Inhibitory effects of transforming growth factor-beta (TGF-β) on certain functions of intraepithelial lymphocytes

Abstract

Human intraepithelial lymphocytes (IEL), CD8⁺ lymphocytes located between epithelial cells, are likely to be influenced by the immunosuppressive cytokine, TGF-β, secreted by epithelial cells. This study evaluates the effects of TGF-β on IEL functions. IEL were derived from proximal jejunum of patients undergoing gastric bypass operations for morbid obesity. Proliferation was determined by ³H-thymidine incorporation; IL-2 production, by ELISA; expression of IL-2 receptor, CD2, HML1, CD16, and CD56, by immunofluorescence; binding, by adherence of radiolabelled cells; and cytotoxicity by ⁵¹Cr-release assay. TGF-β (≥ 1 ng/ml) inhibited the mitosis of IEL to mitogens, IL-7, and stimuli of the CD2 and CD3 pathways. The blocking effect did not target the activation events of IL-2 production and receptor generation. Rather, it reduced cell division after activation when added 24 h after initiating the culture. Antibody neutralization of naturally occurring TGF-β increased IEL proliferation to IL-2, but not to the other stimuli. Of the multiple surface markers tested, only CD2 and HML1 expression increased with TGF-β and decreased with antibody to TGF-β, although the cytokine and the neutralizing antibody had no effects on IEL binding to colon cancer. TGF-β reduced the number of CD56⁺ IEL and the lymphokine-activated killing when co-cultured with IL-7 but not with IL-2 or IL-15. TGF-β inhibits certain IEL functions: the reduction in cell division rather than activation and a decline in IL-7-mediated lysis of colon cancer due to a lowering of the number of natural killer cells.

INTRODUCTION

TGF-β is a multifunctional cytokine composed of two 25-kD subunits linked by a disulphide bond. It is secreted in a latent form, and becomes active with acidification. TGF-β plays a central role in the healing process. Platelets, usually first to appear in a wound, secrete large amounts of TGF-β, which attract neutrophils, T cells, fibroblasts, and monocytes. TGF-β induces fibroblasts to secrete extracellular matrix proteins and enhances integrin expression, promoting cell–cell and cell–matrix adhesion. Sustained TGF-β production leads to continued deposition of extracellular matrix proteins and fibrosis [1].

This cytokine generally exerts a negative effect on immune function, reducing T cell growth (particularly the CD4⁺ T cells). It inhibits the formation of cytotoxic T cells, lymphokine-activated killer (LAK) cells [2], and natural killer (NK) cells [3] and causes a decline in perforin mRNA. However, other reports show augmentation of immune function by TGF-β. It enhances splenic CD8⁺ T cell proliferation with a conversion to the memory (CD45RO⁺) phenotype [4,5]. TGF-β increases CD8 expression even in the absence of cell growth [6]. In addition, it does not change proliferation of phytohaemagglutinin (PHA)-activated human T cells, but instead increases the expression of HML1 and CD2 and augments IL-2 production in response to CD2 and CD28 [7]. Furthermore, it augments allospecific cytotoxic T lymphocyte activity [8].

TGF-β is important in intestinal immunity since: (i) it contributes to B cell switching from IgM and IgG to IgA [9]; (ii) it increases HML1 expression, an integrin found on intraepithelial lymphocytes (IEL) [10]; (iii) it augments CD2 responsiveness, a feature of intestinal lymphocytes [7]; (iv) it regulates adhesiveness of Peyer's patch high endothelial venule cells to lymphocytes [11]; (v) it alters cytokine production, expression of MHC antigens, and barrier function by epithelial cells [12–14]; and (vi) it regulates collagen synthesis by intestinal muscle [15]. TGF-β, then, is found in intestine and may contribute to the reduced functions of human IEL.

