Human intestinal CD3+TCRαβ+CD8+ intraepithelial lymphocytes (IELs) are intimately associated with epithelial cells (ECs) through binding of CD103 to E-cadherin. How these two cell types functionally interact is largely unknown. IEL-EC cross talk was determined using HT-29 cells as the model EC and IL-8 as the readout. IL-8 was derived from both cell types and synergistically increased when the cells were combined. This synergistic effect required active transcription by both IELs and HT-29 cells. Cell contact was required as shown by the loss of the synergistic increase in IL-8 when the two cell types were separated by Transwells. Specifically, IL-8 release required the binding of CD2 on the IELs to CD58 on the HT-29 cells. The association of the CD3/TCR complex with major histocompatibility antigen class I antigens was not involved. Antibody neutralization of tumor necrosis factor-α (TNF-α), but not interferon-γ (IFN-γ), resulted in increased IL-8 production by the coculture. Although both TNF-α and IFN-γ increased IL-8 synthesis and CD58 expression by the HT-29 cells, only IFN-γ reduced IL-8 production by IELs. IL-8 production by either cell type involved phosphorylation of p38 and JNK. In summary, the synergistic synthesis of IL-8 occurs when IELs are stimulated through the CD2 pathway by CD58 on HT-29 cells, resulting in TNF-α release that, in turn, augments IL-8 synthesis and CD58 expression by the HT-29 cells.
- intraepithelial lymphocytes
- epithelial cells
- tumor necrosis factor-α
human intestinal intraepithelial lymphocytes (IELs) are CD3+TCRαβ+CD8+ T cells located between epithelial cells (ECs). IELs possess many unique functional properties not described with circulating human peripheral blood lymphocytes or murine IELs (14). For example, they respond minimally to triggering of the CD3/T cell receptor (TCR) pathway, indicating a limited response to antigen (6). Yet they proliferate briskly through the alternate CD2 pathway (6). In addition, they are stimulated by IL-15 in the absence of CD3/TCR ligation, resulting in proliferation, lysis of colon cancer cells, and production of IL-2, suggesting that they respond to the local cytokine milieu rather than to cognate antigen (7).
The reason, if any, for selective CD2 responsiveness by IELs is unknown. CD2 binding to CD58 in humans and CD48 in rodents facilitates adhesion between T cells and antigen-presenting cells. The resulting intercellular membrane spacing is thought to be optimal for TCR recognition of a peptide Ag bound to major histocompatibility antigen (MHC) (26). However, TCR ligation induces little proliferation or IL-2 production by IELs (6). CD2 ligation also amplifies CD28 signaling and can reverse T cell anergy induced by B7 blockade (3, 4). IELs, however, have few CD28+ cells. CD2 signaling can induce fas-independent apoptosis of activated PB T cells (18). Whether IELs are susceptible to such apoptotic cell death process is unknown. CD2 stimulation can initiate a number of intracellular signaling events, including tyrosine phosphorylation (22) as well as activation of protein kinase C, mitogen-activated protein (MAP) kinase, protein tyrosine kinases, and activation of STAT1 and STAT4 proteins (2, 5, 11, 19). The mechanisms of CD2 signaling events in IELs have not been described.
Chemokines, especially IL-8, are produced by ECs, probably drawing IELs and polymorphonuclear neutrophils (PMNs) into the epithelium (8, 23, 24). Regulation of IL-8 synthesis differs for macrophages compared with ECs. For example, IL-4, IL-10, and TGF-β, which can downregulate IL-8 production by macrophages, have no effect on IL-8 release by ECs (10, 23). Instead, IL-8 produced by ECs is upregulated by TNF-α, IL-1, and IFN-γ and suppressed by IL-15 (10, 16, 23). IL-8 is also produced by peripheral blood CD4+ T cells following activation (12, 25), particularly through the CD2 receptor.
MAP kinases are a group of protein kinases that are activated in response to a variety of extracellular stimuli. They are composed of three subfamilies: c-Jun NH2-terminal protein kinase (JNK), extracellular signal-regulated kinases (ERK), and p38 MAP kinases. All three have been shown to be involved in IL-8 synthesis by ECs (15, 27).
