Immunol. Celt Biol. (1990)68, 387-396

Dual mechanisms of inhibition of the immune response by enterocytes isolated from the rat small intestine Gerald Pang, Robert Clancy and Heather Saunders Department of Pathology, Faculty of Medicine. University of Newcastle, Newcastle. NSIV 2300, Australia (Submitted

26 June 1990. Accepted for publication

9 November

1990.)

Summary Antigen presentation by enterocytes isolated from the rat small bowel was studied by using T cell proliferation, and immunorcgulatory function in an antigen-driven culture system, as indicator systems. Lymph node T cells obtained from rats immunized with ovalbumin (OA) failed to divide when cultured for 4 days in the presence of freshly isolated la+ enterocytes and OA. However, cell division was noted when enterocytes were removed after 18 h by Percoll gradient centrifugation, followed by culture ofT cells for a further 4 days in the absence of antigen. The failure to divide in the primary culture was due to the secretion by enterocytes ofa dialysable non-specific inhibitor. Antigen presentation by enterocytes was specific and was inhibited by monoclonal mouse anti-rat la antibody, 0X6. An epithelial cell line (REC-2) was established from normal rat small intestine. These cells expressed la molecules following incubation with Concanavalin-A stimulated spleen cell supernatant, and were capable of both presenting antigen, and inducing interieukin-2 (lL-2) production, when cultured with primed T cells. Furthermore. la' REC-2 cells functioned as stimulators in a primary mixed lymphocyte reaction (MLR). Both OA-primed T cells activated by enterocytes and antigen, and allogeneic MLR-activated T cells, mediated suppression which was not specific for the initiating antigen. These experiments indicated two mechanisms mediate suppression of cell division in gut mucosa. The contribution of these mechanisms to the control of inflammation at mucosal sites requires investigation.

INTRODUCTION Despite continual exposure to luminai antigens, the gut mocosal immune response is maintained with a minimum of local cell proliferation. Mucosal T cells respond poorly or not at all to mitogens or antigens in vitro (1,2). They are. however, capahle of both cytoxic T cell (3,4) and suppressor activities (5,6). A local immune

Correspondence: Dr Gerald Pang, Department of Pathology, Faculty of Medicine, David Maddison CHnical Science Building, Royal Newcastle Hospital, Newcastle, NSW 2300, Australia. Abbreviations used in this paper: BN rats. Brown Norway rats; Con-A, Concanavalin-A; EDTA, ethylene diamine tetra-acetic acid; Gl, gastrointestinal tract; IEL, intra-epithelial lymphocytes; IL-1, interleukin-1; lL-2, interleukin-2; IMDM, Iscove's modified Delbeceo's medium; KLH, keyhole limpet haemoeyanin; MHC, major histocompatibility complex; MLR, mixed lymphocyte reaction; OA, ovalbumin; OX6, a monoclonal mouse anti-rat la antibody; PEM, peritoneal exudate macrophages; PLN, peripheral lymph node; SC, secretory component.

response to both soluhle and paniculate antigen follows antigen uptake by the specialized epithelium overlying the Peyer's patch follicles (7,8), but the relationship between the epithelum and the intestinal lymphoid system is poorly understood. Class II major histocompatibility complex (MHC) la molecules are expressed on enterocytes (9,10), and recent studies in rat (11) and man (12) have shown that Ia+ enterocytes can present antigen to selectively induce suppressor T cell activity. Poor viability of freshly isolated enterocytes and heterogeneous cell populations have limited interpretation and reproducibility of results in these systems, and studies using MHC incompatible epithelial cell lines as antigen-presenting cells could reflect T cell activation by alloantigens. The present study, therefore, used rat small bowel enterocytes in which la expression could be regulated, to directly study the specific and nonspecific interaction between enterocytes and MHC compatible T cells. The suppressor function and specificity of T cells activated by antigen in the presence of enterocytes were analysed in an antigen-driven culture system.

G.PA^G ETAL.

