Cytolytic Capabilities of Lamina Propria and Intraepithelial Lymphocytes in Normal and Chronically Inflamed Human Intestine

Authors


Prof M.-L. Hammarström, Department of Immunology, Umeå University, SE-90185 Umeå, Sweden. E-mail: marie-louise.hammarstrom@climi.umu.se

Abstract

Cell-mediated lymphocyte cytotoxicity in ileum and colon of patients with ulcerative colitis (UC), Crohn's disease (CD) and controls was investigated. Frequencies of cells expressing perforin and Fas-ligand (FasL) were determined by immunomorphometry. mRNA expression of perforin, granzyme B and FasL in T cells and subsets was assayed by reverse transcriptase-polymerase chain reaction. Cytotoxicity of intraepithelial and lamina propria lymphocytes was analysed without ex vivo activation in three functional assays: (1) anti-CD3-dependent T-cell receptor (TCR)-/CD3-mediated redirected cytotoxicity, (2) Fas-/FasL-mediated TCR-/CD3-independent cytotoxicity and (3) natural killer (NK) cell cytotoxicity. Inflammation in ileum of CD patients caused increased frequency of perforin-expressing cells and enhanced perforin-dependent TCR-/CD3-mediated cytotoxicity. In contrast, lymphocytes in the inflamed colon of UC or Crohn's colitis patients did not display this cytotoxicity nor did lymphocytes of normal colon. Normal colon lymphocytes showed spontaneous Fas-/FasL-mediated cytotoxicity. This activity was retained but not enhanced in inflamed UC colon. In contrast, a significant increase of FasL-expressing cells was seen in situ. Inflammation did not induce NK cell activity in colonic lymphocytes. Intestinal lymphocytes comprise effectors active in two different cytolytic processes. ‘Classical’ cytotoxic T lymphocytes in small intestine and lymphocytes executing TCR-/CD3-independent FasL-/Fas-mediated killing of unknown biological role present throughout the intestinal mucosa. Ongoing normal cytolytic processes seem to be enhanced by chronic inflammation.

Introduction

Ulcerative colitis (UC) and Crohn's disease (CD) constitute the two major chronic inflammatory bowel diseases (IBDs) in human. The inflammation in UC is restricted to the large bowel and mainly localized to the mucosa. CD may be manifested in all parts of the alimentary tract but the ileocecal region is most commonly affected. The inflammation is granulomatous and transmural. The aetiologies of the two diseases are unknown. There are indications that local immune reactions in the intestinal mucosa play important roles in the pathogenesis of both diseases and immunosuppressive drugs are effective in the treatment [1–3]. Studies of genetically manipulated mice strongly suggest an important role for T-lymphocyte-mediated immune mechanisms in IBD [4].

Under normal physiological conditions, T lymphocytes are present in the epithelium [intraepithelial lymphocytes (IELs)] and in the lamina propria (LP) [lamina propria lymphocytes (LPLs)]. At both compartments, they display a heterogeneous surface marker profile indicating functionally different T-cell subpopulations. Moreover, the composition of immune cells differs between small and large intestine [5, 6]. IELs have been suggested to participate in immune protection, surveillance of the epithelium and induction and maintenance of oral tolerance [7]. Moreover, in LP, T lymphocytes control local antibody production. In human small intestine, both IELs and LPLs express cytokines suggesting involvement in cell-mediated immune responses [8–11] and express cytotoxic markers such as perforin and Fas-ligand (FasL) [12–14]. Indeed, freshly isolated normal jejunal IELs and LPLs showed cytolytic activity both in the T-cell receptor (TCR)-/CD3-dependent redirected cytotoxicity assay and TCR-/CD3-independent killing via the Fas/FasL pathway [8, 12]. Cytotoxic T cells were also demonstrated in the murine small intestine [6, 15, 16].

LP of inflamed intestinal mucosa in UC and CD patients is heavily infiltrated by lymphocytes, a large proportion being CD4+αβ T cells. γδ T cells are normally absent or very few in the LP but can be seen at this location in both diseases [17, 18]. Moreover, the colonic LP of UC patients contains basal lymphoid aggregates, a microanatomical structure not found in normal colon. The dominating T-cell subtype in the aggregates is CD4+CD28TCR-αβ+, i.e. cells with a suppressive phenotype [17]. Activated γδ T cells are also present at a significant frequency [17]. T lymphocytes in the inflamed colonic mucosa of UC patients are induced to interleukin-10 (IL-10) production suggesting activation of T-regulatory cells (Tr1) cells [10]. CD seems to be dominated by a Th1-type of immune response, and CD patients have been successfully treated with antibodies directed against tumour necrosis factor-α (TNF-α) [19, 20].

