Interleukin 12 is expressed and actively released by Crohn's disease intestinal lamina propria mononuclear cells

GASTROENTEROLOGY 1997;112:1169–1178

Interleukin 12 Is Expressed and Actively Released by Crohn’s Disease Intestinal Lamina Propria Mononuclear Cells GIOVANNI MONTELEONE, LIVIA BIANCONE, RAFFAELLA MARASCO, GIOVANNI MORRONE, ONORINA MARASCO, FRANCESCO LUZZA, and FRANCESCO PALLONE Dipartimento di Medicina Sperimentale e Clinica, Universita` di Reggio Calabria, Catanzaro, Italy

Background & Aims: Cell-mediated immunity is a feature of Crohn’s disease (CD). The heterodimer interleukin (IL)-12, produced by phagocytes, induces T-cell cytokines, primarily interferon (IFN)-g. This study examined whether CD lamina propria mononuclear cells (LPMCs) express and release bioactive IL-12. Methods: LPMCs were isolated from 13 patients with CD, 9 with ulcerative colitis (UC), and 13 controls. Messenger RNA for p40 and p35 IL-12 subunits was evaluated by reverse-transcription polymerase chain reaction. IL-12 was measured by enzyme-linked immunosorbent assay in LPMC culture supernatants. The INF-g –inducing effect of unstimulated LPMC supernatants was evaluated. Results: Messenger RNA for both IL-12 subunits was detected in LPMCs of 11 of 13 patients with CD, 1 of 9 patients with UC, and 1 of 13 controls (P õ 0.001). IL-12 was measured (10.5 { 2 pg/mL at 24 hours) in unstimulated CD LPMCs and was enhanced by pokeweed mitogen, lipopolysaccharide, and staphylococcal enterotoxin B. No IL-12 was detectable in 8 of 9 patients with UC and 12 of 13 control-unstimulated LPMCs. IL-12 induced by pokeweed mitogen and staphylococcal enterotoxin B in UC was lower than in CD and did not differ from controls. An IFN-g –inducing effect was restricted to unstimulated CD LPMC supernatants and was inhibited by an anti–IL-12 antibody in a dose-dependent fashion. Conclusions: IL-12 transcripts are expressed in CD intestinal tissues. CD LPMCs are up-regulated in their capability of releasing bioactive IL-12. Expression and release of bioactive IL-12 seem to differentiate CD from UC.

I

mmunologic observations and histopathologic data support the concept that cell-mediated immune reactions play a key role in the pathogenesis of tissue damage in Crohn’s disease (CD).1 – 3 Evidence also indicates that T-cell activation is an important feature of the inflammatory response in CD4 – 6 and that T-cell cytokines are involved.7 Suggestions have been provided that a Th1type response takes place in CD and that locally released cytokines induce the preferential differentiation of mucosal lymphocytes into the Th-1 subset.7 – 10 Studies in pa/ 5e1b$$0061

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tients and in experimental animals also indicate that interferon (IFN)-g plays a central role in CD.11,12 IFN-g is expressed and actively released by CD lamina propria mononuclear cells (LPMCs), and the frequency of IFNg–producing cells in the CD diseased tissues is increased compared with controls.9,13,14 Interleukin (IL)-12 is a 70-kilodalton heterodimer consisting of two covalently linked polypeptide chains, 35 (p35) and 40 kilodaltons (p40), encoded by separate genes.15,16 IL-12 is produced mainly by macrophages and monocytes in response to bacteria, bacterial products, or intracellular parasites and modulates T-cell functions. IL12 is a potent inducer of T-cell cytokines, especially IFNg.17,18 Both resting and activated natural killer (NK) cells and T cells are induced by IL-12 to produce IFN-g, although maximal IFN-g messenger RNA (mRNA) accumulation is reached in 2–4 hours in activated T cells or NK cells and in 18–24 hours in resting peripheral blood mononuclear cells (PBMCs).17,19 – 21 Anti–IL-12 antibodies inhibit up to 80% of IFN-g production in response to various stimuli, both in vitro and in vivo, indicating that IL-12 is required for optimal production of IFN-g. Moreover, IFN-g production by activated NK or T cells, in the absence of IL-12–producing cells, is not inhibited by anti–IL-12 antibodies.17 An experimental model of colitis has recently been developed in rodents, mediated by a delayed hypersensitivity reaction and exhibiting a Th1-type cytokine profile.22 In this model, the in vivo administration of adequate doses of anti–IL-12 antibodies proved to prevent colitis, suggesting that IL-12 may play a pivotal role in the pathogenesis of intestinal inflammation. Abbreviations used in this paper: DTT, dithiothreitol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBSS-CMF, Hank’s balanced salt solution, calcium and magnesium free; IFN, interferon; IL, interleukin; LPMC, lamina propria mononuclear cell; LPS, lipopolysaccharide; NK, natural killer; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; PWM, pokeweed mitogen; RT-PCR, reverse-transcription polymerase chain reaction; SEB, staphylococcal enterotoxin B. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00