Because of their intimate association, IELs are likely to have multiple reciprocal interactions with ECs. This may occur through cell contact and soluble factors. Cell contact is made when IELs adhere to EC through CD103 binding to E-cadherin (21). In addition, NKG2D on IELs initiates fas ligand (FL) production and FL-mediated cytotoxicity when contacting MHC class I chain-related gene A (MICA) and MICB on colon cancer cell lines (9). The main soluble factor shown to alter the IEL-EC interaction is IFN-γ, produced constitutively and with stimulation by IELs. IFN-γ, secreted by rat CD4+ IELs, upregulates the expression of MHC class I and II antigens on ECs and their production of nitric oxide (13). It also increases the permeability of the EC barrier and EC apoptosis (17, 28).
Celiac sprue is characterized by numerous IELs in the epithelium, which may be attracted by chemokines produced by the ECs. The gluten-induced IFN-γ production in the lamina propria may alter the IEL-EC interaction.
Human IELs and ECs each have unique features. Their interaction, which is largely unstudied in humans, is likely also to be novel. The present report utilizes the HT-29 cells, which are moderately well-differentiated cells derived from a colon adenocarcinoma, as a model for ECs. Although IL-8 is synthesized by both cell types, a synergistic increase occurs when IELs and ECs are combined. The mechanism of this interaction is dissected out.
Isolation and culture of lymphocytes.
IELs were separated from jejunal mucosa from otherwise healthy patients undergoing gastric bypass operations for morbid obesity after informed consent and permission from the Institutional Review Board at Robert Wood Johnson Medical School. Minced mucosa was treated in a shaking water bath (37°C) for 60 min with 1 mM dithiothreitol (DTT) diluted in RPMI medium containing 10% fetal calf serum, glutamine, and antibiotic-antimycotic solution (all from Sigma Aldrich, St. Louis, MO). The mucosa was then treated in a shaking 37°C water bath with 0.75 mM EDTA (Sigma Aldrich), washed every 45 min with calcium- and magnesium-free Hanks’ balanced salt solution (Biowhittaker, Walkersville, MD) for three cycles. All cells in the supernates were collected and separated by a 60–40% Percoll gradient (Pharmacia, Piscataway, NJ) with 100% Percoll containing 9 parts Percoll and 1 part 10×-phosphate-buffered saline. The IELs, which settled over the 40% solution, were collected. Any preparation containing less than 85% lymphocytes was discarded. After isolation, the viability of IELs was 100% by Trypan blue exclusion and remained over 95% after an 18-h culture in complete medium at 37°C. To remove the CD4+ IELs, cells were labeled with anti-CD4 antibody, washed, then coincubated with magnetic beads coated with goat anti-mouse IgG (Polysciences, Warrington, PA). The unattached CD8+ IELs were collected.
IELs (1 × 105/0.2 ml) were cultured alone or with HT-29 cells (4 × 104/0.2 ml) for 18 h in the presence of antibodies (5 μg/ml) directed against IL-1, IFN-γ, TNF-α, TGF-β, CD11a, CD40, CD54, CD58, CD154, MHC class I, or isotype IgG controls (all from R&D Systems, Minneapolis, MN). In other experiments, IFN-γ, TNF-α, anti-CD3 antibody (R&D Systems), or anti-T112 and anti-T113 (kind gift of Stuart Schlossman, Dana-Farber Institute, Boston, MA), were added at various dilutions. In some experiments, cells were treated for 30 min with metabolic inhibitors of p38 (SB203580) and ERK (PD98059) (both from Sigma-Aldrich), or JNK inhibitor II (SP600125, Calbiochem, La Jolla, CA). This was followed by washes before the 18-h incubation.
Measurement of TNF-α, IFN-γ, and IL-8 production.
IELs and HT-29 cells were cocultured and the supernates collected and tested for TNF-α, IFN-γ, and IL-8 by enzyme-linked immunosorbent assay (ELISA) (R&D Systems).
Single-color staining was accomplished with monoclonal antibody directed against CD58 followed by goat anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC) (Cappel, West Chester, PA). Double-color immunofluorescence began with CD45-phycoerythrin staining. Then the cells were permeabilized with Cytofix Cytoperm (Pharmingen, San Diego, CA) and exposed to biotin-labeled antibody directed against IFN-γ or TNF-α (R&D Systems) or rabbit antibodies against p38, ERK, or JNK (Invitrogen, Carlsbad, CA). This was followed by strepavidin conjugated to FITC or goat anti-rabbit IgG conjugated to FITC. Fluorescence was detected by a Beckman Coulter FC500 flow cytometer (Beckmann Coulter, Miami, FL) and expressed as relative fluorescence intensity or the fold increase in intensity of staining compared with an IgG-FITC control.