388

MATERIALS AND METHODS Animate Male SPF Wistar and Brown Norway (BN) rats were obtained from Ihe Animal Resource Centre, Perth, Western Australia, and were used at 6-10 weeks of age. Media Cell cultures were established in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal calf serum (FCS), 2 X lO'^ mol/L 2- mereaptoethanol, 50 mmol/L glutamine, 50 iig/mL gentamicin, 100 U/mL penicillin, 100 |ig/mL streptomycin and 0-25 jig/mL amphotericin (Commonwealth Serum Laboratories [CSL], Melbourne, Australia). Cell preparation T cells Ovalbumin (OA)- or keyhole limpet haemocyanin (KLH)-primed T cells were isolated from the popliteal lymph nodes of rats 10 days after immunization with OA (1 mg/mL) or K.LH (0-5 mg/mL) (Sigma Chemical Co., St Louis. Missouri) emulsified in complete Freund's adjuvant. The T cell preparation was obtained by passing peripheral lymph node (PLN) cells twice through nylon wool columns (13). Enterocytes Enterocytes were isolated from the small intestine of a non-immunized rat. In brief, the entire small intestine was quickly removed and thoroughly washed in cold phosphate buffered saline (PBS). It was inverted on to a paediatric F5G feeding tube (Indoplas. Sydney, Australia), inflated byfillingit with cold PBS, and then ligated at both ends. The tissue was washedbriefly twice with 100 mL of cold PBS and then incubated in 5 mmol/L ethylene diamine tetra-acetic acid (EDTA). and 1% glueose in PBS at room temperature for 15 min on a magnetic stirrer. The cell population was depleted of intra-epithelial lymphocytes by centrifugation at 400 g for 30 min at room temperature on a two-step Percoll (Pharmacia. Sodertalje, Sweden) gradient prepared from 40% and 70% dilutions of an isotonie Percoll stock solution. Enterocytes recovered in the top layer were washed and quickly dispersed into single cells in complete IMDM before culture. Cell viability using trypan blue dye exclusion test was between 35 and 50%. Enterocyte cell line For propagation of cell lines, enterocytes were isolated from the small intestine of Wistar rats by four eonseeutive 10 min incubations at room temperature as described above. Calcium chloride was added to a concentration of 10 mmol/L. After centrifugation the ceils were suspended in complete IMDM containing 50 U/mL of insulin and 5% FCS, and cultured at 2 X 10' viable cells per mL in a 25 mL plastic flask (Corning. NY). After 48 h, non-adherent cells were removed and replaced with fresh medium. Thereafter, the cultures were fed twice weekly for 2-3 weeks with complete medium. Monolayer cultures consisting of fibroblasts and epithelial cells were isolated by rinsing the cell layer with warm PBS, incu-

bated with Versene buffer containing trypsin (CSL, Australia) for 1 min at room temperature, before the excess was removed. The flasks were incubated at 37° C for 2-3 min and then 10 mL of warm complete IMDM was added. The mixed cell population was cloned by limiting dilution in 96-well. tlat-bottomed microtitre plates (0 3 cells/well). The cloned cell line REC-2 used in experiments described in this paper has been maintained in continuous culture for more than 8 months. Preparation of j\broblasts Dermal fibroblasts were established from outgrowth of rat dorsal skin exptants in tissue culture dishes (Corning. NY) containing complete IMDM supplemented with insulin (50 U/mL). Cultures were grown at 37°C in humidified air containing 5% CO2 and fed twice weekly with 2 mL of growth medium. When confluent, the cells were detached by treating with Versene/trypsin solution, washed and subcuUured. Peritoneal exudaie macrophages (PEMj: Peritoneal exudate cells were obtained by intraperitoneal (i.p.) injection of naive rats with 20 mL of thioglycollate. The exudate was collected 7 days later hy lavage. The cell suspension was layered onto a Percoti gradient (1-07 g/mL) followed by centrifugalion at 2000 r/min for 20 min at room temperature. Macrophages (>95% purity) recovered from the interface were used as accessory ceils after treatment with mitomycin-C (100 Hg/mL). Lymphoproliferation Antigen-primed T cells (2 X lOVwell) were stimulated with antigen and graded numbers of enterocytes orPFM from naive rats, in a total volume of 200 jiL, in 96-well, flat-bottomed microtitre plates (Nunc. Roskilde, Denmark) at 37°C in an atmosphere of 5% CO; and air. Triplicate or quadruplicate cultures were pulsed with tritiated thymidine (0 5 ^Ci/well) for the final 18 h of culture, before harvesting on glass filter pads (Flow Lab., UK) for counting. Incorporated -'HTdR was measured by a scintillation counter (LKB Instruments, Sweden). Separation of T cells following culture with enterocytes and antigen T cells were stimulated with graded numbers of enterocytes and antigen for 18 h and then separated from the enterocytes by centrifugation on a Percoll density gradient prepared from 40% and 70% dilutions of an isotonie Percoll stock solution in Hank's balance salt solution. After centrifugation at 2000 r/min for 20 min at room temperature, viable T cells (>90%) were recovered from the interface. The cells were washed twice with IMDM containing 2% fetal calf serum (FCS), diluted to 2 X lO*- cells/mL in complete culture medium and then seeded at 2 X 10* cells/well in triplicate cultures. After 4 days, the cultures were pulsed with [^H]-thymidine (0 5 nCi/well) and harvested as described above.