METHODS

Isolation of lymphocytes

IEL were isolated from jejunal mucosal specimens, averaging 7 × 4 cm, obtained from healthy individuals undergoing gastric bypass operations for morbid obesity. In brief, the minced mucosa was treated for 30 min at 37°C with l mm dithiothreitol followed by three 45-min incubations in a shaking water bath with 0.75 mm EDTA; supernatant cells were collected. After purification by Percoll density gradient centrifugation, IEL, averaging 100 × 10⁶ IEL per specimen, were 100% viable by trypan blue exclusion and > 90% lymphocytes that were 94 ± 5% CD2⁺, 5 ± 5% CD4⁺ and 89 ± 2% CD8⁺ [16], in agreement with immunohistochemical staining of tissue sections.

To isolate lamina propria lymphocytes (LPL), the treated tissue received three more 45-min incubations with EDTA, and the released cells were discarded. The remaining tissue was digested for 3 h at 37°C in 20 U/ml collagenase, then pressed through a wire mesh sieve to disperse the cells. LPL (about 70 × 10⁶ obtained from each specimen) were also purified using a Percoll density gradient and were 55 ± 10% CD4⁺, 35 ± 11% CD8⁺. PBL were isolated by Ficoll density gradient centrifugation.

T lymphocytes were separated in CD4⁺ and CD8⁺ subsets by negative selection using antibodies to CD8 and CD4, respectively. The cells bearing these markers were bound by MoAb, then by goat anti-mouse IgG, attached to magnetic beads, and the magnetic particle–cell complexes removed by application of a magnet as detailed elsewhere [17]. After separation, lymphocytes were < 1% positive for the depleted phenotype.

Measure of proliferation and IL-2 production

Lymphocytes (1 × 10⁵/0.1 ml) were cultured in triplicate with one or two of the following agents: PHA (1 μg/ml; Murex Diagnostics, Norcross, GA), preservative-free MoAb to CD3-ɛ (1 μg/ml; Immunotech, Westbrook, ME), IL-2 or TGF-β (10 ng/ml; R&D Systems, Minneapolis, MN), staphylococcal enterotoxin B (SEB; 100 ng/ml; Sigma Chemical Co., St Louis, MO), and the mitogenic T11₂ and T11₃ antibodies (1:500 dilution, kind gift from Dr E. L. Reinherz, Dana-Farber Cancer Institute, Boston, MA). Each stimulus was added at the start of the culture; after a 3-day incubation, proliferation was determined by ³H-thymidine uptake and expressed as a stimulation index (SI), calculated as the fold-increase in ct/min compared with an unstimulated control.

To measure IL-2 production, supernates of lymphocyte cultures stimulated as above were collected after 24 h and tested by IL-2 ELISA (Immunotech).

Flow cytometric analysis

Lymphocytes were stained by indirect immunofluorescence with antibody to IL-2R, β₁, CD2, CD3, CD8, CD16, CD25, CD44, CD56, HML1, and MHC class I, followed by fluorescein-conjugated goat anti-mouse IgG (Immunotech). Fluoresence was measured on a Coulter Profile analytical flow cytometer with a 25-mW argon laser. Data were analysed using Overton's Cumulative Subtraction program in the Cytologic Flow Cytometry Analysis Software. Results are given as percentage of positive cells and density of marker expression in units of relative fluorescence intensity (RFI) or fluorescence intensity compared with a control stained with fluorescein-conjugated goat anti-mouse IgG alone.