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Taken together, these observations suggest that IL-12 may be involved in mediating the inflammatory response in CD. Preliminary data from our laboratory showed that IL-12 was detectable in culture supernatants of LPMCs isolated from actively involved CD intestinal tissues. The present study was therefore designed to explore whether IL-12 is expressed and produced in CD. The specific aims of the study were (1) to show IL-12 transcripts in CD LPMCs; (2) to determine whether IL-12 is spontaneously released by CD LPMCs and whether IL-12 released by LPMCs induces T-cell cytokines; and (3) to explore whether LPMC can be induced to release IL-12. We report data indicating that (1) bioactive IL-12 is expressed and spontaneously released by CD LPMC; (2) IL12 released by CD LPMCs is capable of inducing IFNg; (3) macrophages in CD inflammatory infiltrates are up-regulated in their ability to produce IL-12; and (4) LPMC IL-12 expression and release differentiate CD from ulcerative colitis (UC).

Materials and Methods Patients and Samples Mucosal samples were obtained from the diseased areas of intestinal specimens of 13 patients with CD (12 surgical and 1 biopsy). The primary site of involvement was ileal in 6, ileocolonic in 5, and colonic in 2 patients. In all patients, the disease was active as defined by a Crohn’s Disease Activity Index (CDAI) of ú200 and positive laboratory test results.23,24 Indication for surgery was medical intractability in all patients. At the time of surgery, 6 patients were receiving corticosteroids and 6 patients were taking mesalazine. The patient whose biopsy samples were studied was undergoing mesalazine and antibiotic therapy. Additional mucosal samples were also available from macroscopically and microscopically unaffected areas of 9 of the 12 CD surgical specimens (4 ileal and 5 colonic). Controls included (1) mucosal samples from the inflamed and noninflamed areas of colectomy specimens from 4 patients with UC, (2) multiple colonoscopic biopsy specimens (4–5) from the involved and uninvolved colon of 5 patients with UC, (3) mucosal samples from macroscopically and microscopically unaffected areas of 10 surgical colon specimens of colon cancer, and (4) multiple (4–5) colonic specimens of 3 patients with irritable bowel syndrome. As additional controls, mucosal specimens were obtained from 1 patient with amebic colitis undergoing colectomy for toxic megacolon and from 4 patients with diverticular disease undergoing subtotal colectomy for drug refractory diverticulitis. In the UC group, disease activity was defined by clinical criteria25 supplemented by endoscopic and histopathologic data.26,27 All 9 patients had active disease at the time of study. In the 4 patients who underwent colectomy, indication for surgery was a chronic active course poorly responsive to medical management.

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In all 4 patients, preoperative endoscopy showed moderate to severe changes. Disease extent was left-sided in 1 and substantial in the remaining 3. In no cases were dysplasia or extraintestinal manifestations the indication for surgery. In the 5 patients whose biopsy samples were studied, colitis was leftsided in 2 and substantial in 3 patients. In all patients, the disease was moderately active according to the clinical classification, whereas endoscopic and histological changes were moderate in 3 and severe in 2 patients. Three patients were undergoing corticosteroid therapy and the other 2 mesalazine therapy. Autologous PBMCs were obtained from 7 of the patients with CD, 5 of the patients with UC, and 5 noninflamed control patients. PBMCs from 10 healthy subjects were also available. The study was approved by the Department Ethical Committee.