ENTEROCYTE INHIBITION OF THE IMMUNE RESPONSE

Mixed lymphocyte reaction (MLR) For assessment of allogeneic primary MLR, REC-2 cells were used as stimulators. In brief, REC-2 cells were cultured in a 96-wetl. flat-bottomed plate (Nunc, Denmark) in the presence or absence of ConcanavalinA (Con-A) stimulated spleen cell supernatant (50%) for 24-48 h. The cells were washed three times with warm medium and then treated with mitomycin-C (100 ng/mL) for 2 h. Following three washes with medium, nylon-wool purified T cells (2X105/well) from naive Wistar or BN rats were added. After 4 days, the cultures were pulsed with 0 5 ^Ci [-^H]-thymidine per well for the final 18 h before harvesting and counting. Suppressor T cell assay The suppressor activity of MLR-activated T cells or of T cells isolated by Pcrcoll gradient eentrifugation after stimulation with graded numbers of enterocytes and antigen for 18 h was measured. Activated T cells were co-cultured with antigen-primed indicator T cells {2 X lOVweli) plus mitomycin-C treated PEM (lOOOO/well) as accessory cells in wells of a 96-wel!. flat-bottomed plate. Cultures were stimulated with antigen for 4 days and pulsed for the final 18 h with [^H]-[hymidine. before harvesting and counting. lnterteukin-2 production IL-2 production was used as an indicator of aetivation of T cells following antigen presentation by cultured enteroeytes. In brief, REC-2 cells (IO'*/weII) were grown to contluency in the presence or absenee of Con-A stimulated spleen cell supernatants in a 96well. flat-bottomed microtitre plate (Nunc. Denmark). The monolayers were washed three times with medium before incubation with primed T cells (2 X lOVwell) in the presence of antigen. After incubation for 24 h. the supernatant was collected, eentrifugcd. filtered, and stored at -20°C until assay for lL-2 production using an IL-2-dependent T cell line (NK-7) (14). MLA-144 supernatant which eontained 3000 U/ml, lL-2 was used as a standard, Expression of la molecules, secretory component (SC) and cytokeratin by enterocytes la expression was assessed by incubating either eytospin slide preparation of freshly isolated enterocytes or REC-2 cell monolayers with monoclonal mouse 0X6 anti-rat la antibody. This antibody recognizes a rat non-polymorphic determinant which is a homologuc of the mouse I-A gene product (I 5) (SeraLab, UK). Bound mouse antibody was detected by staining with peroxidase-labelled anti-mouse antibody using Vectastain ABC kit (Vector Laboratories, USA). To determine whether REC-2 cells express SC, they were grown on an 8-well glass slide (Flow Lab., UK), fixed with cold acetone for 10 mm and then incubated with rabbil immunogiobulin G (IgG) anti-SC (kindly provided by Associate Professor G. Jackson. University of NSW, Australia) for 30 min at room temperature. After washing three times with PBS, the cells were stained with peroxidasc-conjugaled sheep anli-rabbit

