Human colonic intraepithelial lymphocytes regulate the cytokines produced by lamina propria mononuclear cells

Using an in vitro autologous human system, the immunomodulatory function of colonic intraepithelial lymphocytes (IEL) on cytokine production by lamina propria mononuclear cells (LPMNC) has been investigated. In contrast to LPMNC, colonic IEL produced only low amounts of IL-10, interferon-γ and interleukin-2. However, co-culture experiments (IEL + LPMNC) have shown that IEL can enhance the PHA-induced synthesis of IL-2 and interferon-γ, but not IL-10 by LPMNC. Using a transwell filter culture system apparatus, this effect was shown not to require a cell-to-cell interaction. Thus, IEL in vitro may modulate the cytokine synthesis of LPMNC, through the production of soluble factors. This may prove highly relevant in the in vivo immune activation of the gastrointestinal mucosa.


Introduction
Because of their unique location within the epithelium, their peculiar morphological and phenotypic features (different from those of peripheral blood and lamina propria lymphocytes) and their oligoclonal expression of ab T cell receptors, many studies have focused on IEL function. Although their precise role in vivo remains enigmatic, it has been shown in vitro that, under certain conditions human IEL (hIEL) exert various patterns of cytotoxicity, are capable of proliferation, and perform some regulatory functions. 1 9 It is believed that they may interact with the adjacent epithelial cells and underlying lamina propria cells. In particular, we have previously shown that colonic hIEL can inhibit the proliferation of autologous LPL. 10 Moreover, hIEL were also able to suppress IgA synthesis. 11 Inhibitory mechanisms were shown to occur through soluble factors, possibly cytokines.
While some cytokines have a critical role in inducing in ammation, others are crucial in reducing in ammation and mediating the healing process. Most studies on murine IEL (mIEL) have used lymphocytes from the small intestine. Freshly isolated mIEL produce interferon-c, and IL-5. 12 Subsequently, it has been shown that both TCR-ab and cd mIEL could produce interferon-c, IL-2, IL-3 and IL-6 but not IL-4. 13,14 However, the amounts of cytokines released by cd mIEL were much lower compared with ab mIEL. In addition, culture supernatants from anti-TCR antibody-activated cd or ab mIEL trigger synthesis of TNF-a, TGF-b and GM-CSF that in uence epithelial function. 14 16 Murine colonic IEL expressed similar levels of IL-1, interferon-c and TNF-a mRNA, and signi cantly more IL-2, IL-4, IL-5 and IL-10 mRNA when compared with small intestine mIEL. 17 Recently, human IEL were analysed for cytokine mRNA expression: freshly isolated hIEL expressed high levels of IL-1b, IL-8, IL-2R and IFN-c mRNA, but signi cantly less IL-1a, IL-6, IL-7, TNF-a and TNF-b mRNA. After in vitro polyclonal activation with phytohaemagglutinin (PHA) and PMA, IL-2 and IFN-c mRNA were signi cantly increased, whereas IL-1 mRNA was signi cantly decreased. 18,19 Furthermore, hIEL can produce small amounts of IL-2 and IFN-c in response to PHA even though there is little proliferation. 1,6,20 The production of these cytokines by hIEL is markedly increased by addition of sheep red blood cells to PHA or after stimulation through the CD2 molecule. 1 In hIEL, PHA induced no IL-4 production while superantigen stimulation resulted in detectable IL-4. 9,21 It is thus reasonable to consider the possibility that IEL may in uence LPL function through the secretion of soluble factors. Therefore, colonic hIEL were tested for their ability to produce IL-2, IFN-c and IL-10. Furthermore, the putative immunoregulatory function of IEL on cytokine secretion by autologous LPL was examined.

Subjects
Normal mucosa from colonic specimens was used from 10 control patients who were undergoing resection for a colonic carcinoma. The specimens were taken at least 5 cm away from any macroscopic lesion.