LPMC and PBMC Isolation and Culture LPMCs were isolated by the dithiothreitol (DTT)–ethylenediaminetetraacetic acid (EDTA)–collagenase sequence as previously described in detail.5 Briefly, the dissected intestinal mucosa was freed of mucus and epithelial cells in sequential steps with DTT and EDTA and then digested with collagenase (Sigma Chemical Co., St. Louis, MO). Modifications for small samples were used in processing the biopsy specimens, as previously described.28 After collagenase digestion, the medium containing the mononuclear cells was collected and centrifuged at 400g for 10 minutes. After two washings in Hank’s balanced salt solution, calcium and magnesium free (HBSS–CMF) (Sigma), the pellet was resuspended in a 40% Percoll solution (Pharmacia, Uppsala, Sweden). An isotonic Percoll solution, consisting of nine parts of Percoll and one part 101 HBSS–CMF (pH 7.4, 290 mOsm), was used to prepare dilutions with HBSS–CMF. In a glass tube, 2 mL each of 100%, 60%, 40%, and 30% Percoll were layered. The tube was centrifuged at 400g for 25 minutes, and LPMCs at the 60%–40% Percoll layer interface were collected. The isolated cells were counted and checked for viability using 0.1% trypan blue (viability ranged from 86% to 94%). PBMCs were isolated by density gradient centrifugation (Lymphoprep; Nycomed Pharma, Oslo, Norway) from 10 mL heparinized blood samples. Cells were resuspended in RPMI 1640 supplemented with 10% fetal calf serum, 1% L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (all from Sigma) at a concentration of 1 1 106 cells/mL and cultured in 24-well culture plates (Falcon Plastic) with and without the initial addition of phytohemagglutinin (PHA; 1 mg/mL), lipopolysaccharide (LPS; Escherichia coli; 1 mg/mL), pokeweed mitogen (PWM; 1 mg/mL) (Sigma), or staphylococcal enterotoxin B (SEB; 1 mg/ mL; supplied by Dr. M. R. Capobianchi, Institute of Virology, University La Sapienza, Rome, Italy). After 0, 24, 48, and 72 hours, culture supernatants were collected and stored at 0807C until tested.

Colonic Tissue Homogenate Preparation Colonic biopsy specimens were also available for RNA analysis on freshly obtained whole tissue. Three biopsy speci-

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mens were taken from both involved and spared colonic areas of 3 patients with UC and 3 patients with colonic CD. In addition, tissue samples were taken from 3 patients with diverticular disease undergoing colonoscopy for recurrent abdominal pain. Biopsy specimens were separately placed in guanidine thiocyanate buffer on ice, homogenized using a tissue homogenizer (Ystral GmbH, D-7801; PBI International, Dottingen, Germany), and immediately used for RNA extraction.

RNA and Complementary DNA Preparation Total RNA was extracted from both unstimulated and stimulated LPMC cultured for 0, 6, 24, 48, and 72 hours. For RNA preparation, cells were lysed in 1 mL of guanidium thiocyanate buffer and subjected to phenol/chloroform extraction according to Chomczynsky and Sacchi.29 The sample obtained was quantitated by absorbance at 260 nm. RNA integrity was assessed by electrophoresis on a 1.5% agarose gel. Complementary DNA (cDNA) was synthetized from 0.5–1 mg of total RNA using 0.2 U of murine leukemia virus reverse transcriptase (Promega, Madison, WI), 2.5 mmol/L random hexamers (Boehringer–Mannheim, Mannheim, Germany), 1 mmol/L deoxynucleoside triphosphate (Boehringer–Mannheim), and 2 U ribonuclease inhibitor (Promega) in a total volume of 20 mL. The reaction was performed at 377C for 60 minutes.

Reverse-Transcription Polymerase Chain Reaction Before examining transcripts for IL-12, sample cDNA content was normalized on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal. For this purpose, varying amounts of cDNA were incubated in a reverse-transcription polymerase chain reaction (RT-PCR) for 18, 20, 21, 22, 23, and 25 cycles with GAPDH specific primers. IL-12/p40 and IL-12/p35 primers were assayed on all samples by incubating an equivalent amount of cDNA for 35 cycles. RT-PCR reactions were performed in a total volume of 50 mL in presence of 1 U of Taq DNA Polymerase (Boehringer–Mannheim), 200 mmol dNTPs (Boehringer–Mannheim), and 25 pmol/L 5* and 3* primers. Reactions were incubated in a Robocycler thermal cycler (Stratagene, La Jolla, CA) (denaturation 1 minute at 947C, annealing for 1 minute at 557C, and extension for 1 minute at 727C). PCR primers (Genosys, Cambridge, England) were as follows: p35, 5*-GAGAGAGACACAGAAGGAGA-3* and 3*-GAGGCCAGGCAACTCCCATTAG-5*; p40, 5*-CATTCGCTCCTGCTGCTTCAC-3* and 3*-CAGAGGGGACAACAAGGAGTA-5*; and GAPDH, 5*-CACCATCTTCCAGGAGCGAG3* and 3*-TCCGGGAAACTGTGGCGTGA-5*. To exclude the amplification of genomic DNA contaminating the samples, experiments were also performed using RNA as substrate for RTPCR assay. Ten-microliter RT-PCR products were combined with 1 mL of loading buffer and electrophoresed on a 1.5% agarose gel (in Tris-EDTA buffer). A 123–base pair (bp) ladder was

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used to assess sample size. Specificity of RT-PCR products was confirmed by specific restriction enzymes (Mae II for IL-12/ p40, Boehringer–Mannheim; and EcoRV for IL-12/p35, Promega). As a positive control, IL-12–producing Epstein–Barr virus-transformed human B-cell lines were also tested.