Binding assay

The cells—KD (lip fibroblasts) or HISM (jejunal smooth muscle) and HT29 (colonic adenocarcinoma cells) (American Type Culture Collection (ATCC), Rockville, MD)—were seeded into tissue culture micowell plates 18 h and 3 days, respectively, before the assay so confluence was reached. The ECM proteins—collagen types I and IV, fibronectin, laminin (Sigma)—were added to microwells 2 h before the assay and incubated at 37°C. The excess protein was washed out and the plate air-dried before use. Lymphocytes were assayed for binding activity after a 3-day culture with IL-2 in the presence or absence of TGF-β. These prestimulated cells were labelled with ⁵¹Cr-sodium chromate, and then 2 × 10⁵ cells were added to each well containing the adherent cell monolayers or ECM proteins. After incubating for 2 h at 37°C, non-adherent cells were removed by several gentle washes accomplished by adding and decanting warm complete medium. A 2% cetrimide solution was then added to lyse adherent cells; the contents of each well were collected and the radioactivity counted. After adjusting for the spontaneous release of radiolabel, the number of lymphocytes bound was calculated as a percentage of the total added [17].

LAK activity

LAK cells were generated by culturing IEL for 3 days with IL-2, IL-7, or IL-15 with or without TGF-β (all at 10 ng/ml). The cells were then washed and incubated in triplicate at various ratios with ⁵¹Cr-labelled HT-29 cells (ATCC). The percentage of cytotoxicity was calculated in relation to the spontaneous and maximal releases by target cells in medium and 2% cetrimide solution (Fisher Scientific, Fair Lawn, NJ), respectively, as detailed previously [18].

Serine esterase content

Lymphocytes, cultured with medium, IL-2, IL-7, or IL-15 for 3 days, were lysed by freeze-thawing, and the supernatants tested for the amount of granule secretion using the N-α-benzyloxycarbonyl-l-lysine thiobenzyl ester (BLT) assay [19].

Statistical analysis

For each set of data, a mean and s.e.m. were calculated. Pairs of data sets were analysed by Student's t-test for paired or independent variables.

RESULTS

Effects of TGF-β on proliferation

TGF-β, an inhibitory cytokine, is secreted by epithelial cells (EC), which are adjacent to IEL. It has variable effects on the proliferation of peripheral blood (PB) T lymphocytes depending upon their state of activation. It inhibited responsiveness of IEL to PHA, SEB, IL-7, and ligation of CD2 and CD3, but not to IL-2 or IL-15 (Fig. 1). While it inhibited responsiveness of LPL to all stimuli, it reduced only IL-2-induced proliferation by PBL (Fig. 1). At least 1 ng/ml of TGF-β was required to inhibit PHA-induced responses by IEL (Fig. 2). Increasing the concentration of TGF-β 100-fold increased the inhibition up to 45% of control; this levelling off suggests that TGF-β will not completely block blastogenesis. Spontaneous proliferation of LPL was unchanged by TGF-β but increased by antibody to TGF-β from 1026 ± 225 ct/min to 4143 ± 1188 ct/min (P < 0.05, paired Student's t-test, n = 4), suggesting that endogenous TGF-β reduced this baseline proliferation. Antibody to TGF-β, however, had no effect on baseline proliferation of IEL or PBL (Fig. 1).

Fig. 1.

Effect of TGF-β on proliferation of intraepithelial lymphocytes (IEL) (a), lamina propria lymphocytes (LPL) (b), and peripheral blood lymphocytes (PBL) (c). Lymphocytes were cultured with various stimuli and TGF-β (10 ng/ml) for 3 days; proliferation was determined by ³H-thymidine incorporation. *Values reduced by TGF-β (n = 8, P < 0.05, Student's t-test, paired variables).

Fig. 2.

Inhibitory effect of various amounts of TGF-β on phytohaemagglutinin (PHA)-induced proliferation of intraepithelial lymphocytes (IEL). IEL, cultured for 3 days with PHA (1 μg/ml) and various concentrations of TGF-β, were tested for resulting proliferation. *Proliferation with TGF-β that is less than proliferation with no TGF-β (n = 5, P < 0.05, Student's t-test, paired variables).