389

IgG (Vectastain ABC kit). Asa positive control, frozen sections of rat gut were used to indicate anti-SC staining of crypt cells. Cytokeratin expression was determined using mouse monoclonal anti-cytokeratin antibody (Amersham, UK) followed by peroxidase staining as described above. All photographs were taken on Kodak Pan technical film. Statistical Analysis Significance of differences between proliferation of T cells and enteroeyte-activated T cells was determined using the Student's /-test. RESULTS Characterization of enterocytes To determine whether rat enterocytes express la molecules, freshly isolated enterocytes from the small intestine were stained with a motioclotial antibody. 0X6. which recogtiizes a monomorphic determinant of rat la. As cati be seen in Fig. 1 (a), la molecules were expressed on the surface of enterocytes, with staining most intense in the basal region. la expression was visible at 24 h, at a stage when the eells were no longer viable. In contrast. REC-2 cells were negative for la (Fig. l(b)). unless they were exposed to a 5% Con-A conditioned spleen cell supernatant for 24-48 h, when la expression was induced on a proportion of cells {Fig. Uc)). To confirm that REC-2 enterocytes were mucosal epithelial cells, they were tested for SC and cytokeratin staining. As can be seen in Fig. l(d) and l(e), REC-2 enterocytes showed positive staining for SC and keratin using rabbit anti-Sc and mouse monoclonal anticytokeratin; control slides using normal rabbit and mouse IgG were unstained (result not shown). Antigen-presenting cell ability of enterocytes Primed T cells were stimulated with graded numbers of freshly isolated enterocytes or peritoneal macrophages from naive rats in the presence of OA for 4 days (Table 1). Macrophages but not rat enteroc>tes were capable of presenting antigen to T cells as detected by cell proliferation in the read-out system. Supernatants collected from 24 h enterocyte cultures inhibited OA-stimulated PLN cell cultures, compared with supernatants obtained from cultured tibroblasts (Table 2). The inhibitory factor was not secreted by T-cells as tbe supernatant collected from enterocytes cultured alone was inhibitory. To overcome this inhibitory effect, enterocytes were removed from T cells by Percoll gradient eentrifugation after 18 h of culture. T cells recovered from the interface were washed and re-cultured for 4 days, without further addition

390

G. PANG ETAL.

Fig. 1. Enterocytes. (a) Immunoperoxidase staining of freshly isolated enterocytes for la molecules with mouse monoclonal 0X6 anti-rat Ia antibody (X 250), (b) and (c) REC-2 cells stained for la before and after incubation with Con-A stimulated rat spleen cell supernalant (X400). (d) Stained for SC (X400). (c) REC-2 ceils stained with mouse monoclonal anti-keratin antibody (X400).

of antigen. A typical experiment is shown in Table 3. Enterocytes were capable of stimulating OA-primed T cells as shown by tbe proliferative response which occurred in those cultures from which enterocytes were removed after 18 b. Similar DNA synthesis was not seen in continuous cultures. Tbe response was antigen-specific, as no response was detected in cultures stimulated with enterocytes and an irrelevant antigen, keyhole limpet haemocyanin (KLH). If 0X6 anti-rat Ia monoclonal antibody was added to T cell cultures stimulated with enterocytes in tbe presence of OA, the proliferative response of T cells was inhibited in a dose-dependent manner,

indicating tbat Ia expression on enterocytes is probably required for antigen presentation (Fig. 2). To determine whetber REC-2 cells are capable of presenting antigen, the method of Ransom el a/. (16) based on tbe production of lL-2 was used as variable cell division results were obtained due to a high concentration of an inbibitory factor secreted by the REC-2 cells (unpublished data). In this assay, REC-2 cells were grown to confluency in the absence, or presence, of Con-A stimulated spleen cell supernatant, in a flat-bottomed 96-well microtitre plate. After several washes in medium, OA-primed T cells and OA