Little is known about the role of cytotoxic lymphocytes in the pathogenesis of IBD. The presence of functional cytotoxic T lymphocytes (CTLs) in the inflamed mucosa was suggested by demonstration of effector cells in anti-TCR-/CD3-dependent redirected cytotoxicity in mouse colitis models [21, 22] while murine, normal colonic mucosa did not harbour CTLs as estimated by this assay [6]. In addition, cytotoxic γδ T cells were shown to exacerbate colitis in murine IBD [23].

The aim of this study was twofold: (1) to determine the cytolytic capacities of normal colonic T cells, and (2) to investigate whether intestinal lymphocytes from CD and UC patients show increased cell-mediated cytotoxicity contributing to the pathogenesis. Frequencies of cells expressing perforin and FasL were determined by immunomorphometry analyses of normal and inflamed ileum and colon, and mRNA expression for perforin, granzyme B (GrB) and FasL was determined in T cells isolated from the same tissues. Three functional cytotoxicity assays were used: (1) Anti-CD3-dependent redirected cytotoxicity as a model of TCR-/CD3-mediated perforin/Gr exocytosis, (2) spontaneous killing of the Fas-expressing human T cell line Jurkat as a model of TCR-/CD3-independent, Fas-/FasL-mediated cytotoxicity and (3) spontaneous killing of K562 cells as an indicator of functional NK cells. Effector cells were not subjected to ex vivo stimulation, thus on-going cytolytic activity was measured.

Materials and methods

Source of intestinal tissue.  Apparently normal human small and large intestine tissue specimens were obtained from patients undergoing bowel resection for gastrointestinal cancer (n = 32) or benign conditions (n = 11). Two specimens were endoscopic biopsies from patients undergoing investigation for anaemia or diarrhea. The colonic samples were from 12 men and 10 women [64 (51–84) years (median and range)], and ileal samples were from eight men and six women [72 (34–87) years]. The samples were distant to any macroscopically detectable lesion. Colonic and ileal samples were from the same patient in four cases.

Inflamed ileal specimens were obtained from nine patients suffering from CD. All were obtained by surgical resection. Samples were from four male and five female patients [48 (19–56) years]. In three cases, both colonic and ileal samples were obtained from the same patient. Two of the colonic samples were apparently normal while one was inflamed. Two additional colonic samples were from Crohn's colitis patients (one patient with inactive disease and one with active disease). All CD patients received long-term azathioprine treatment.

Inflamed colonic specimens were also obtained from 27 patients suffering from UC. Fifteen of the specimens were obtained by surgical resection and 12 of the specimens were endoscopic biopsies. Samples were from 21 male and six female patients [34 (19–72) years]. UC patients received prednisolone alone or in combination with 5-aminosalicylic acid or azathioprine or received 5-aminosalicylic acid only.

Cytotoxicity experiments were performed using cells from surgically resected tissue. Endoscopic material was only utilized for mRNA analyses. All patients undergoing bowel resection received a single intravenous dose of antibiotics 2 h prior to surgery according to preoperative standard procedure. None of the control patients were or had been subjected to radio- or chemotherapy, long-standing antibiotic or steroid treatment. The ethics committee of the Medical and Odontology Faculty of Umeå University Hospital, Umeå, approved this study and the patients gave their informed consent.

Antibodies.  The following mouse monoclonal antibodies were used: antiepithelial antigen monoclonal antibody BerEP4, anti-CD45 monoclonal antibodies, a mixture of clones 2B11 and PD7/26, all immunoglobulin (Ig)G1 (Dakopatts, Glostrup, Denmark); anti-CD3 monoclonal antibody OKT3, IgG2b (American Tissue Culture Collection, Rockville, MD, USA); the inhibitory anti-Fas monoclonal antibody ZB4, IgG1 (MBL/Nordic Biosite, Täby, Sweden); anti-FasL monoclonal antibodies G247-4 and NOK-1, both IgG1 (Pharmingen, Heidelberg, Germany); antiperforin monoclonal antibody δG9, IgG2b, a kind gift from Prof E. R. Podack, Department of Microbiology and Immunology, University of Miami, Florida.

Paramagnetic beads used for cell fractionation were Dynabeads M-450 (Dynal, Oslo, Norway) coupled with anti-CD2, anti-CD4, anti-CD8 or goat antimouse IgG charged with anti-CD3 monoclonal antibody OKT3 or antiepithelial antigen monoclonal antibody BerEP4.

Isolation of lymphocytes.  IELs and LPLs were isolated from fresh, resected intestinal specimens and from endoscopic biopsies as previously described [9, 12, 17, 24]. In all cases, contaminating epithelial cells (ECs) were removed from the IEL and LPL fractions by treatment with monoclonal antibody BerEP4-charged magnetic beads.

T cells and subpopulations thereof were obtained by positive selection of IELs and LPLs binding to magnetic beads charged with monoclonal antibody specific for CD2, CD3, CD4 or CD8 as described [8–10]. The isolation procedure was performed at 4 °C, and positively selected cells were frozen within 1 h after exposure to monoclonal antibody.

Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll–Isopaque density gradient centrifugation.