To determine whether TGF-β affected cell activation or division, the cytokine was added on days 0, 1, or 2 of a 3-day incubation of IEL with PHA (Fig. 3). Inhibition occurred even when TGF-β was added 24 h after initiating the culture, indicating that it acted on cell division rather than activation. Similarly, other early events—IL-2 production and IL-2 receptor generation—were unchanged by TGF-β. IL-2 production by PHA-stimulated IEL, measured by ELISA on culture medium after a 1-day incubation, was the same in the presence (210 ± 20 pg/ml) or absence (205 ± 15 pg/ml) of TGF-β (n = 3, NS). Similarly, the percentage of IL-2 receptor-expressing IEL after 1 day in PHA was comparable whether or not TGF-β was added: 69 ± 22% and 67 ± 26%, respectively (n = 3, NS). These studies show that TGF-β inhibits IEL mitosis rather than the activation events.

Fig. 3.

Effect of TGF-β added on different days to a 3-day culture of intraepithelial lymphocytes (IEL) and phytohaemagglutinin (PHA). IEL, cultured for 3 days with PHA, were supplemented on days 0, 1, and 2 with TGF-β (10 ng/ml) and proliferation was measured on day 3. *Values significantly less than the control value (n = 6, P < 0.05, Student's t-test, paired variables).

Since TGF-β lowers the IL-2-induced response of LPL (composed mainly of CD4⁺ T cells) but not IEL (mainly CD8⁺), it may have a greater effect on the IL-2-stimulated proliferation of the first subset over the other. To test this, each subset was isolated by negative selection from LPL and cultured with IL-2 for 3 days in the presence or absence of TGF-β. This cytokine more effectively blocked the IL-2-induced responses of unseparated LPL and the CD4⁺ T cell subset (79 ± 4% and 73 ± 5%, respectively) than the CD8⁺ T cell subset (30 ± 10% inhibition, n = 5, P < 0.05), indicating that TGF-β acts primarily on CD4⁺ LPL during IL-2 stimulation. Similar experiments were carried out with PHA-activated IEL. The proliferative responses by unseparated IEL, the CD4⁺ IEL, and the CD8⁺ IEL were reduced proportionately by TGF-β (Fig. 4, n = 4). For LPL, too, TGF-β inhibited the PHA-induced proliferation of unseparated LPL, the CD4⁺ T cells, and the CD8⁺ LPL to an equal extent (n = 4). The effect of TGF-β, then, depends on the stimulus, with CD4⁺ T cells being blocked in the presence of IL-2 and both CD4⁺ and CD8⁺ T cells in the presence of PHA.

Fig. 4.

Effects of TGF-β on phytohaemagglutinin (PHA)-induced proliferation of intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) subsets. IEL and LPL were divided into CD4⁺ and CD8⁺ subsets by immunomagnetic sorting and then stimulated with PHA for 3 days, with and without TGF-β. *Values significantly reduced by the cytokine (n = 5, Student's t-test, paired variables).

The low cell division of IEL with various stimuli may be due to endogenous production of TGF-β. To test this, the effects of antibody to TGF-β (5 μg/ml) on IEL proliferation were measured using a range of stimuli (Fig. 5). Antibody neutralization of TGF-β increased blastogenesis of IEL to IL-2 only. IL-2 may cause IEL to produce TGF-β and develop responsiveness to TGF-β so that proliferation is augmented by specific antibody but unaffected by exogenous cytokine (Fig. 1).

Fig. 5.

Effect of antibody to TGF-β on proliferation of intraepithelial lymphocytes (IEL) in response to various stimuli. IEL, cultured for 3 days with IL-2, antibody to CD3, or phytohaemagglutinin (PHA) in the presence or absence of antibody to TGF-β (5 μg/ml), were tested for their proliferative response on day 3. *Values increased in the presence of this cytokine (n = 8, P < 0.05, Student's t-test, paired variables).