IL-12 Enzyme-Linked Immunosorbent Assay IL-12 was measured in LPMC and PBMC supernatants using a sensitive enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). According to the manufacturer directions, the minimum detectable IL-12 concentration was 5 pg/mL.

IFN-g –Inducing Effect of LPMC Supernatants To determine whether the immunoreactive IL-12 detected in LPMC supernatants was functionally active, the IFNg–inducing effect was examined as described by Kobayashi et al.30 and Aragane et al.31 Freshly isolated normal human PBMCs were cultured in 96-well plates (2 1 105 cells/well). Triplicate wells were added on time 0 of either 100 mL of complete medium or 100 mL conditioned medium. Conditioned medium consisted of unstimulated 24-hour supernatant from CD, UC, and control LPMC cultures run as indicated above. After 24 hours of incubation, cell-free supernatants were collected from each well and IFN-g was measured as international units per milliliter. A commercially available ELISA assay (Medgenix Diagnostics, Fleurus, Belgium) was used for measuring IFN-g with a minimum detectable concentration of 0.03 IU/mL. Aliquots of the 24-hour LPMC supernatants used in these experiments were tested for their IFN-g content. IFN-g was detectable in CD LPMC supernatants at a concentration of 1.31 { 0.48 IU/mL. To explore whether the IFN-g–inducing effect of LPMC supernatants was attributable to IL-12, parallel cultures were added of graded dilutions of a neutralizing anti–IL-12 antibody (Sigma) (ratios, 1:200, 1:800, 1:1600, and 1:3200). In three experiments, an anti–IL-4 antibody (R&D Systems) was used at the same dilutions as a nonrelevant control antibody. In three experiments, the IFN-g–inducing effect of LPMC supernatants was compared with that of known concentrations of IL-12. Triplicate normal PBMC cultures were run in complete medium additioned of human recombinant IL-12 (Sigma) at final concentrations of 1, 10, 100, and 1000 pg/mL.

Statistical Analysis The Student’s t test and the Fisher’s Exact Test were used as appropriate for the statistical analysis of the data.

Results IL-12 Transcripts GAPDH was consistently detected in all samples tested. In freshly LPMCs isolated transcripts for IL-12/ WBS-Gastro

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Table 2. IL-12/p40 mRNA in Unstimulated and Stimulated LPMCs and PBMCs From CD, UC, and Controls LPMC

Unstimulated PHA LPS PWM SEB

Figure 1. Agarose gel stained with ethidium bromide showing RT-PCR products for IL-12/p40 (268 bp), IL-12/p35 (534 bp) (after 35 cycles), and GAPDH (368 bp) (after 22 cycles) in freshly isolated LPMCs. Cells were isolated from a patient with ileocolonic CD from whom ileal and colonic samples, either macroscopically involved or uninvolved, were available (lanes 2–5 ). LPMCs from a patient with UC (lane 6) and one control subject (lane 7 ) were examined. Lane 1, 123-bp ladder; lane 2, CD LPMCs from an uninvolved ileal area; lane 3, CD LPMCs from an involved ileal area; lane 4, CD LPMCs from an uninvolved colonic area; lane 5, CD LPMCs from an involved colonic area; lane 6, UC LPMCs from an uninvolved area; lane 7, LPMCs from a control subject; and lane 8, negative control tube.

p40 (Figure 1) were detected in 11 of 13 (84.6%) samples from patients with CD (Table 1). In LPMCs freshly isolated from the actively involved CD intestinal tissues IL12/p40, mRNA was detected in 9 of 11 ileal and 7 of 7 colonic samples. In addition, transcripts for IL-12/p40 were detected in LPMCs isolated from the macroscopically and microscopically unaffected areas of 3 of 4 ileal and 5 of 5 CD colonic surgical specimens (Figure 1). IL-12/p40 mRNA was consistently detected in tissue homogenates from both involved and uninvolved colonic areas of 3 patients with CD. IL-12/p40 mRNA was found in 1 of 9 (11.1%) samples from patients with UC and in 1 of 13 (7.7%) control LPMCs ( P Å 0.00155 and 0.0002, respectively, vs. CD). No transcript for IL-12/ p40 was found in the homogenized tissue samples of patients with UC and diverticular disease. No IL-12/p40 mRNA was detected in LPMC from both the patients with amebic colitis and diverticular disease. No tran-