Cell lines.  The murine mastocytoma cell line P815, the human erythroleukaemia cell line K562, and the human T cell lines Jurkat and MOLT-4 were all grown in RPMI1640 containing 5% fetal calf serum (FCS) and antibiotics.

Cytotoxicity assays.  Anti-CD3-mediated redirected cytotoxicity was measured in a 4 h 51Cr-release assay as previously described [8, 12]. Briefly, freshly isolated IELs and LPLs were pretreated with anti-CD3 monoclonal antibody or sham treated for 1 h at room temperature, and thereafter mixed with Na2[51Cr]O4-labelled P815 cells, Jurkat cells or K562 cells in a 4 h assay. Different effector : target (E : T) cell ratios were set up in triplicates, and radioactivity was measured in both the supernatant and the cell pellet. The proportion of released and cell-bound radioactivity was calculated for each tube and expressed as mean percentage 51Cr-release of triplicates. Spontaneous 51Cr-release was estimated in parallel tubes in which effector cells were replaced with the corresponding number of MOLT-4 cells.

The spontaneous 51Cr-release was <10% in all three assays. Results are presented as percentage of specific lysis and as lytic units (LUs). Specific lysis was calculated as mean percentage 51Cr-release at a particular E : T cell ratio subtracted with mean percentage 51Cr-release from target cells incubated with MOLT-4 cells at the same ratio. The number of LU(20%)/106 cells was calculated from the number of effector cells needed to lyse 20% of the target cells: LU = {106/[E/T(20%)]} × T, where E : T(20%) is the E : T ratio needed to get 20% specific 51Cr-release and T is the number of target cells [12, 25].

Ca2+-dependent cytotoxicity was blocked by addition of ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)/Mg2+, and Fas-/FasL-mediated cytotoxicity was blocked by addition of anti-FasL monoclonal antibody NOK-1 or anti-Fas monoclonal antibody as described [12]. The reagents were added at the start of incubation and were present throughout.

Immunoflow cytometry.  Perforin and FasL-expressing cells were stained as described [12]. Briefly, cells were fixed with paraformaldehyde and permeabilized with saponin, incubated with monoclonal antibody followed by fluorescein isothiocyanate-conjugated secondary antibody. Finally, 10,000 cells were analysed on a fluorescence-activated cell scanner (Becton Dickinson, Montain View, CA, USA) without light-scatter gate using the cellquest software program. Cells incubated with isotype and concentration-matched irrelevant monoclonal antibody served as negative controls.

Immunomorphometry.  Fresh tissue was rinsed in cold phosphate-buffered saline, snap frozen in isopentane precooled in liquid nitrogen and stored at −80 °C. Cells expressing perforin (1 : 2000-fold dilution of ascites fluid), FasL (clone G247-4, 2 µg/ml) and CD45 (4.5 μg/ml) were visualized using the polyclonal Mirror Image Complementary Antibodies (polyMICA) detection system (The Binding Site, Birmingham, UK) as described [9, 12]. The sections were developed using 0.05% 3,3′-diaminobenzidine tetrahydrochloride and 0.03% H2O2 and counter stained with methyl green. Sections incubated with isotype and concentration-matched irrelevant monoclonal antibody served as negative controls. Morphometry analyses were performed on immunohistochemically stained tissue sections using a ×40 objective and the Leica Q500MC computer image analysis system. Frequencies of marker-expressing cells in colonic LP outside follicles and aggregates as well as in aggregates of UC were counted according to Weibel and colleagues as described [12, 17, 26]. Eight to 15 randomly chosen ocular fields were counted. Results are given as percentage marker-positive cells of all LP cells and as

image

Frequencies of marker-expressing IELs were determined by counting the number of positive IELs through the number of ECs in 12–15 randomly chosen ocular fields [9, 12]. Initial counting gives the frequency of marker-positive cells/1000 ECs. Results are thereafter calculated as percentage marker-positive cells of all cells within the epithelium, i.e. ECs plus CD45+ intraepithelial cells as well as percentage marker-positive cells of IELs estimated as CD45+ intraepithelial cells.

RNA extraction and reverse transcription-polymerase chain reaction.  Total RNA was extracted from freshly isolated intestinal T cells and subsets thereof as described [8, 9, 12]. Analyses for perforin, GrB and FasL mRNAs were performed using the primers and protocols described in Melgar et al. [12]. To allow a semiquantitative comparison, each sample was also analysed for mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) (P-E Biosystems, Norwalk, CT, USA). The same concentration of GAPDH mRNA was used per RT-PCR reaction. A pool of RNA from anti-CD3-stimulated PBMCs served as positive control in all RT-PCR assays. PCR products were analysed by electrophoresis in 2% agarose gel and visualized by ethidium bromide staining.

Statistical analysis.  Mann–Whitney's ranking test was used for statistical analysis of differences in LUs between samples. Student's t-test was used to compare frequencies of cytotoxic markers. Two-tailed analyses were used throughout. A P-value <0.05 was regarded as statistically significant.