Isolation of mucosal lymphocytes
IEL were isolated as previously described. 10 Brie y, to isolate colonic IEL, mucosal strips were immersed in RPMI-supplemented with fetal calf serum (FCS), antibiotics (gentamycin 50 mg/ml, penicillin 100 U/ml, streptomycin 100 mg/ml). The washed specimens were then stirred in the same medium with the addition of 1 mM dithiothreitol (Sigma) for 10 min. After further washing, they were then incubated with calcium-and magnesium-free HBSS and 0.75 mM ethylenediaminetetraacetate (EDTA) in a shaking water bath at 378 C for three periods of 30 min each at 140 oscillations per minute. This step was repeated twice and the resulting supernatants were stored overnight at 48 C. Further puri cation was achieved by passing this crude preparation down a glass wool column. The ltrate was then centrifuged at 800 g for 30 min through a discontinuous 44 67 5% Percoll bilayer. The IEL were recovered from the interface. To isolate the lamina propria mononuclear cells (LPMNC), the treated tissue was submitted to three more 45-min incubations with calcium-and magnesium-free HBSS and 5 mM EDTA. The mucosal strips were digested overnight at 378 C with 100 ml RPMI 1640 supplemented as above plus 30 mg Clostridium histolyticum collagenase (Boehringer Mannheim, Germany). The digestate was passed through a 100 mm nylon mesh lter. After separation on Ficoll-paque gradient, the LPL were washed and resuspended in RPMI-supplemented as above. The viability of the cells was assessed using 0.1%trypan blue.
Preparation of cytokine-containing cell culture supernatants hIEL or LPMNC at a concentration of 1 3 10 6 were cultured in 1 ml aliquots in 24-well plates with or without 10 mg/ml PHA for 72 h at 378 C in 5% CO 2 . The supernatants were then harvested, centrifuged and passed through a 0.22 mm lter and stored at 208 C for subsequent cytokine analysis.
In¯uence of IEL on the cytokine production by autologous LPL LPMNC (10 6 ) were cultured with IEL (10 6 ) for 72 h with or without PHA (10 mg/ml) in 24-well plates in a nal volume of 1 ml at 378 C in 5% CO 2 . The control cultures consisted of IEL or LPMNC alone, with or without PHA. At the end of the culture, the supernatants were harvested, centrifuged, passed through a 0.22 mm lter and stored at 208 C for subsequent cytokine analysis (IL-2, IFN-c and IL-10). In addition, IEL were co-cultured with LPMNC in a Transwell apparatus (Costar Cambridge, MA) (6.5 mm diameter, 3 mm pore size) which physically separated IEL from LPMNC. The purpose of this series of experiments was to investigate whether the immunoregulatory function of IEL is mediated by a soluble factor or requires interaction between IEL and LPMNC.

Cytokine assays
Gamma-interferon, interleukin-10 (IL-10) and interleukin-2 (IL-2) were measured by a sensitive enzyme ampli ed immunoassay (Medgenix Diagnostics, Fleurus, Belgium). Optical densities were measured by an automated dual beam ELISA reader at 450 nm for c-IFN, IL-10 and IL-2. Concentrations of these cytokines were determined by reference to the serially diluted standards included in each plate. The minimum detectable concentrations of these assays were: c-FN 0.03 IU/ml; IL-10 1 pg/ml; IL-2 0.1 IU/ml. In these assays, duplicate values varied by , 5% from their mean value.
In¯uence of IEL on the cytokine production by autologous LPMNC After 3 days of culture, the effect of IEL on cytokine synthesis in co-culture with autologous LPMNC was studied. Addition of 10 6 IEL only minimally affected IL-10 production. The median secretion of IL-10 secreted by PHA-stimulated LPMNC was 4 pg/ml (range 1± 100). Following the addition of IEL, the concentrations increased to 7.5 pg/ml (range 2± 340). In contrast, they enhanced signi cantly the IL-2 and IFN-c production by LPMNC. After 3 days of co-culture, IL-2 and IFN-c levels were respectively 39.5 IU/ml (range 8± 450) and 104 IU/ml (18± 500) (Figs 1, 2 and 3). Using the Transwell culture apparatus, similar results were obtained (Table 1).