Table 1. IL-12/p40 and IL-12/p35 mRNA in Freshly Isolated LPMCs and Autologous PBMCs From CD, UC, and Controls CD

LPMC PBMC

UC

Controls

p40

p35

p40

p35

p40

p35

11/13 0/5

12/13 5/5

1/9 0/5

3/9 5/5

1/13 0/5

6/13 5/5

NOTE. Numbers indicate proportion of positive samples.

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PBMC

CD

UC

Controls

CD

UC

Controls

11/13 5/5 5/5 3/3 3/3

1/9 0/5 0/5 3/3 3/3

1/13 1/5 1/5 3/3 3/3

0/5 3/3 3/3 3/3 3/3

0/5 3/3 3/3 3/3 3/3

0/5 3/3 3/3 3/3 3/3

NOTE. Numbers indicate proportion of positive samples after 1-day culture.

script for IL-12/p40 was observed in freshly isolated PBMC from either disease groups or controls. As shown in Table 2, the exposure of LPMCs from UC and controls to PHA or LPS did not seem to influence IL-12/p40 transcript detection, whereas PWM and SEB stimulation induced the expression of IL-12/p40 mRNA in all LPMC samples (Figure 2). IL-12/p40 mRNA was detectable as early as 6 hours after exposure to PWM or SEB. As shown in Figure 2, when stimulation with mitogens, LPS, or SEB was provided to PBMCs, IL-12/ p40 mRNA was detected in all samples from both disease groups and controls (Table 2). IL-12/p35 mRNA was detected in freshly isolated LPMCs from 12 of 13 (92.3%) patients with CD, 3 of 9 (33.3%) patients with UC, and 6 of 13 (46%) controls (P Å 0.006 and 0.03, respectively). Again, PWM and SEB stimulation induced IL-12/p35 mRNA in all LPMCs tested. In all LPMC samples expressing mRNA for IL-12/p40, transcripts for IL-12/p35 were also detectable. IL-12/p35 mRNA expression was consistently observed in the homogenized tissue samples from patients with CD, UC, and diverticular disease and in freshly isolated PBMC samples from either disease groups and controls. IL-12 Production by LPMCs and PBMCs IL-12 was measured by ELISA in the supernatants of 11 CD LPMC samples expressing IL-12/p40 mRNA. In the 24-hour supernatants of unstimulated CD LPMC, IL-12 concentrations ranged between 8.5 and 12.5 pg/ mL (mean, 10.5 { 2 pg/mL) and did not increase over the culture period (11 { 0.9 at 48 hours, 12 { 0.5 at 72 hours) (Figure 3). No difference was observed in terms of spontaneous IL-12 release by cultured LPMC between diseased ileal and colonic areas (10.1 { 1.8 vs. 11.0 { 0.6 pg/mL at 24 hours; 9.8 { 0.4 vs. 12.0 { 2.0 pg/mL at 48 hours; and 12.0 { 0.4 vs. 11.9 { 0.8 pg/mL at 72 hours). ILWBS-Gastro

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12 production by LPMCs isolated from the involved areas (either ileal or colonic) did not differ from that of LPMC isolated from the uninvolved intestine (Table 3). No IL-12 was detected in the supernatants of LPMCs from 8 of 9 UC samples (either involved or spared) and 12 of 13 controls. Spontaneous production of IL-12 was detected in the supernatants of 1 UC sample and 1 noninflamed unstimulated LPMC sample. The amount of IL12 released in these two samples was 5.6 and 5.0 pg/ mL, respectively, after 24 hours of culture. In both samples, IL-12/p40 mRNA was present. When CD LPMCs were stimulated with LPS, PWM, or SEB, the 24-hour IL-12 release was significantly higher than in unstimulated cultures (Figure 3). As with unstimulated LPMCs, there was no further increase in IL-12 concentration over the culture period (Figure 3). At each time of the culture, the amount of IL-12 in the SEB-stimulated LPMC supernatants was significantly higher than that of PWM- or LPS-stimulated cultures

Figure 3. IL-12 release in 3-day cultures of both unstimulated and stimulated CD LPMCs. At each time interval, the amount of IL-12 in LPS-, PWM-, and SEB-stimulated cultures was significantly higher than that in unstimulated samples (*P õ 0.0005), whereas there was no difference between unstimulated and PHA-stimulated cells. At each time of the culture, SEB-induced IL-12 release was significantly higher than that measured in PWM- or LPS-stimulated cultures (SEB vs. PWM, P õ 0.0001 at all time intervals; SEB vs. LPS, P õ 0.0001 at 24 hours, P õ 0.05 at 48 and 72 hours). No difference was found between PWM- and LPS-stimulated LPMCs. Columns indicate the mean of all experiments, and vertical bars represent 1SD.