Results

Normal ileal and colonic mucosa contain lymphocytes expressing perforin and FasL

The frequencies of IELs and LPLs expressing perforin and FasL in normal intestine were estimated by morphometry analyses of immunohistochemically stained sections of ileum and colon. Perforin-positive and FasL-positive cells were found intraepithelially and in the LP of both ileum and colon. Intraepithelially, they were situated both at the top of the villi or the free luminal surface and in the cryptal area. No positive cells were detected in the follicles. The staining was mostly cytoplasmic or granular. Membrane or peripheral cytoplasmic staining was only seen on a few FasL+ cells. Table 1 summarizes the results. FasL+ cells were more frequent than perforin+ cells constituting 12 and 28% of the CD45+ cells in LP in ileum and colon, respectively, and about 4% of IELs at both locations. Perforin-positive cells constituted about 4% of the CD45+ cells in LP of both ileum and colon but were more abundant among ileal IELs than among colonic IELs (2.6 and 1.1%, respectively).

Table 1.  Frequencies of perforin and FasL-expressing lymphocytes in normal human ileum and colon as determined by immunomorphometry
 Marker-positive cells in lamina propriaMarker-positive cells in epithelium
Cytotoxic protein% of all cells*% of CD45+ cellsn% of all cells§% of CD45+ cellsn
  • *Frequency of marker-positive cells as determined by morphometry according to Weibel (26). Values are given as mean ± 1 SD.

  • Calculated as: inline image

  • n = number of samples analysed.

  • §

    Frequency of marker-positive cells of all cells in the epithelium (epithelial cells plus intraepithelial lymphocytes).

  • Calculated as: inline image

Ileum
 Perforin0.7 ± 0.43.9 ± 2.630.4 ± 0.12.6 ± 0.23
 FasL2.4 ± 1.211.8 ± 4.530.6 ± 0.14.0 ± 0.53
Colon
 Perforin0.7 ± 0.53.8 ± 3.150.1 ± 0.11.1 ± 1.36
 FasL5.0 ± 1.727.8 ± 9.750.3 ± 0.24.4 ± 3.66

Frequencies of perforin-expressing cells are increased in LP of inflamed intestine in both UC and CD patients whereas the frequency of FasL expressing cells is increased only in UC patients

CD ileum and UC colon were subsequently stained for perforin- and FasL-expressing cells. As was the case in normal intestine, perforin-positive and FasL-positive cells were detected both in the epithelium and in the LP, and the staining was mostly intracellular (Fig. 1A–D). The frequencies of perforin-positive and FasL-positive cells in LP are shown in Fig. 2. Because the colonic mucosa of UC patients contains two separate compartments, i.e. the basal lymphoid aggregates and LP outside the aggregates [17], these compartments were counted separately (Fig. 2A,B).

Figure 1.

Immunohistochemistry staining for perforin and FasL-expressing cells in inflamed intestinal mucosa. (A) Colonic mucosa of an ulcerative colitis (UC) patient stained with anti-perforin monoclonal antibody δG9. Several perforin-positive cells are seen in lamina propria. One perforin-positive cell is also seen inside a blood vessel. (B) Ileal mucosa of a Crohn's disease (CD) patient stained with anti-perforin monoclonal antibody δG9. Two perforin-positive cells are seen in lamina propria and one within the epithelium (arrowhead) (C) Colonic mucosa of an UC patient stained with anti-FasL monoclonal antibody G247-4. Numerous FasL-positive cells are seen in lamina propria while only few FasL-positive cells are seen in the basal lymphoid aggregate. The latter are all located in the outer rim of the aggregate. (D) Ileal mucosa of a CD patient stained with anti-FasL monoclonal antibody G247-4. Several FasL-positive cells are seen in lamina propria of a villus. One FasL-positive IEL is also seen (arrowhead). A, basal lymphoid aggregate; CR, crypt; E, epithelium; LP, lamina propria; V, blood vessel. Original magnification: ×220 in (A), (B), (D) and insert in (C), and ×55 in (C).

Figure 2.

Frequencies of perforin-positive (A) and FasL-positive (B) cells in normal and inflamed ileum and colon as determined by immunomorphometry. Inflamed ileum of Crohn's disease (CD) patients and inflamed colon of ulcerative colitis (UC) patients were compared to normal ileum and colon of controls (Ctrs), respectively. The lamina propria (LP) outside follicles was counted (Ctr, CD, UC-LP). The basal lymphoid aggregates found in UC but not in control colon were counted separately (UC-Agg). Filled circles indicate the percentage of stained cells of all cells in individual samples and the horizontal bars indicate mean values. Statistically significant differences between inflamed and the corresponding normal intestinal mucosa are given.