Effect of TGF-β on surface markers and binding

TGF-β augments expression of certain markers on PB T cells and IEL, particularly those involved in adhesion [20]. To evaluate this, IEL were cultured for 3 days with IL-2 in the presence or absence of TGF-β and then tested for intensity of expression of β₁, CD2, CD3, CD8, CD44, HML1, and MHC class I (Table 1). Only intensity of expression of CD2 and HML1 increased with TGF-β and decreased with specific antibody, indicating selectivity in its effect. There were no changes in the numbers of IEL expressing each of these markers.

Table 1.

Effect of TGF-β on marker expression of intraepithelial lymphocytes (IEL)

IEL, treated with IL-2 for 3 days with or without TGF-β, were then tested for binding to monolayers of colon cancer (HT29) and fibroblasts (KD), ECM proteins (collagen types I and IV, laminin, fibronectin), and plastic. The cytokine did not alter binding of IEL to these surfaces (n = 3, not shown).

Effect of TGF-β on cytotoxicity

IEL were cultured with IL-2, IL-7, or IL-15 in the presence or absence of TGF-β (Table 2). This cytokine decreased cytotoxic activities of IEL effector cells that developed in IL-7 but not in IL-2 or IL-15, paralleling its effects on proliferation. Since TGF-β is known to lower NK activity, its effect on the number of NK cells in IEL after culture with IL-2, IL-7, or IL-15 was measured using immunofluorescent staining with specific antibodies and enumerating positive cells by flow cytometry (Table 3). TGF-β significantly reduced the number of CD56⁺ IEL cultured with IL-7, but not with IL-2 or IL-15, correlating with its effects on cytotoxic activity. There was a near significant TGF-β-induced decline in the number of CD16⁺ cells cultured in IL-7 (P = 0.08).

Table 2.

Effects of TGF-β and specific antibody on IL-2, IL-7, and IL-15-induced cytotoxicity by intraepithelial lymphocytes (IEL)

Table 3.

Effect of TGF-β on natural killer (NK) markers on intraepithelial lymphocytes (IEL)

TGF-β also reduced the content of serine esterase in lymphocytes after a 3-day culture with IL-2, IL-7, or IL-15. IEL in IL-2 developed 130 ± 6% more serine esterase than IEL in medium (defined as 100%). With IL-2 and TGF-β, the content dropped to 116 ± 9% (P < 0.05, n = 6). Similarly, TGF-β reduced the content that developed with IL-7 (from 120 ± 10% to 101 ± 5%, P < 0.01) and IL-15 (185 ± 28% to 164 ± 29%, n = 6, P < 0.01). Antibody to TGF-β had no effect on the development of LAK activity, the number of NK cells, or the serine esterase content (not shown). The TGF-β-induced reduction in cytotoxicity with IL-7 was associated with a decline in both the number of NK cells and the serine esterase release. Since the reduction in serine esterase release by TGF-β did not affect cytotoxicity induced by IL-2 or IL-15, the decline in the number of NK cells with IL-7 may be the critical change causing reduced cytotoxicity with this cytokine.

DISCUSSION

TGF-β is produced by the colonic epithelial cells and LPL, sources that are adjacent to IEL. Continual presence of this cytokine in the epithelium may account for the low level functions of IEL. However, the effect of TGF-β on IEL is not readily predictable, since deviation from its predominantly immunosuppressive actions occurs with memory CD45RO⁺ PB T lymphocytes, which IEL phenotypically resemble. Its effects depend upon various factors, such as the degree and duration of lymphocyte activation, the type of stimulus, the phenotype of the lymphocyte, the memory state, and the in vivo exposure to TGF-β. Naive PBL show no decline in proliferation with the addition of TGF-β except with IL-2 stimulation ([7] and present study).

TGF-β reduced the already low proliferation of IEL to mitogens, stimuli of CD3, and IL-7, but also the high cell division with CD2 ligation. This block occurred during the progression rather than the initiation of proliferation: IL-2 production and receptor generation were not affected, and the cytokine effect occurred even when added 24 h after beginning cultures. TGF-β has been shown to preserve the production of IL-2 and interferon-gamma (IFN-γ) by PB T cells while reducing production of IL-4 and IL-5 [21]. In other situations, it reduces IL-2 production [22].