(P õ 0.04), whereas there was no significant difference in terms of IL-12 release between PWM- and LPS-stimulated CD LPMC. The exposure to PHA did not affect the capability of CD LPMCs to release IL-12. No IL-12 was measured in the supernatants of LPMCs from patients with UC and controls after PHA or LPS stimulation. All UC and control LPMC samples were, however, induced to release IL-12 by either PWM or SEB. The amount of IL-12 measured after exposure to either PWM or SEB in CD LPMC was significantly higher than that measured in UC and control LPMC (P õ 0.007), whereas no difference in terms of amount of released IL12 was observed between UC and controls (Figure 4). In both UC and control LPMCs, IL-12 release induced by SEB was significantly higher than that induced by PWM, at each time interval (P õ 0.01) (Figure 4). No IL-12 was measured in the supernatants of unstimulated PBMC cultures from either disease groups or con-

Table 3. Spontaneous IL-12 Release in 24-Hour Culture Supernatants of CD LPMCs LPMCs Figure 2. Agarose gel stained with ethidium bromide showing RT-PCR products for IL-12/p40 (268 bp), IL-12/p35 (534 bp) (after 35 cycles), and GAPDH (368 bp) (after 22 cycles) in (A ) LPMCs and (B ) autologous PBMCs from control subject cultured with or without stimulation. Lane 1, 123-bp ladder; lane 2, unstimulated cells; lane 3, PHA (1 mg/mL)stimulated cells; lane 4, LPS (1 mg/mL)-stimulated cells; lane 5, PWM (1 mg/mL)-stimulated cells; lane 6, SEB (1 mg/mL)-stimulated cells; and lane 7, negative control tube.

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Involved area (pg/mL) Uninvolved area (pg/mL)

Ileal

Colonic

10.6 { 1.0 9.5 { 1.8

11.05 { 1.3 9.82 { 0.8

NOTE. Numbers indicate mean values of IL-12 produced by LPMC isolated from either involved (ileal and colonic) or uninvolved (ileal and colonic) areas of three surgical samples.

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alone. In contrast, when normal PBMCs were cultured in the presence of CD LPMC supernatants (conditioned medium), IFN-g was detected in the supernatants at concentrations ranging between 1.56 and 4.76 IU/mL (mean, 3.16 { 1.6) (Figure 5). These concentrations were comparable to those detected in PBMC supernatants after exposure to 10 pg/mL of recombinant human IL-12 (Figure 5). It is worth noting that the mean IL-12 concentration in CD supernatants used for conditioned medium was 10.5 pg/mL. To provide further evidence that the IFN-g–inducing effect of CD LPMC supernatants was mediated by IL-12, graded dilutions of neutralizing anti–IL-12 antibodies were added to the conditioned medium. As shown in Figure 6, neutralizing anti–IL12 antibodies seemed to inhibit the CD supernatantinduced IFN-g release in a dose-dependent fashion. A 50% inhibition of IFN-g release was observed with an anti–IL-12 1:800 dilution, whereas a 1:200 dilution virtually abrogated the IFN-g response. No inhibition was observed using a nonrelevant anticytokine monoclonal antibody (anti–IL-4) (Figure 6).

Discussion

Figure 4. Kinetics of IL-12 (pg/mL) in vitro release by LPMCs cultured for 72 hours after stimulation with (A ) PWM and (B ) SEB. LPMCs were isolated from 3 patients with CD ( ), 3 patients with UC (rrr), and 3 controls (– – –). Each point on the curve is the mean of all experiments. Vertical bars indicate 1SD. At each time interval, IL-12 released in response to either PWM or SEB was higher in CD LPMCs than in UC and controls ( P õ 0.006).