In the LP of inflamed ileum of CD patients, the frequency of perforin-positive cells was significantly increased as compared to controls while the frequency of FasL-positive cells remained at the same level as in LP of control ileum (Fig. 2A,B). In the LP of the inflamed colon of UC patients, the frequency of perforin-positive cells outside the aggregates was significantly increased compared to controls, with means of 2.6 versus 0.7% in LP of UC and control colon, respectively. However, the frequencies of perforin-positive cells were the same in the aggregates (mean: 0.7%) as in LP of controls. A similar pattern was seen for FasL-positive cells in UC (Figs. 1C and 2A,B). The mean values for FasL-positive cells were 15.4% in LP outside the aggregates and 2.9% in the aggregates of UC and 5.0% in LP of control colon. Thus, the frequencies of both perforin and FasL-expressing cells were significantly increased in LP outside the aggregates in UC compared to LP in control colon (Fig. 2A,B). As approximately 25% of the cells in the LP outside the aggregates in UC are CD45+ cells [17], this implies that approximately 10 and 60% of the CD45+ cells express perforin and FasL, respectively.

Immunoflow cytometry analyses performed on freshly isolated colonic LPLs of UC patients revealed that 5.5 ± 0.7% (n = 2) and 9.3 ± 5.0% (n = 3) of the cells were positive for perforin and FasL, respectively. The difference in frequencies between immunoflow cytometry and immunomorphometry analyses is mainly explained by the fact that LPLs from UC is a mixture of lymphocytes from aggregates, which can occupy up to 50% of the LP [17] and contains low frequencies of perforin-positive and FasL-positive cells, and from LP outside the aggregates with higher frequencies of positive cells.

Perforin, GrB and FasL mRNAs are all expressed in T cells of inflamed intestine

Expression of perforin, GrB and FasL mRNAs was determined in the total T-cell population (CD3+ cells) retrieved by positive selection from freshly isolated LPLs and IELs of normal and inflamed ileum and colon. The frequency of perforin mRNA expressing CD3+ LPL samples of normal colon was low. All other T-cell populations expressed the three mRNA species at high frequency [Table 2].

Table 2.  Expression of mRNAs for proteins involved in cytotoxicity in intraepithelial and lamina propria T lymphocytes of normal and inflamed ileum and colon
  Cytotoxic protein*
TissueSource of T lymphocytesPerforinGranzyme BFas-Ligand
  • *

    mRNA for the indicated protein was determined by reverse transcriptase-polymerase chain reaction. mRNA for CD45 was detected in all samples.

  • CD3+ cells were retrieved by treatment of freshly isolated intraepithelial lymphocytes and lamina propria lymphocytes with anti-CD3 monoclonal antibody-coated magnetic beads.

  • Results are expressed as number of positive samples/number of samples analysed.

Normal ileumEpithelium4/55/55/5
 Lamina propria5/54/43/4
Crohn's disease ileumEpithelium4/44/43/4
 Lamina propria4/44/53/4
Normal colonEpithelium4/63/55/5
 Lamina propria1/55/54/5
Ulcerative colitis colonEpithelium7/99/104/6
 Lamina propria7/98/95/6

Colonic T cell subsets were also analysed. CD8+CD4 and CD2+ DN LPLs were the main sources of all three mRNA species in UC colon [Table 3]. Perforin and GrB mRNAs were expressed in all three T-cell subset analysed (CD4+, CD8+CD4–, and CD2+ DN LPLs) in UC. Expression levels in controls appear to be lower particularly in CD4+ LPLs [Table 3].

Table 3.  Expression of mRNAs for proteins involved in cytotoxicity in lamina propria T-cell subsets of the inflamed colon of ulcerative colitis (UC) patients and the normal colon of controls (Ctrs)
 Perforin*Granzyme BFas-Ligand
T-cell subsetUCCtrUCCtrUCCtr
  • ND, not done.

  • *

    mRNA for the indicated protein was determined by reverse transcriptase-polymerase chain reaction. mRNA for CD45 was detected in all samples.

  • Freshly isolated lamina propria lymphocytes from UC and control (Ctr) colon were subjected to sequential positive selection by treatment with magnetic beads coupled with anti-CD4 monoclonal antibody (CD4+), followed by beads with anti-CD8 monoclonal antibody (CD8+CD4) and finally beads with anti-CD2 monoclonal antibody (CD2+ DN). The CD2+ DN fraction contains γβ T cells that are present in lamina propria of UC but not normal colon (17).

  • Results are expressed as number of positive samples/number of samples analysed.

CD4+4/41/43/42/40/41/4
CD8+ CD43/42/44/44/43/42/4
CD2+ DN4/4ND4/4ND3/4ND

Ileal but not colonic IELs and LPLs can exert TCR-/CD3-mediated cytotoxicity

The cytotoxic potential of IELs and LPLs from inflamed and normal ileum and colon was analysed in a redirected cytotoxicity assay using the FcγR-expressing murine mastocytoma cell line P815 as target cells. Table 4 summarizes the results.

