PATIENTS AND SAMPLES

Intestinal biopsy specimens were obtained from 33 control adult patients (mean age 49 years, range 33–66) undergoing intestinal endoscopy for investigation of dyspeptic complaints or for cancer screening. Duodenal biopsy specimens from 19 patients and right colonic biopsy specimens from 14 patients were analysed. Only the specimens taken from macroscopically unaffected mucosa and with no evidence of microscopic inflammation after histological analysis were included in this study. For each patient, seven biopsy specimens were taken, and transported within one hour in Ca⁺⁺/Mg⁺⁺ free Hank’s buffered salt solution (HBSS, Gibco, Life Technology, Paisley, Scotland, UK) to the laboratory. One specimen was fixed in formalin for routine histological examination, and the other six were processed for IEL and LPL isolations. Venous blood samples were collected from 20 patients.

Informed consent was obtained from all patients and this study was approved by the Research Ethics Committee of the Medical Faculty of the Université Libre de Bruxelles.

ISOLATION OF IEL, LPL, AND PERIPHERAL BLOOD MONONUCLEAR CELLS

The biopsy specimens were first extensively washed in Ca⁺⁺/Mg⁺⁺ free HBSS containing 0.02% penicillin-streptomycin and 40 μg/ml gentamycin. Epithelial cells (IEL and enterocytes) were then obtained using a modification of a previously described protocol.14 Briefly, the biopsy specimens were incubated for one hour under stirring at 37°C with 1 mM EDTA (Sigma Chemical Co., St Louis, Missouri, USA) in Iscove’s medium (Gibco) supplemented with 40 μg/ml gentamycin, 10% fetal calf serum (FCS), and 50 μM 2-mercaptoethanol. Extracted cells (IEL plus enterocytes) were collected by decanting them after tissue fragments had settled; they were washed once in HBSS at 600 g for 10 minutes, and resuspended in supplemented Iscove’s medium. Histological examination of the remaining fragment revealed that the villous and lamina propria structures were still preserved at that time.

Subsequently, lamina propria cells were obtained by a standard procedure in which the remaining biopsy fragments were first cut into approximately 1 × 1 mm fragments and then incubated while stirring at 37°C for 45 minutes in Iscove’s medium with 1 mg/ml collagenase-dispase (Boehringer Mannheim, Mannheim, Germany).2 The undigested tissue was separated from the cell suspension by sedimentation and the cells were resuspended in supplemented Iscove’s medium after being washed at 600 gfor 10 minutes.

For both suspensions, the numbers of mononuclear cells (MNC) were counted under a Bürker chamber, and the percentage of lymphocytes was estimated by counting them on May-Grünwald Giemsa (azur-eosine-methylene blue solution) stained cytospin smears. On average, the dispersion procedure yielded 3.7 × 10⁶nucleated cells (range 1.3–6.2 × 10⁶) per pool of six duodenal biopsy specimens, comprising 8–25% lymphocytes in the epithelium, and 2.8 × 10⁶ nucleated cells (range 1.3–6.1 × 10⁶) with 20–40% lymphocytes in the lamina propria. For six colonic biopsy specimens, the number of nucleated cells recovered was 2 × 10⁵ (range: 1.2–5 × 10⁵) with 9–27% lymphocytes in the epithelium, and 8 × 10⁵(range 2.5–1.4 × 10⁵) with 12–46% lymphocytes in the lamina propria. Viability, as determined by trypan blue exclusion, was greater than 95% both for IEL and LPL, from the duodenum and from the colon.

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (760 g, 20°C, 30 minutes) on Lymphoprep (Nycomed, Oslo, Norway).

