitis compared with controls. Immunohistochemical staining showed that inflamed gut mucosa in Crohn's disease is characterized by an increase in the absolute number of CD3+ cells. The absolute number of Th1 cells in the inflammatory infiltrate seems to remain unchanged. Thus, the increase in CD3+ cells in IBD is caused by non Th1 cells, resulting in a proportional decrease of Th1 cells in IBD.
Earlier studies on the expression of IFN-γ and IL-2 in the intestinal mucosa revealed contradictory results. In several studies, increased levels of IFN-γ mRNA have been reported in mucosal lesions of patients with IBD compared with normal controls [25–27]. Fuss et al. [28] cultured cells with stimuli after which supernatants were collected and assayed for cytokines using ELISA kits. The LP T cells were stimulated via the CD2/CD28 pathway which led to a significant increased IFN-γ secretion in Crohn's disease LP CD4+ cells compared with control LP CD4+ cells. In contrast, other studies using ELISA and ELISPOT techniques, showed decreased levels of IFN-γ in the lamina propria of patients with IBD compared with controls [29–31]. In the study of Desreumaux et al. [32] a decrease in IFN-γ mRNA values was detected in early ileal lesions during 3 months after surgery compared with normal ileal mucosa of patients with CD and with the control group.
Similarly to IFN-γ the results on IL-2 are conflicting. In several studies using RT-PCR, increased levels of IL-2 mRNA have been reported in mucosal lesions of patients with IBD, compared with normal controls [25,26,33,34], but decreased production of IL-2 has been observed in studies of mitogen stimulated LP cells in patients with IBD [28,35,36].
These contradictory results can partially be explained by the technical discrepancies pointed out earlier in the discussion. Alternatively, a high proportion of patients used azathioprine alone or in combination with sulphasalazine, mesalazine or methylprednisolone. Although, it is known that azathioprine strongly affects cytokine secretion [37], it seems that the proportional decrease of IFN-γ and IL-2 producing CD3 colonic LP cells was not due to the use of azathioprine since this decrease was also present in the non‐azathioprine treatment group.
In a recent study on patients with spondyloarthropathy (SpA), also the proportion of Th1 cells among gut lamina propria lymphocytes was decreased [18]. It has been shown that some SpA patients have a subclinical form of CD [38,39].
Several hypotheses have to be considered to evaluate the biological relevance of the observed proportional decrease of IFN-γ and IL-2 in CD and UC. (i) It cannot be excluded that the stimulation in vitro does not reflect the physiological behaviour of the T cells in situ. The type of stimulation (PMA–ionomycin) could theoretically influence the cytokine profile. Another possibility is that T cells are exhausted by a sustained Th1 cytokine production in vivo resulting in an incapacity to produce large amounts of these Th1 cytokines after restimulation in vitro. (ii) The observed proportional decrease of IFN-γ and IL-2 producing cells could be explained as a consequence of chronic exposure to the high, but not T cell derived, TNF-α levels which were reported using ELISA and RT-PCR [40,41].
In summary, the present study shows a proportional decrease of IFN-γ and IL-2 producing CD3+ cells in gut in patients with Crohn's disease and ulcerative colitis. Although the in vitro situation of lymphocytes does not necessarily reflect the behaviour in vivo, these data suggest that the Th1 paradigm in Crohn's disease should be interpreted carefully and that a more subtle Th1/Th2 dysequilibrium could be present.
Acknowledgments
This work was supported by a concerted action grant GOA12050196 and GOA12051501 of Ghent University, Belgium and by FWO Vlaanderen grant 3.022.96. Dominique Baeten is a FWO Vlaanderen Research Assistant.
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