Table 4.  Cytotoxic capacities of intraepithelial (IELs) and lamina propria lymphocytes (LPLs) isolated from normal and inflamed human intestine
 Cytotoxic capacity*[LU(20%)/106 cells]
Effector cellsAnti-CD3-mediated redirected cytotoxicitynPSpontaneous anti-CD3-independent cytotoxicity§n
  • IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte; LU, lytic unit; CD, Crohn's disease; ND, not determined.

  • *

    Cytotoxic capacity was estimated as LUs(20%)/106 IEL and LPL calculated from the number of effector cells needed to lyse 20% of the target cells.

  • Freshly isolated IELs and LPLs were pretreated with anti-CD3 monoclonal antibody and cytotoxicity measured in a 4 hr

  • 51

    Cr-release assay using P815 cells as targets.

  • P = P-value obtained in statistical analyses comparing IELs and LPLs in normal ileum (•) and colon (•) with the corresponding cell type in inflamed ileum and colon, respectively, in anti-CD3-mediated redirected cytotoxicity.

  • §

    Freshly isolated IELs and LPLs were mixed with

  • 51

    Cr-labelled Jurkat cells and

  • 51

    Cr-release measured after 4 hr incubation at 37 °C.

  • Median LU(20%)/106 cells. Values within parenthesis give the range.

  • ††

    IELs were from five colon samples of control patients and two samples of apparently normal colon of CD patients. LPLs were from seven colon samples of control patients and three samples of apparently normal colon of CD patients.

  • ‡‡

    Ileal samples were from CD patients and colonic samples were from UC patients.

Normal intestine
 Ileal IELs6.4 (0–22.9)7ND 
 Ileal LPLs7.7 (0–28.2)8ND 
 Colonic IELs††1.9 (0.8–4.9)716.3 (9.6, 23.1)2
 Colonic LPLs††1.7 (0.4–6.1)104.7 (1.5–11.3)3
Inflamed intestine‡‡
 CD ileal IELs27.9 (1.6–32.1)30.2ND 
 CD ileal LPLs20 (4.1–44.9)40.18.0 (6.1, 9.9)2
 UC colonic IELs2.3 (0–4.4)51.08.3 (3.3–14.6)4
 UC colonic LPLs2.5 (2.3–2.7)30.510.0 (2.9–11.6)3

The cytotoxicity detected in the redirected cytotoxicity assay using IELs and LPLs of control ileal samples as effector cells was variable. Half the number of the samples contained cells that exhibited significant cytotoxicity the other half did not (Fig. 3A and Table 4). This is in line with the large variation in frequencies of perforin-positive cells in control ileum [Table 1]. In contrast, ileal IELs and LPLs of CD patients exhibited strong cytotoxic activity in all cases but one (Fig. 3C,D and Table 4). On average, this cytotoxicity tended to be increased compared to IELs and LPLs of controls [Table 4]. These results are consistent with the increase of perforin-positive cells in ileal LP of CD patients (Fig. 2A). Colonic IELs from the same CD patient exhibited only marginal cytotoxicity in the redirected assay (Fig. 3C). No spontaneous cytotoxicity against P815 cells was induced by inflammation in either ileum or colon (Fig 3C) (n = 7). The anti-CD3-dependent cytotoxicity of ileal lymphocytes in CD patients was inhibited by EGTA. The inhibition was approximately 65% and remained at that level over a range of E : T cell ratios (Fig. 3D and data not shown). This value is comparable to the results obtained by effector cells of normal small intestine, i.e. 60% in IELs and 50% in LPLs of one ileal sample at an E : T ratio of 100 (data not shown) and approximately 75% for jejunal IELs and LPLs [12].

Figure 3.

Ileal but not colonic T lymphocytes of controls and Crohn's disease (CD) patients exhibit a significant Ca2+-dependent T-cell receptor/CD3-mediated cytotoxicity. Freshly isolated intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were treated with anti-CD3 monoclonal antibodies or left untreated and analysed for cytotoxicity against 51Cr-labelled P815 cells at different E : T cells ratios. Cytotoxicity is given as percentage specific lysis (mean + 1 SD) calculated as mean 51Cr-release of triplicates at the different E : T cells ratios and corrected for the corresponding spontaneous 51Cr-release from target cells. (A) IELs of one ileal control sample. Black bars indicate anti-CD3-treated cells and gray bars untreated cells. (B) IELs of one colonic control sample. Black bars indicate anti-CD3-treated cells and gray bars untreated cells. (C) IELs of one ileal and one colon sample from the same CD patient. Black bars indicate anti-CD3-treated ileal IELs, striped bars indicate untreated ileal IELs, grey bars indicate anti-CD3-treated colonic IELs, and hatched bars indicate untreated colonic IELs. (D) LPLs of one ileal CD sample. Black bars indicate anti-CD3-treated cells and striped bars anti-CD3-treated cells incubated in the presence of EGTA.