PHENOTYPIC CHARACTERISATION OF IEL AND LPL

Flow cytometric analysis was performed on cells directly resuspended at 1 × 10⁶/ml in Iscove’s medium supplemented with 10% FCS and incubated for 30 minutes at 4°C with 10 μl anti-CD3 (Leu-4), anti-CD4 (Leu-3a), anti-CD8 (Leu-2a), or anti-CD69 (Leu-23) (Becton Dickinson, San José, California, USA). The monoclonal antibodies were directly conjugated either to fluorescein isothiocyanate (FITC), phycoerytrin (PE), or piridin-chlorophyll-a-protein (PerCP). Cells incubated with isotype matched irrelevant monoclonal antibody served as negative controls. For this phenotypic characterisation, LPL were obtained by incubation of the mucosal fragments with collagenase type I (Sigma Chemical Co.) for 150 minutes at 37°C while stirring continuously. After staining, the cells were washed, and at least 5000 cells were analysed by flow cytometry (FACScan, Becton Dickinson). Spectral overlap between the three fluorochromes used was eliminated by electronic compensation. Lymphocyte populations were gated by forward sideward scatter light and by gating for CD3⁺ T cells.

CYTOKINE SPECIFIC ELISPOT ASSAY

Cell suspensions were assayed for numbers of cells spontaneously secreting IFN-γ or IL-4 by a two site reverse enzyme linked immunospot (ELISPOT) technique using epitope specific mouse antihuman IFN-γ or IL-4 monoclonal antibodies as capture and developing reagents (IFN-γ: Chromogenix, Stockholm, Sweden; IL-4: Mabtech AB, Stockholm, Sweden). Briefly, individual wells of nitrocellulose bottomed 96 well millititre HA plates (MAHAN 4550, Millipore, Eschborn, Germany) were coated at 37°C for three hours with 100 μl of a 10 μg/ml solution of anti-IFN-γ (clone 1/D1K) or anti-IL-4 (clone 82–2). After the plates were washed five times with phosphate buffered saline (PBS), blotted dry, blocked with 100 μl/well PBS containing 1% (wt/vol) bovine serum albumin (BSA), 10% FCS, and 2% (wt/vol) milk powder for one hour at 37°C, washed again five times with PBS, and blotted dry again, 100 μl of different concentrations of cell suspensions (5 × 10⁴ to 20 × 10⁴ MNC/well) in complete medium was added into the coated wells. The plates were incubated in 5% CO₂ for 20 hours at 37°C. After incubation, the plates were thoroughly washed 10 times with PBS-0.05% Tween 20 to remove the cells, and then incubated for three hours at room temperature with 100 μl/well of biotinylated anti-IFN-γ (3 μg/ml; clone 7/B6/1) or anti-IL-4 (1 μg/ml; clone 12–1) in PBS-Tween. After washing the plates at least five times with PBS-Tween, extravidin-horseradish peroxidase (Sigma; 2 μg/ml in PBS-0.05% Tween) was added for one hour. After washing five times with PBS, the enzyme-substrate solution was added (3-amino-9-ethylcarbazole, AEC; Sigma). Spots appeared after a few minutes, and the reaction was stopped by briefly washing the plates with tap water. Spots were counted under low magnification (×40) and recorded as the number of secreting cells (SC) per 10⁵lymphocytes.

PREPARATION OF T LYMPHOCYTE SUBPOPULATIONS

To deplete the intestinal MNC suspensions of cell subsets, different cell suspensions were prepared by negative or positive selection techniques using monoclonal antibodies attached to immunomagnetic beads. CD4⁺ or CD8⁺ lymphocytes were selected with magnetic microspheres directly coated with antibodies to either CD4 or CD8 (Dynabeads M-450 CD4 and Dynabeads M-450 CD8, Dynal, Oslo, Norway). The intestinal MNC were incubated for 30 minutes at 4°C with the antibody coated bead preparations. Cells attached to beads and free beads were then retained by applying a magnetic field. Unbound cells were washed twice and resuspended in complete medium. CD4⁺ or CD8⁺ lymphocytes attached to the beads were then detached using immunomagnetic beads coated with antimouse IgG (Detachabead, Dynal, Oslo, Norway). Intestinal MNC suspensions underwent this depletion procedure twice, yielding MNC suspensions that contained less than 5% and less than 15% of residual contaminating CD4⁺ or CD8⁺cells, respectively, in the positively selected and in the depleted subpopulation, as controlled by FACS analysis.