Discussion
The present data con rm our previous work showing that colonic IEL produce very low amounts of interferon-c. 20 Although there was a signi cant increase in interferon-c production following stimulation with PHA, the absolute amounts of interferon-c were persistently low. Sperber et al. reported similar ndings using IEL stimulated with PHA or superantigens. 9 This was not due to the failure of lectin binding. 22 The amount of interferon-c produced by stimu-   Median (range) cytokines production, n 5.
lated hIEL was of suf cient quantity to induce MHC class II expression molecules on HT-29 cells. 20 Class II bearing epithelial cells can in turn cause further activation of hIEL. 23 IL-2 is a central regulator of immune and in ammatory responses. It affects T cell proliferation as well as B cells and the function of macrophages, natural killer cells and lymphokine activated killer cell. PHA-stimulated colonic hIEL produce similar low amounts of interleukin-2, as do PHA-stimulated jejunal hIEL. 6,20 Conversely, Sperber et al. showed that IL-2 production was not observed with PHA-or superantigen-stimulated IEL. 9 Interestingly, induction of low levels of IL-2 does not correlate with the induction of in vitro proliferation. However, addition of exogeneous IL-2 does not restore a normal proliferative response. 6 IL-10 is a powerful suppressor of cytokines produced by macrophages and Th2 subpopulations of CD4 T cells. 24 In addition, IL-10 inhibits antigen presentation but supports B cell differentiation and IgA secretion. 25,26 To the best of our knowledge, this is the rst observation that human colonic IEL produce small amounts of IL-10 (as do colonic lamina propria cells). However, the precise action of this cytokine in the intestinal mucosa remains to be de ned although a colitis occurs in mice in which there has been targeted deletion of the IL-10 gene. 27 Teitelbaum et al. have shown that neutralizing antibody to IL-10 did not block the inhibitory activity of rat intestinal intraepithelial lymphocytes on the proliferative response of allogeneic spleen-thymus cells stimulated with concacanavalin A. 15 Murine helper T cells can be classi ed in relation to their cytokine production pro le. Thus, Th1 cells produce large quantities of IL-2, IFN-c and TNF-b while Th2 cells produce preferentially IL-4, IL-5, IL-6 and IL-10. 24 It has been suggested that a subset of murine CD8 IEL can produce some type 1 and 2 cytokines. 12,13 Furthermore, it has been shown that some human CD8 clones may secrete both patterns of cytokines. 28,29 It thus appears that these two subsets (Th1 and Th2) are mutually antagonistic. Th1 cells may in uence Th2 cell function through these cytokines and vice versa. For example, interferon-c may be able to downregulate a Th2 response while IL-10 has powerful inhibitory effects of the synthesis of Th1 cytokines.
The crucial role of cytokines in the mucosal response is well illustrated by a number of animal models of in ammatory bowel disease. IL-2-, IL-10 and TGF-b1 knockout mice develop chronic intestinal in ammation. 27,30,31 The local functions of these cytokines secreted by colonic hIEL have not been fully investigated. The data presented in this paper indicate that colonic hIEL can enhance the PHA-induced synthesis of IL-2 and interferon-c by human colonic LPMNC. The upregulated LPMNC cytokine synthesis in hIEL LPMNC co-cultures revealed that IEL did not contain cells inhibiting synthesis of these cytokines. Furthermore, absolute amounts of IL-10 produced by PHA-stimulated hIEL and LPMNC cultured alone or in co-culture were persistently low. Thus, the increased release of the Th1-cytokines may prove of importance in the modulation of subsequent immunopathological reactions including both activation of in ammatory cells and expression of class II molecules on epithelial cells.
Additional work must be done to understand the hIEL inhibitory activity previously described. 10,11 As IL-4 is virtually not produced by hIEL, further studies must be done in order to determine other inhibitory cytokines produced by IEL such as TGF-b and other inhibitory factors (e.g. neuropeptides).
It is important to note that cytokines and neuropeptides might in turn in uence hIEL function. It has been suggested that interleukin-7 (IL-7) might be produced in the mucosa by intestinal epithelial cells, especially globlet cells. 8 Interleukin-7 is a B-and T-cell growth factor. It affects the proliferation of activated but not unstimulated human T cells. Recent reports have shown that IL-7 stimulates hIEL proliferation. 7,8 In contrast, IL-9 or IL-12 did not stimulate the proliferation of IEL. 7 IL-4 secreted by activated lamina propria cells suppresses IEL proliferation and LAK activity. 21 Finally two neuropeptides, substance P and vasoactive intestinal peptide induce no proliferation nor spontaneous cytotoxicity by hIEL. This was due to an absence of substance P and vasoactive intestinal peptide receptors. 32,33 In conclusion, human colonic IEL may in uence the cytokines produced by LPMNC. This in vitro observation may be of importance in the local in vivo immune activation of the gastrointestinal tract.