IELs and LPLs of normal colonic samples exhibited no, or very low, activity in the redirected cytotoxicity assay (Fig. 3b and Table 4). Moreover, inflammation did not induce cytotoxicity in colonic lymphocytes of UC patients [Table 4]. Similarly, no significant activity was monitored when colonic IELs and LPLs of two CD patients with colitis were used as effector cells in the same assay [median LU(20%)/106 cells values were 3.0 and 3.2 for IELs and LPLs, respectively]. Thus, inflammation in ileum of CD patients but not colon of UC or CD patients enhanced T-cell cytotoxic activity.

Lymphocytes of inflamed intestine exhibit spontaneous Fas-/FasL-mediated cytotoxicity against Jurkat T cells

We have recently reported that jejunal T lymphocytes spontaneously can act as effector cells in a TCR-/CD3-independent manner using the Fas/FasL pathway [12]. To investigate whether intestinal T cells from inflamed small and large intestine retain or increase this ability, freshly isolated IELs and LPLs were assayed for cytotoxicity against the Fas-expressing Jurkat cells in a 4 h 51Cr-release assay. The results are summarized in Table 4. All UC samples, except one sample of IELs, exhibited significant spontaneous killing of Jurkat cells with LU(20%)/106 cells values >5, i.e. the same degree of killing as exhibited by IELs and LPLs of control colon [Table 4]. Addition of anti-FasL monoclonal antibody or anti-Fas monoclonal antibody inhibited the cytotoxicity (37–39% inhibition at an E : T ratio of 33, n = 2). CD8+ LPLs seem to be important effector cells as depletion of CD8+ cells reduced the cytotoxicity to approximately 50% of that executed by the total LPL population (n = 2). This result is consistent with the presence of FasL mRNA in CD8+ LPLs but not in CD4+ LPLs of UC [Table 3]. The cytolytic activity of LPLs from inflamed ileum of CD patients was similar to that of jejunal and colonic lymphocytes (Table 4 and [12]). Anti-CD3 pretreatment did not enhance the cytotoxic activity against Jurkat cells of intestinal lymphocytes in either UC or CD patients (n = 8).

Lymphocytes in normal and inflamed human colon do not have NK cell activity

The NK cell activity of freshly isolated IELs and LPLs from inflamed UC and control colon was assayed using K562 cells as targets. Neither lymphocyte from the inflamed colonic mucosa nor from control colon was cytotoxic against K562 cells. Treatment with anti-CD3 monoclonal antibody did not induce cytotoxicity against K562 cells in lymphocytes either from UC or control colon (n = 3 and 5, respectively).

Discussion

This study shows that: (1) ileal mucosa harbours effector cells for TCR-/CD3-mediated cytotoxicity. The frequency of these cells was increased in inflammation; (2) lymphocytes in colonic mucosa do not function as effector cells in TCR-/CD3-mediated cytotoxicity, although the colonic T cells express perforin and GrB. Cytolytic activity via this pathway is not induced by inflammation, although the frequency of perforin-expressing lymphocytes increased significantly in UC; (3) both ileal and colonic mucosa harbours effector cells in the TCR-/CD3-independent, Fas-/FasL-mediated cytotoxicity. This cytotoxicity was retained but not increased in either inflamed CD ileum or in inflamed colon of UC patients, and 4) colonic mucosa does not harbour NK cells, and NK cell activity is not induced by inflammation at this site.

The increased cytotoxicity in CD ileum is in line with previous studies reporting increased frequency of perforin mRNA-expressing T cells and elevated cytotoxicity in the ileal mucosa of CD patients [27, 28]. The cytotoxicity was inhibited by EGTA, a well-known blocking agent of the perforin/granzyme exocytosis pathway, and T cells expressed mRNA for perforin and GrB both in normal and inflamed ileum. This is in accordance with our study [12] showing anti-CD3-dependent redirected cytotoxicity by jejunal IEL in which CD8+αβ T cells constitute the effector cells. Taken together, these results suggest that ‘classical’ CTLs are the effector cells in the TCR-/CD3-mediated cytotoxicity also in ileum and that the inflammatory process in CD ileum involves T-cell activation, which generates additional CTLs. Enhanced CTL differentiation is compatible with the Th1 cytokine profile in CD [19, 28]. The observation that spontaneous cytotoxicity against epithelial tumour cells is enhanced in small intestinal IELs of CD patients is compatible with our results even though the cytolytic mechanism was not investigated [29].

Inflammation in colon did not generate effector cells in the anti-CD3-dependent redirected cytotoxicity assay, neither in UC patients nor in the two CD patients with colitis. This was in spite of the fact that the frequency of colonic T cell samples expressing perforin mRNA was higher in UC than in controls. Analyses of T-cell subsets suggest that this increase reflects two parallel events, namely induction of perforin expression in CD4+ cells and relocation of perforin-expressing γδ T cells (CD4CD8 double negative CD2+ cells) to LP. Increased perforin mRNA expression in colonic LPL of UC has been noted once before [27].