FLOW CYTOMETRIC ANALYSIS OF INTRACELLULAR IFN-γ

Analysis was performed on cells resuspended at 1 × 10⁶/ml in Iscove’s medium supplemented with 10% FCS and prestimulated with phorbol myristate acetate (PMA; 10 ng/ml; Sigma) and calcium ionophore A23187 (100 ng/ml; Eurobiochem, Belgium) in the presence of brefeldin A (10 μg/ml; Sigma) for four hours at 37°C in a humidified incubator at 5% CO₂. For each sample to be analysed (100 μl of cell suspension), 5 μl of anti-CD3 or anti-CD8 (Becton Dickinson) was added, incubated for 20 minutes (4°C), and washed, followed by incubation with 80 μl of reagent A (Fixation Medium, Cell Permeabilisation Kit, AN DER GRUB, Austria) (15 minutes at 20°C). Reagent B (80 μl; Permeabilization Medium) was then added with 5 μl of anti-IFN-γ (clone 25723.11, Becton Dickinson), and the mixture was incubated for 15 minutes at 20°C; control isotypes were tested in parallel. Cells were finally resuspended in PBS for immediate analysis by flow cytometry (FACScan, Becton Dickinson). Lymphocytes were gated by forward sideward scatter light and by gating for CD3⁺ T cells.

STATISTICAL ANALYSIS

Tests for significance of differences were performed using the Mann-Whitney U test.

PHENOTYPIC CHARACTERISATION OF IEL AND LPL SUSPENSIONS

FACS analysis was used to enumerate CD4⁺, CD8⁺, and CD69⁺ cells among CD3⁺lymphocytes retrieved from the epithelium (IEL) and the lamina propria (LPL) of normal duodenum and colon. Most CD3⁺ cells, both among IEL and among LPL, expressed the early activation antigen CD69, especially in the duodenum. However, the phenotypic features of CD3⁺ IEL were slighly different in the colon compared with the duodenum as the proportion of CD4⁺ IEL was higher within the colonic mucosa (table 1).

SPONTANEOUS IFN-γ AND/OR IL-4 SECRETION BY INTESTINAL LYMPHOCYTES

As assessed by a two site reverse ELISPOT technique, significant numbers of cells spontaneously secreting IFN-γ and/or IL-4 were found among intestinal cell suspensions (fig 1). The proportion of secreting cells was always significantly higher among duodenal lymphocytes than among colonic lymphocytes for both cytokines (IFN-γ or IL-4) and for the two sites (IEL or LPL) considered. Whereas 3.6% of IEL and 1.9% of LPL secreted IFN-γ among duodenal lymphocytes, only 0.2% of colonic lymphocytes, both IEL and LPL, spontaneously secreted IFN-γ. Interleukin 4 was spontaneously secreted by 1.3% of IEL and by 0.7% of LPL in the duodenum, and by only 0.1% of colonic lymphocytes (IEL and LPL).

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Figure 1

Numbers of IFN-γ and/or IL-4 secreting cells per 10⁵ lymphocytes within IEL and LPL cell suspensions obtained from normal human intestinal duodenal and colonic mucosae. Individual results are shown and horizontal bars indicate the medians.

In the duodenum, the numbers of IFN-γ SC were significantly higher than the numbers of IL-4 SC both among IEL and LPL (IEL: p<0.001; LPL: p<0.05) (fig 1). The ratio (median) between IFN-γ SC and IL-4 SC was 3.25 (range 0.7–23) among IEL and 1.90 (range 0.4–33) among LPL, indicating that the physiological balance comprises a higher number of IFN-γ SC compared with IL-4 SC in the majority of healthy individuals, even though there is a wide individual variation in the numbers of cytokine SC.

In contrast to the intestinal lymphocytes, the numbers of cells spontaneously secreting IFN-γ among PBMC were extremely low (median 12 per 10⁵ cells, range 1–27) while IL-4 SC were undetectable. The secretion of cytokines by intestinal lymphocytes in the absence of any added exogenous stimulus was not induced by the cell dispersion procedure as treatment of PBMC with EDTA and collagenase-dispase did not modify the numbers of IFN-γ and/or IL-4 SC (data not shown).