Statistical analysis was performed using the Mann-Whitney Wilcoxon rank sum test or the Student's t-test depending on the distribution of the data. For more than two data sets, analysis of variance was used with the Tukey's test to evaluate pairs within the group.
IL-8 production is synergistically increased in the IEL-EC coculture.
IEL-EC cross talk was determined by using HT-29 cells as the model EC and IL-8 as the readout. Both IELs and HT-29 cells produce IL-8 (Fig. 1). Cultures containing 2.5-fold more IELs than HT-29 cells resulted in equivalent amounts of IL-8. When the cells were combined, IL-8 production was significantly greater than an additive effect, indicating synergy. When either one of the cell types was treated with actinomycin D, their production of IL-8 was reduced. In addition, the synergistic increase was also lost, indicating that RNA synthesis by both IELs and ECs is required for the synergistic effect (Fig. 2). Removing the small CD4+ IEL subset made no difference in the synergistic effect, indicating that it was due to the interactions between CD8+ IELs and the ECs.
Synergistic increase in IL-8 synthesis in IEL-EC coculture is mediated through the CD2-CD58 interaction.
To determine whether cell contact is needed, IELs were cultured in an insert overlying a well to which HT-29 cells were attached (Fig. 3). IL-8 release increased only additively, not synergistically, and was less than the release induced when the cells were cultured together without an insert. This suggests that cell-cell interactions are necessary for the synergistic effect.
To define further the molecular nature of this interaction, IELs and HT-29 cells were cocultured with specific blocking antibodies (Fig. 4A). These experiments showed that the synergistic increase in IL-8 declined significantly when the CD2-CD58 interaction was blocked by anti-CD58 antibody, but not by antibodies against CD11a, CD154, or CD40. Anti-CD58 antibody had no effects on IL-8 production by IELs or HT29 cells when cultured alone (Fig. 4B) or when the two cell types were separated by Transwells (Fig. 3). Additionally, disarming the TCR-MHC class I pathway with anti-MHC class I antibody made no difference in the IL-8 release in cocultures, indicating that antigen recognition was not involved (Fig. 4). Other interactions, such as CD154-CD40 or CD11a-CD54, were also not involved according to antibody inhibition experiments (not shown).
TNF-α production by IELs increases with CD2 stimulation and with HT-29 cells.
Since CD58 mediates IL-8 production, the reactivity of its ligand, CD2, was investigated. Previous studies have documented that when IELs are stimulated by the monoclonal antibodies, T112 and T113, which activate the CD2 receptor, their proliferation and IL-2 production greatly exceed those seen with antibody triggering the CD3/TCRαβ receptor (6). Experiments were conducted to determine whether this distinction holds true for IFN-γ and TNF-α release (Fig. 5). Both cytokines were upregulated to a greater extent with triggering of the CD2 rather than the CD3/TCRαβ receptor.
The possibility that HT-29 cells augment TNF-α or IFN-γ production by IELs was determined by ELISA (Fig. 6). HT-29 cells slightly increased TNF-α, but not IFN-γ, release. When the cytokines were detected by intracytoplasmic staining (Fig. 7), similar results were obtained. In both cases, the increase in TNF-α was too low to definitively determine whether it could be inhibited by anti-CD58 antibody.
TNF-α and IFN-γ stimulate IL-8 release and CD58 expression by HT-29 cells whereas only IFN-γ decreases IL-8 release by IELs.
The effects of TNF-α and IFN-γ on IL-8 production by HT-29 cells or IELs were determined by ELISA (Fig. 8). IL-8 released by HT-29 cells increased to the same extent with either TNF-α or IFN-γ supplementation. IL-8 release by IELs, in contrast, was unchanged by TNF-α and declined with IFN-γ.
The effects of TNF-α and IFN-γ on CD58 expression on HT-29 cells were determined by immunofluorescence (Fig. 9). Both cytokines increased expression of this marker to the same extent.