The present study was designed to explore whether IL-12 is expressed and produced in CD. We show that IL-12 is expressed and released by unstimulated CD LPMCs, providing evidence that IL-12 expression and spontaneous release clearly differentiate CD from UC and controls. Data of our study are supported by a recent study showing that IL-12 synthesis is enhanced in CD tissue.32 IL-12 has an unique structure consisting of two disul-

trols. However, all stimuli tested proved to be able to induce IL-12 production in the PBMC samples examined. The IL-12 production induced by the exposure of PBMC to LPS and SEB was statistically higher than to PHA or PWM stimulation in both inflamed and noninflamed samples at each culture time (P õ 0.03). No difference was observed in terms of IL-12 production between CD and control stimulated PBMC samples. IFN-g –Inducing Effect of LPMC Supernatants To test whether IL-12 produced in CD was biologically active, supernatants of unstimulated LPMCs were examined for their ability to induce IFN-g in normal PBMCs. After 24 hours of culture, no IFN-g was detected in supernatants of normal PBMCs cultured with medium / 5e1b$$0061

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Figure 5. In vitro IFN-g release in 1-day normal PBMCs. PBMCs were cultured with medium alone or with conditioned medium (supernatant of 24 hours CD LPMC unstimulated cultures) or with recombinant human IL-12 (rh-IL-12) at concentrations of 1, 10, and 100 pg/mL. Data are shown as the mean { SD of 3 representative experiments.

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Figure 6. In vitro IFN-g release in 1-day normal PBMCs cultured in conditioned medium with the addition of graded dilutions of a neutralizing anti–IL-12 antibody. No inhibition was observed using a nonrelevant anticytokine monoclonal antibody (anti–IL-4). Data are shown as the mean { SD of 3 representative experiments. Readings with each IL-12 antibody dilution were significantly lower than those with no IL-12 antibody. Differences between readings with the various IL12 antibody dilutions were all significant ( P õ 0.05).

fide-linked subunits (p35 and p40) encoded by two separate genes located on different chromosomes.15,16 We detected transcripts of the p35-subunit gene in PBMC and LPMC samples not secreting immunoreactive IL-12, whereas immunoreactive IL-12 was measured only in cell samples expressing transcripts of both the p35 and p40 subunit genes. These observations are in agreement with the notion that transcripts of the p35 subunit gene are constitutively expressed in different cell types, whereas the expression of the p40 gene is restricted to IL-12– producing cells, and that the production of both subunits is required to form the biologically active p70 heterodimer.15 – 18 The physiological significance of the p40 homodimer is not fully understood. In the mouse, the recombinant p40 subunit inhibits the biological activity of the p70 heterodimer,33 suggesting that p40 may act as a physiological antagonist of IL-12. No evidence has been provided for the release of a p40 homodimer with binding capability to IL-12 receptors and antagonist function in the human intestinal lamina propria. However, our data are not in contrast with the hypothesis that LPMCs may release a p40 homodimer capable of binding to IL-12 receptors without signaling agonist activity.34 Transcripts of the p35 subunit gene were detected in nearly half of the unstimulated control LPMCs and were not enhanced by LPS, whereas transcripts of the p40 gene were not detected in control unstimulated LPMCs and / 5e1b$$0061

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were not induced by LPS. Stimulation with PWM or SEB seemed to efficiently induce p40 and enhance p35 expression. Accordingly, control LPMCs released IL-12 only after exposure to PWM or SEB but not to LPS, a potent IL-12 inducer both in vivo and in vitro.35 These data suggest that in the normal human intestinal mucosa, IL-12 production is a down-regulated function, as also suggested by the observation that IL-12/p40 is poorly expressed in human intestinal epithelial cell lines after bacterial stimulation.36 The finding of a defective IL-12 release in response to LPS is consistent with the observation that other cytokines are poorly released by LPSstimulated LPMCs37 and is supported by the recent finding that human LPMCs lack expression of the LPS receptor CD14.38 Our data also suggest that, as with other cell types, in human LPMCs, the expression of p40 and p35 is differently regulated, and that in human LPMC levels of p35, expression may to some extent influence IL-12 production, as recently shown in other cell systems.39 Virtually all freshly isolated CD LPMCs expressed both p40 and p35 mRNA and released immunoreactive IL-12. No p40 transcript was detected and no IL-12 was measured in unstimulated autologous CD PBMCs, whereas these cells were fully capable of expressing p40 and releasing IL-12 after appropriate stimulation, suggesting that spontaneous IL-12 expression and release is compartmentalized in CD as shown in other disease states.40 The IL-12 expression and enhanced release by LPMCs in CD was not dependent on the cells’ sampling site. LPMCs from either ileal and colonic mucosa were equally capable of expressing and releasing IL-12, suggesting that neither mucosal microenvironment nor variation in the luminal content were involved. Moreover, in CD, IL-12 was expressed and relased by LPMCs from spared intestinal samples from either ileum or colon, indicating that IL-12 production may not be an epiphenomenon of active inflammation and suggesting that IL12 up-regulation occurs as a result of disease-specific stimuli. The amount of IL-12 released by unstimulated CD LPMCs was relatively small but consistently measurable. Immunoreactive IL-12 concentrations similar to those observed in our CD LPMCs were detected in other human cells and in vitro systems.41 Furthermore, when IL-12 was measured by the ELISA used in the present study, levels not exceeding 30 pg/mL were found in human PBMC supernatants after maximal mitogen stimulation. Thus, the amount of IL-12 we measured in CD LPMCs may indicate a sustained activation of IL-12. This seems further suggested by the observation than even minute WBS-Gastro