To determine whether the ex vivo secretion of cytokines requires de novo protein synthesis, experiments using cycloheximide (CHX) were performed. Addition of CHX (100 μg/ml; Sigma Chemical Co.) for four hours before and during the incubation in the ELISPOT plates reduced the numbers of IFN-γ SC and IL-4 SC by only 12–39% compared with the control without CHX (table 2). In contrast, the numbers of IFN-γ and/or IL-4 SC among PBMC stimulated with PHA (10 μg/ml; Wellcome, Murex Diagnostics Limited, Dartford, UK) were reduced by more than 90% in the presence of CHX (data not shown). Taken together, these data indicate that de novo protein synthesis is not required for the in vitro detection of cytokine SC among intestinal cells, suggesting that most cytokine synthesis occurred in vivo.

IFN-γ AND/OR IL-4 SECRETION BY INTESTINAL LYMPHOCYTES IN RESPONSE TO IN VITRO POLYCLONAL STIMULATION

To determine whether in vitro activation of the cells could further increase the numbers of detectable cytokine SC, duodenal cell suspensions from three patients were either directly processed within the ELISPOT plates or stimulated in vitro with PMA and calcium ionophore A23187 (10 ng/ml and 100 ng/ml respectively) before and during the ELISPOT procedure. Preliminary experiments have shown that optimal in vitro activation of the cells required a four hour preincubation with the stimulant. Results shown in table 3 indicate that the numbers of IFN-γ SC were significantly increased by an in vitro polyclonal activation of the cells, both within IEL and LPL. In contrast, the numbers of IL-4 SC did not increase after in vitro polyclonal stimulation. Control experiments performed on cells preincubated for four hours in the absence of any stimulation showed undetectable or very low numbers of spontaneous cytokine SC compared with cell suspensions directly processed within the ELISPOT plates, further suggesting that most cytokine synthesis detected in the absence of in vitro stimulation reflects the in vivo situation.

PHENOTYPIC CHARACTERISATION OF CYTOKINE SECRETING CELLS

The phenotype of the intestinal cells spontaneously secreting cytokines was characterised by applying the ELISPOT technique on intestinal cell suspensions depleted or enriched in different subpopulations by using immunomagnetic beads coated with monoclonal antibodies. In preliminary experiments, combined removal of CD4⁺ and CD8⁺ cells from the intestinal MNC suspensions resulted in the disapperance of IFN-γ and/or IL-4 SC both among IEL and LPL, indicating that the secreting cells were T cells (table 4).

Further experiments on CD4⁺ or CD8⁺ cell depleted suspensions were performed to determine the respective roles of CD4⁺ and CD8⁺ cells in the secretion of cytokines. Depletion of CD4⁺ cells completely abrogated the detection of cytokine SC except for IFN-γ SC among IEL. In contrast, the CD8⁺ cell depletion procedure only slightly reduced the number of cytokine SC. To confirm that CD4⁺ cells represent the major source of cytokines in the basal state, we also quantified cytokine SC among CD4⁺ cell enriched LPL. Both IFN-γ and IL-4 SC were recovered within the CD4⁺ cell enriched LPL (table 4). This experiment could not be performed on IEL because of the low numbers of CD4⁺ cells.

The phenotype of intestinal IFN-γ SC in response to in vitro polyclonal stimulation could be analysed by flow cytometry, a technique which was not sensitive enough to detect the cytokine secretion in the basal state. In response to stimulation with PMA ionophore, IFN-γ was synthesised by both CD4⁺ and CD8⁺ lymphocytes. CD8⁺ lymphocytes were the major source of IFN-γ within IEL, whereas within LPL, IFN-γ was mainly synthesised by CD4⁺ lymphocytes (fig 2). Within IEL, 27% of CD3⁺ lymphocytes produced IFN-γ: 16% were CD3⁺/CD8⁺ and the other 11% were CD3⁺/CD8⁻. Within LPL, 65% of CD3⁺ lymphocytes produced IFN-γ: 22% were CD3⁺/CD8⁺ and the other 43% were CD3⁺/CD8⁻.

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Figure 2

Dot plots from three colour flow cytometric analysis for anti-IFN-γ on CD3⁺ and on CD8⁺ or CD8⁻ cells for IEL and LPL. The cells were cultured for four hours with PMA and ionophore A 23187 in the presence of brefeldin A. The fluorescence plots shown are representative of one of three different experiments and cells analysed were gated for CD3⁺ cells so that results are given as percentage of CD3⁺ cells. Control isotype was used to differentiate positive from negative immunofluorescence.