ENTEROCYTE INHIBITION OF THE IMMUNE RESPONSE

Table 1. Antigen-presenting function of cnterocytes and macrophages in OA-stimulated T cell cultures. Proliferative response^ (mean ct/min [^H]-thymidine incorporation + s.e.m.) Enterocytes Macrophages

No. of APC added

None 58±19 122 ±20 200 000 41±9 266 ±32 100 000 181 ± 72 372 + 80 50 000 175 ±45 5 490±2242 25 000 366 ±49 9 559±1339 12 500 186±73 12393±727 6 250 251±97 8 096 ±4678 3 000 517±270 222 ±82 Nylon wool purified T cells (2 X lOVwell) from Wistar rats immunized with OA were stimulated in flat-bottomed microtitre wells containing OA (200 tig/mL) and graded numbers of enterocytes or peritoneal macrophages from naive rats for 4 days. Cultures were pulsed with [^H]-thymidine (0.5 |iCi/well) for the final 18 h before harvesting. Each value represents the mean of triplicate cultures ± s.e.m.

391

were added and cultured for 24 h. Culture supertiatants were collected, dialysed against medium and assayed for the production of IL-2. As shown in Fig. 3, increased IL-2 activity was detected in cultures of REC-2 cells pre-treated with spleen cell supernatant but not in untreated cultures, indicating that the REC-2 enterocytes were capable of antigen presentation, which in turn depended on la expression. No significant IL-2 activity was detected when T cells were cultured with spleen cell supernatant stimulated REC-2 cells if they were first treated with OX6 anti-rat la antibody (results not shown). In another series of experiments, REC-2 cells were stimulated for 4 days with Con-A spleen cell supernatant to induce la expression followed by treatment with mitomycin-C. to test their capacity to stimulate a primary allogeneic MLR response against la molecules on the REC2 cells. As shown in Table 4, Con-A spleen cell supernatant stimulated REC-2 cells induced

Table 2. Kinetics of release by enterocytes of a factor which inhibits T cell proliferation.

Time (h)

Proliferative response^ (mean ct/min [^HJ-thymidine incorporation ± s.e.m.) Enterocyte supernatant Enterocyte-T cell supernatant Fibroblast-T cell supernatant

0

31 386 ±1026 3 906 ± 349 873±28 389±112 235 ±57 35 ± 9

'/l

1

2 6

24

32 828 ±1006 5 051 ±549 I 469 ± 52 132±23 122±5O 66 ±30

33 308 ±1856 26 683±141l 31 683 ±876 32 063 ±5744

30 263 ±4743 3 PLN cells (2X lOVwell) from Wistar rats immunized with OA were cultured with OA (2OO|4g/mL) in Hat-bottomed microtitre wells for 4 days in the presence of supernatant (50%, v/v) collected over a 24 h period from cultures of enterocytes alone or T cells stimulated with enterocytes or fibroblasts at a ratio of 5 • I in the presence of OA (200 ^g/mL). Cultures were pulsed with ^H-thymidine (0 5 nCi/well) for the final 24 h before harvesting. Eaeh value represents the mean of triplicate cultures ± s.e.m. Table 3. Effects of removing enterocytes on the proliferative response of primed T cells to ovatbumin.

T:EC ratio* 2.5 5 10 20

Proliferative response (mean ct/min [-^H]-thymidinc incorporation ± s.e.m ) OA-stimulated KLH-stimulated Continuoust Remove* Continuous Removed 73 ±35 54±4 186±73 252 ±97

533 ±184 1O3O±123 3873 ±363 5O87±719

51±12 46±19 39±9 56±7

100±15 I25±22 175 ±45 181 ±72 Nylon-woo! purified OA-primed T cells (2 X lOVwell) from Wistar rats were stimulated with enterocvles (EC) from naive rats at various ratios in the presence of OA (200 ng/mL) or KLH (500ug/mL) in tlai-b'ottomed microtitre wells. + Cultures were incubated for 4 days. * Cultures were incubated for 18 h, after which enterocytes were removed by Percoll gradient centritugation and activated T cells recovered from the interface were further cultured for 4 days in the absence of antigen Cultures were pulsed with [^HJ-thymidine (0 5 jiCi/well) for the final 18 h before harvesting. Each value represents the mean of triplicate cultures±s.e.m.