To identify soluble mediators that may be required for IL-8 production, antibodies neutralizing IL-1, TGF-β, TNF-α, or IFN-γ were added to cultures containing both IELs and/or HT-29 cells (Fig. 4). Anti-IL-1, -IFN-γ, or -TGF-β antibodies had no effects. Although anti-TNF-α antibody did not alter IL-8 release by each cell type cultured separately, it decreased IL-8 production by IELs and HT-29 cell combined. This is likely to be due to neutralization of IEL-derived TNF-α which has an upregulatory effect on IL-8 from HT-29 cells (Fig. 10). Anti-IFN-γ antibody, in contrast, increased IL-8 production by IELs alone (Fig. 4), consistent with the inhibitory action of IFN-γ on their IL-8 release (Fig. 8). Similarly, anti-IFN-γ antibody had either no effect (n = 4) or increased (n = 6) IL-8 production by the coculture.
Phosphorylation of the MAP kinases does not change whether IELs and HT-29 cells are cultured alone or combined.
Since MAP kinases contribute to IL-8 production by intestinal ECs (15, 27), experiments were conducted to determine whether either HT-29 cells or IELs utilize these pathways. To do this, IELs were cultured with or without HT-29 cells for 4 or 18 h and phosphorylation of MAP kinases determined by immunofluorescence, double-staining with CD45 to identify the IELs. There were no differences in the levels of phosphorylation of p38, ERK, or JNK whether cells were cultured alone or combined (Fig. 11). However, when the cells, either alone or combined, were treated with inhibitors of the three MAP kinases, inhibitors of p38 and JNK, but not ERK, partially reduced IL-8 production (Fig. 12).
IELs and ECs each produce IL-8 constitutively (Fig. 10). There is a synergistic increase when both cell types are combined, an effect that requires active transcription. This synergy depends on the CD2/CD58, but not the CD3/TCR-MHC class I, interaction. TNF-α and IFN-γ are produced by IELs with stimulation of the CD2 receptors by activating antibody. Only TNF-α release is augmented with addition of HT-29 cells to IELs. Both cytokines upregulate IL-8 production and CD58 expression by the ECs. IFN-γ, however, has an inhibitory effect on IL-8 from IELs. Therefore, neutralizing TNF-α reduces IL-8 production by the IEL-EC coculture whereas neutralizing IFN-γ either had no effect or increased IL-8. The coculture did not change MAP kinase phosphorylation although IL-8 release partially involved phosphorylation of p38 and JNK.
No studies have examined IL-8 synthesis by IELs. IL-8 production by peripheral blood T cells is a property of the CD4+ T cell subset and occurs only following activation. Interestingly, this occurred with stimulation of the CD2 receptor in one report (12, 25). This differs from IELs, which are CD8+ T cells and secrete IL-8 constitutively, although they also utilize the CD2 receptor in combination with HT-29 cells. Both p38 and ERK have been shown to be involved in IL-8 production by activated T cells (12). IELs along with HT29 cells produce IL-8 in conjunction with p38 and JNK although the levels of phosphorylated proteins do not change during interaction with HT-29 cells.
IELs produce both TNF-α and IFN-γ with CD2 stimulation. Both cytokines had similar effects on the HT-29 cells, causing an increase in both IL-8 production (1) and CD58 expression. When TNF-α and IFN-γ are combined, however, apoptosis of HT-29 cells has been reported to occur (1). In addition, IFN-γ increases the susceptibility of HT-29 cells to spontaneous cytotoxicity by IELs (9). However, at the IEL-to-HT-29 cell ratio of 2.5:1 established in these studies, there was little apoptosis or necrosis occurring in the coculture as demonstrated by annexin and propidium iodide staining (not shown).
IFN-γ, but not TNF-α, inhibits IL-8 synthesis by the IELs. This could be due to the promotion of activation-induced cell death which has been reported to occur with IFN-γ (20). However, constitutive production of IFN-γ is associated with little apoptosis of IELs.
IEL and HT29 cross-talk involves the CD2/CD58 interaction. Similarly, proliferation (6) and production of TNF-α and IFN-γ are upregulated through CD2 but not through the CD3/TCR complex. It would be of interest to determine whether other IEL-EC interactions depend on CD2 activation.
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