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amounts of IL-12 are biologically active42 and that, on a molar basis, IL-12 is very potent in inducing its effects.17 We have shown that IL-12 in CD LPMC supernatants was biologically active as indicated by the marked IFN-g–inducing effect. It is unlikely that the IFN-g detected in PBMC cultures after incubation with CD LPMC supernatants was not induced because the baseline IFN-g concentration in the CD LPMCs was much lower and, in control experiments, did not increase over the culture time. We provided evidence that the IFN-g– inducing effect of CD LPMC supernatants was dependent on their content in IL-12. An anti–IL-12 polyclonal antibody inhibited the effect in a dose-dependent fashion, whereas nonrelevant cytokine antibodies were not effective. In our experiments, the amount of IFN-g induced by CD LPMC supernatants was similar to that induced by 10 pg/mL human recombinant IL-12 (i.e., a concentration of IL-12 similar to that measured in CD LPMCs). In CD, LPMC IL-12 release was significantly enhanced by LPS, PWM, or SEB, and the response to these stimuli was higher in CD than in UC and control LPMCs. This is in agreement with other studies showing that macrophages in the intestinal inflammatory infiltrates undergo further in situ activation and up-regulation in their capability of releasing cytokines.14,43 – 45 SEB was more potent than other stimuli in enhancing IL-12 in CD LPMCs, suggesting that SEB-induced mediators, including IFNg, may potentiate in situ macrophage activation and IL12 release.13,35,46 An IFN-g–mediated positive feedback stimulation of IL-12 may therefore occur in CD tissues. An IL-12–enhancing effect for locally released IFN-g has been shown during cytokine-mediated granuloma formation, and IL-10 and IL-4 were potent inhibitors.47 The recent finding of an impaired IL-4 production in CD LPMCs suggests that a defective suppression of IL-12 may also take place.48 Taken together, these observations indicate that the IL-12 expression and release in CD tissue reflect a local immune dysregulation related to disease-specific stimuli and are dependent on an imbalance of locally released regulatory cytokines. In our study, no IL-12 was detectable in unstimulated LPMCs from patients with UC, clearly differentiating UC from CD. Different cytokine profiles have been shown in CD and UC, suggesting different pathogenic mechanisms. Derangements in cell-mediated immunity are likely to be complex in both conditions, although evidence from studies both in patients and in experimental animals suggest that, in CD, the local immune response is predominantly Th1 in type, whereas in UC, Th2-mediated phenomena predominate. Our data add support to this view and suggest that in CD tissues, IL/ 5e1b$$0061

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12 may facilitate the generation of Th1-cell clones.49,50 It seems therefore that IL-12 is a key mediator of the inflammatory reaction in the intestine of patients with CD, suggesting that IL-12–blocking agents (e.g., anti– IL-12 antibodies) have potential therapeutic use in these patients.22

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49. Trinchieri G. Interleukin-12 and its role in the generation of Th1 cells. Immunol Today 1993;14:335–338. 50. Parronchi P, Giannarini L, Sampognaro S, Garcea MR, Salvadori G, Maggi E, Romagnani S. Accumulation of Th1 cells in gut biopsies of Crohn’s disease (abstr). Ital J Gastroenterol 1995;27:A190.

Received July 23, 1997. Accepted November 5, 1997. Address requests for reprints to: Francesco Pallone, M.D., Catte-

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dra di Gastroenterologia, Dipartimento di Medicina Sperimentale, Policlinico Universitario, Via T. Campanella, 88100 Catanzaro, Italy. Fax: (39) 961-775595. Supported by grant CNR 96.03133 CT04 from the Italian National Research Council. Presented partly at the annual meeting of the American Gastroenterological Association, San Francisco, California, May 18–23, 1996, and published in abstract form (Gastroenterology 1996;110:A972).

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