392

G. PANG

OA-Ptimed T cells + REC-2 cells 1/160 dilution OA-Pfimed T cells * REC-2 cells^ + OA 1/80 diluilon

OA-Primed T cells * la* REC-2 cells'' + OA

5 10 000

20 000

10

15

20

25

30

30 000

ct/min

IL-2 pfoducljon (U/mL)

Fig. 2. Effect of mouse monoclonal OX6 anti-rat la antibody on antigen-presenting function of enterocytes. OA-primed Wistar rat T cells (2X lO^/well) were stimulated with enterocytes from naive rats at 10: 1 ratio in the presence of OA (200 (Jg/mL) and mouse monoclonal 0X6 anti-rat la at various dilutions in flat-bottomed microtitre wells for I8h. After removal of enterocytes by PercoH gradient eentrifugation, activated T cells (2XI0Vwe!I) recovered from the interface were cultured for 4 days in the absenee of antigen. Control represents cultures stimulated in the absence of OX6 antibody. Results shown are the mean c.p.m. of triplicate cultures ± s.e.m.

Fig. 3. Antigen presenting function of REC-2 cells. REC-2 cells (10''/well) were cultured for 24-48 h in the (a) absence or (b) presence of Con-A stimulated spleen cell supernatant (50%) in a 96-welI. Hat-bottomed plate. After treatment with mitomycin-C(IOO^g/mL), OA-primed Wistar rat T cells (2X10Vwclt) were added to each well and stimulated with OA (200 lag/mL) for 24 h. Supernatants collected were assayed for lL-2 activity using NK-7 IL-2-dependent cell line. Results shown are tbe mean ct/min of five replicate cultures ± s.e.m.

allogeneic BN T cells to proliferate in a dosedependent manner; no ceil proliferation was seen with autologous Wistar T cells. Unstitnulated REC-2 cells had no effect (data not shown), indicating that the allogeneic response required la expression.

cells showed similar results. The reverse was also true (Fig. 4(b)). Furthermore, non-specific suppression was observed with T cells obtained from MLR cultures using REC-2 enterocytes as stimulators (Fig. 4(a) and 4(b)). In every case, cellular proliferation was required for suppressor function, as mitomycin-C abolished the suppressive effect (results not shown).

Activation of suppressor T cells by enterocytes To determine whether enterocytes modulate regulatory T cell functioti, OA-primed T cells were co-culutred with enterocytes at various ratios. After an initial 18 h stimulation, activated T cells were separated frotn enterocytes by Percoll gradient eentrifugation, and then co-cultured with primed indicator T cells at various ratios in the presence of OA. As shown in Fig. 4a. T cells activated in the absence of enterocytes had an enhancing effect on the proliferative response when co-cultured with indicator T cells, compared with T cells stimulated with enterocytes. The suppressive effect induced by enteroeyte-activated T ceils in the presence of OA was dose dependent and non-specific as the addition of KLH-primed enteroeyte-activated T

DISCUSSION The experiments reported here demonstrate that freshly isolated Ia+ enterocytes can present antigen to primed T cells, that an enterocyte cell line was capable of presenting antigen provided la molecules were expressed, and that a dialysable factor spontaneously secreted by enterocytes inhibited T cell proliferation. Suppressor T cells were induced following antigen presentation by enterocytes. which were non-antigen specific in their effector role. The observation that primed T cells were not stimulated by antigen in the presence of enterocytes, compared with macrophages, is con-

ENTEROCYTE INHIBITION OFTHE IMMUNE RESPONSE

393

Table 4. Stimulation of allogeneic T cells by REC-2 cells. Proliferative response (mean ct/min [^Hj-thymidine incorporation ± s.e.m.) No of REC-2 cells seeded per well' 105 E.xpl 1