LPL proliferation to all stimuli was reduced by TGF-β. By studying the T cell subsets in LPL separately, CD4⁺ T cells were more vulnerable to the inhibitory effects of TGF-β using IL-2 than were the CD8⁺ T cells, explaining why the LPL, which comprise a greater number of CD4⁺ T cells than the IEL, were blocked by TGF-β to a greater extent. In contrast, with PHA both T cell subsets, whether taken from IEL or LPL, were equally susceptible to inhibition by this cytokine. In other systems, TGF-β inhibits the CD4⁺ T cells more than the CD8⁺ T cells [23] or both to an equal degree [24], and in some situations it preferentially increases CD8⁺ T cell mitosis [25,26]. The degree of inhibition by TGF-β is usually partial [27], perhaps because it does not arrest all cell types.

Adding antibody to TGF-β increases the IL-2-mediated proliferation of IEL. This would occur if IEL were already suppressed by endogenous TGF-β, so antibody to this cytokine would reverse the suppression. The IEL may produce less TGF-β when stimulated with PHA or antibody to CD3, explaining why these responses are lowered by exogenous substance and not raised by antibody to TGF-β. The effect of TGF-β is certainly dependent on cell type and stimulus.

The binding of IEL to adjacent cell types (epithelial cells and fibroblasts) and to ECM proteins was not altered by exposure to TGF-β, nor was the expression of β₁ involved in these events. Expression of HML1 on IEL that binds E-cadherin on epithelial cells [17,28] is increased by TGF-β, but IEL binding to colon cancer monolayers was not affected, probably since this integrin is already strongly expressed.

TGF-β reduced the LAK activity and number of CD56⁺ IEL that developed with IL-7, but not IL-2 or IL-15. This correlates with the TGF-β-mediated reduction in IL-7-induced proliferation. Again, one could hypothesize that IL-2 and IL-15 induce more endogenous TGF-β than does IL-7, so that exogenous cytokine would have no effect. The primary response to TGF-β may be the reduction by half in the number of CD56⁺ IEL in IL-7, since this phenotype is largely responsible for the LAK activity [18]. In the peripheral blood, most effector cell activities are depressed by TGF-β, such as NK, LAK, and CD3-redirected lytic activity [2,3,25]. The maintenance of IL-2- and IL-15-induced LAK activity by IEL in the presence of this suppressive cytokine may be important in the immune surveillance against colon cancer and intestinal infections.

TGF-β enhanced expression of memory or late activation antigens on naive PB T cells and on IEL. The IEL response to TGF-β, however, differed from that of memory (CD45RO⁺) CD8⁺ T lymphocytes from PB. TGF-β suppresses proliferation of IEL but enhances proliferation of memory lymphocytes [26]. In contrast to the sustained lytic activities by IEL with TGF-β, those of memory cells were reduced. Also in contradistinction to memory cells, IEL have no increased density of adhesion molecules and no heightened recall response to stimuli [29].

IEL are probably derived from a subset of circulating memory CD8⁺ T cells and migrate to the intestine by utilizing MadCAM (α₄β₇) to bind to high endothelial venules on Peyer's patches. The IEL then migrate toward IL-8 produced by epithelial cells [30], and bind through HML1 to E-cadherin on the basolateral surfaces of epithelial cells [17]. Here, they are probably continually bathed by TGF-β secreted by epithelial cells. The drop in CD2 and HML1 expression on IEL cultured with antibody to TGF-β suggests that these markers have already been up-regulated by TGF-β and are likely to be important in IEL function. Certainly, CD2 is a pathway that leads to strong IEL proliferation and lymphokine production. The memory PB CD8⁺ T lymphocytes probably differ from IEL by their lack of intensive TGF-β exposure.