A role for γδ T cells in UC was indicated by our previous study demonstrating that Vδ1+γδ T cells redistribute to LP and that activated γδ T cells with downregulated surface TCR expression are present in the basal lymphoid aggregates of the inflamed mucosa in UC [17]. In the present study, mRNAs for perforin and GrB were demonstrated in these cells. The failure to trigger these cells to cytotoxicity in vitro may at least partly be explained by the low surface expression of the TCR/CD3 complex [17]. Alternatively, costimulatory signals may also be required. Established Vδ1+γδ T cell clones of intestinal origin are cytotoxic to cells expressing the stress-induced major histocompatibility class I-like molecules MICA/MICB [30, 31]. In vitro stimulation of jejunal IEL with IL-15 induced expression of the NKG2D, the receptor for MICA/MICB and capacity to kill in an anti-NKG2D-mediated redirected cytotoxicity assay [32]. NKG2D was shown to function as a costimulatory molecule for murine intraepithelial γδ T cells in cytotoxicity against tumour cells expressing Rae-1, the corresponding molecule to MICA in mice [33]. T cells expressing NK receptors, so-called NKT cells, have been demonstrated both in human and murine small intestine [34, 35] and also in murine large intestine [36]. NK receptors have been demonstrated both on αβ- and γδ T cells. We hypothesize that the perforin-expressing cells in human colon may be NKT cells and that these cells may require dual stimulation through the TCR/CD3 complex and an NK-receptor to be triggered in functional in vitro assays.

Poor NK cell activity seems to be a feature of lymphocytes in the large intestine. No killing of the NK cell target K562 could be detected in IELs or LPLs of normal or inflamed colon. These results are in line with previous studies on NK cell activity of small intestinal IELs. Cerf-Bensussan et al. [37] showed that freshly isolated IELs do not have NK cell activity, and Léon et al. [38] demonstrated that a NK cell-like IEL subset gained NK cell activity only after in vitro stimulation with IL-2. A corresponding NK cell-like population has not yet been identified in human colon. Given the very low IL-2 production in the inflamed colonic mucosa of UC patients [10], activation of such NK cells would not be expected.

We have recently demonstrated the presence of FasL-positive cells in the upper small intestinal mucosa and shown that the small intestinal lymphocytes exert cytotoxicity via Fas/FasL interaction without requirement of ex vivo stimulation [12]. When ileum of CD patients was analysed, we found that inflammation did not influence the Fas-/FasL-mediated cytotoxicity, the frequency of FasL-expressing LPLs or the FasL mRNA expression by T cells. In contrast, the inflamed colon of UC patients showed a significant increase of FasL-expressing cells in LP when only the area outside the aggregates was considered. However, FasL+ cells were few in the aggregates. Thus, the frequency in the total immune cell population is difficult to predict, as it depends on the proportion of LP occupied by aggregates. We have earlier demonstrated that basal lymphoid aggregates can occupy between 7 and 45% of the colonic LP in UC [17]. Thus, our demonstration of unchanged Fas-/FasL-mediated cytotoxicity in UC can be explained on this basis. One previous study reported increased frequency of FasL mRNA-expressing cells in colonic LP of UC [39]. The increased FasL expression by LPL may reflect an attempt for downregulation of T-cell responses in UC through activation-induced cell death (AICD). It is noteworthy that FasL mRNA was not expressed in CD4+ cells in UC. Intracellular FasL staining was found in UC and control colon suggesting that some FasL may be secreted. Soluble FasL has been ascribed inhibitory actions on Fas-/FasL-induced apoptosis [40] and chemotactic effects on neutrophil granulocytes [41]. This agrees with two phenomena described in UC, namely that the frequency of apoptotic cells is decreased in UC colonic mucosa [42] and that infiltration of neutrophils and formation of abscesses is commonly seen.

A weak spontaneous cytotoxic activity by small intestinal IEL against colon carcinoma cell lines has been reported [43]. Whether this phenomenon is related to the cytotoxicity described here remains to be elucidated.

In summary, chronic inflammation leads to increased perforin-mediated cytotoxicity in the small intestine and increased FasL expression in colon. Thus, it seems that already ongoing normal processes are enhanced by the inflammation. Increased antigen burden in the intestinal tissue might activate the local T cells to enhance their normal responses, e.g. defense against intracellular pathogens in small intestine and homeostasis through AICD. Our preliminary data suggest that CD8+ cells are the main players in both responses at both locations.

Acknowledgments

The skillful technical assistance of Elisabeth Granström and Marianne Sjöstedt is gratefully acknowledged. This work was supported by grants from the Swedish Science Research Council-NT (M.-L. H.), the Swedish Research Council-M (Å. D.), the Swedish Cancer Society (S. H.), Bengt Ihre's Fund (Å. D.) and the Medical Faculty Research Fund of Umeå University (Å. D.).

Ancillary