None Autologous"^ Allogeneic*' Kxpl. 2 None Autologous* Allogeneic^

— 246 ±111 3I6±17

166 ±26 1215±245 8977 ±508

— 186±25 307 ±107

n.d. 21O±22 503 ±51

290 ±102 505 ±105 23 720±122 n.d. 298 ±49 I 743 ±153

143 ±4 375 ±65 16 587 ±3220

n.d. 187±25 3 657±I348 * Graded numbers of Wistar rat REC-2 cells were cultured for 24 h in wells of a flat-bottomed microtitre plate containing 10% Con-A stimulated spleen cell supernatant to induce la expression. + Nylon-wool purified T cells (2 X 10-Vwell) were added to REC-2 cells treated with mitomycin-C (100 tig/mL). Cultures were stimulated for 4 days and then pulsed with -^H-thymidine (0 5 nCi/well) for the final 18 h before harvesting. *-ST cells from naive Wistar and BN rats. Results represent means of triplicate cultures±s.e.m.) (n.d.= no data available).

sistent with the previous findings of Bland and Warren (11). The lack of response was not due to culture conditions or a poor viability of T cells as removal of etiterocytes from these cultures was followed by an enterocyte-dependent T cell proliferation. Thus in the initial experiment cell division was masked by the secretion of an inhibitory factor of cell division. No similar factor was secreted by fibroblasts. The dialysable factor and its synthesis was not inhibited by cycloheximide or indomethacin (unpubl. data). Inhibition of cell division was dependent on the continued presence ofthe factor. The nature of this factor is unknown but it may have a physiological role to restrict the proliferation of intra-epithelial lymphocytes. This would explain why isolated intra-epithelial lymphocytes fail to proliferate in viiro when stimulated with T cell mitogens or allogeneic antigens (1,2). The establishment of an enterocyte cell line from a normal small intestine has enabled both a more quantitative assessment of enterocytelymphocyte interactions, and a better definition of conditions for enterocyte differentiation in vitro. The cultured enterocyte was characterized by morphology, the presence of both secretory component and cytokeratin, and is probably derived from unditTerentiated dividing crypt enterocytes. A requirement for Ia expression in antigen presentation was supported by the ability of cultured enterocytes to induce IL-2 production by antigen-primed T celis following treatment with Con-A stimulated spleen cell supernatant. In addition, these cells were effec-

tive stimulators of an allogeneic MLR, provided Ia was expressed (data not shown). These results are similar to those using freshly isolated differentiated Ia* enterocytes. The activation of T cells depends both on the ability of an antigen-presenting cell to process protein to produce peptide in an immunogenic form that can be recognized by T cells, and on the density of Ia molecules expressed by the presenting cells (17,18). Whether or not enterocytes are capable of processing antigen into an immunogenic form is unclear. Although human enterocytes can apparently process antigen this way (12), we have not been able to reproduce these results with poorly viablefreshly isolated rat enterocytes. However, longlived and metabolically active cultured enterocytes should be a better model to study antigen processing. Antigen presentation by a variety of cell types including macrophages and dendritic celis (19), thymic epithelial cells (16), vascular endothelial cells (20), and liver sinusoidal lining cells (21), requires the production of IL-1 which in turn induces T cells to produce IL-2. An enhanced IL-2 responsiveness of T cells exposed to enterocytes, involves an enterocyte-derived factor which acts by inducing the expression of lL-2 receptors on T cells (11). It is possible that cultured enterocytes produce a variety of mediators which have yet to be characterized. Indeed, we have shown that cell lines established from human gut epithelial tumours secrete IL1-like and B cell differentiating factors (unpubl. data).

G. PANG ETAL.

394 (a)

OA-Bpeclfic proiiterative rosponse

CO-CULTURE^

Indicator Tl* cells alone

+ TC cells OA

* EC-act T''cells OA Indicator T Cells

Activated T Cells

.* EC-act T^ cells KLH

* EC-act T« cells MLR 30 000

(b) KLH-specilic proliteratiwe response

CO-CUUTUnE"

Indicator "^^ cells slone

cells KLH

+ EC-act T

Dual mechanisms of inhibition of the immune response by enterocytes isolated from the rat small intestine.

Antigen presentation by enterocytes isolated from the rat small bowel was studied by using T cell proliferation, and immunoregulatory function in an a...
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