Up-Regulation of the IL-12 Receptor β2 Chain in Crohn’s Disease

Patients and samples

Mucosal samples were taken from freshly obtained intestinal resection specimens of four patients with CD. The primary site of involvement was ileal in one and ileocolonic in three patients. The disease was active in all patients, as defined by a CD activity index >200 (20). At the time of surgery two patients were taking corticosteroids (CS) and two were taking mesalazine plus antibiotics. In all patients indication for surgery was a chronic active course poorly responsive to medical treatment. From patients with ileocolonic involvement, mucosal samples were taken from involved (gross lesions) and spared, ileal and colonic areas.

Additional mucosal samples were taken during endoscopy from 13 CD patients. In these patients the primary site of involvement was ileal in five, ileocolonic in three, and colonic in five. At the time of endoscopy, 11 patients had active disease. Five patients were taking CS, five were taking mesalazine, one was taking mesalazine plus antibiotics, and the remaining two were not receiving treatment.

Controls included colonic mucosal samples taken from involved areas of nine ulcerative colitis (UC) patients undergoing endoscopy, 13 patients with irritable bowel syndrome (IBS) undergoing endoscopy for recurrent abdominal pain, and one patient with diverticular disease. Both IBS and diverticular disease patients were categorized as the non-IBD control group. In addition, biopsy specimens were taken from three patients with infectious (salmonella) colitis. All UC patients had active disease at the time of the study, as defined by clinical criteria (21) supplemented by endoscopic and histopathologic data (22, 23). Disease activity was moderate in two and mild in seven patients. Disease extent was substantial in four and distal in five patients. Two patients were taking CS, five were taking mesalazine, and the remaining two were not receiving treatment.

Autologous PBMC were obtained from three CD and three UC patients, and three non-IBD controls. Biopsy specimens from the distal duodenum of four patients with untreated celiac disease and four normal controls were obtained during upper gastrointestinal endoscopy. Diagnosis of celiac disease was made according to the original or revised European Society for Pediatric Gastroenterology and Nutrition criteria for celiac disease (24, 25). The histopathologic diagnosis was based on typical mucosal lesions with crypt cell hyperplasia, villous atrophy, and increased number of intraepithelial lymphocytes. All celiac disease patients were positive for anti-endomysial and anti-gliadin Abs. Control patients were under investigation for gastrointestinal symptoms, but had normal histology and were anti-endomysial and anti-gliadin Ab negative. Additional samples were taken from the gastric antrum of three patients with documented diagnosis of Helicobacter pylori (HP)-associated gastritis and three normal controls (HP-negative subjects). Biopsy specimens were snap-frozen in liquid nitrogen and stored at −80°C until used.

The study was approved by the department ethical committee.

Lamina propria mononuclear cell (LPMC) isolation and culture

LPMC were isolated by the DTT-EDTA-collagenase sequence as previously described (2, 5, 23). The isolated cells were counted and checked for viability using 0.1% trypan blue (viability ranged from 90–94%). PBMC were isolated by density gradient centrifugation (Lymphoprep, Nycomed Pharma, Oslo, Norway) from 10-ml heparinized blood samples.

To analyze which cells express IL-12Rβ2, CD3⁺ lamina propria lymphocytes (LPL) were isolated from biopsy specimens of three patients with CD and three normal controls. CD3⁺ LPL were purified incubating LPMC with immunomagnetic beads armed with mAb for CD3 according to the instructions of the manufacturer (Dynal, Oslo, Norway). Purified cells were >93% as determined by FACS analysis.

To investigate whether IL-12 enhances IL-12Rβ2 expression, normal LPMC were resuspended in complete medium (RPMI 1640 supplemented with 10% FCS, 1% l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin; all from Sigma, St. Louis, MO) at a concentration of 2 × 10⁶ cells/ml and cultured in 24-well plates in the presence of PHA (1 μg/ml; Sigma) for 12 h. After that, nonadherent LPMC were collected, washed twice with 1× PBS, and cultured in the absence or the presence of graded doses of recombinant human IL-12 (Sigma; final concentration ranging from 0.1–10 ng/ml) for 6 h. To parallel LPMC cultures were added 10 ng/ml IL-12 and either a neutralizing IL-12 or IFN-γ Ab (both used at a final concentration of 1 μg/ml; both purchased from Sigma). To verify the efficiency of the anti-IFN-γ Ab, nonadherent LPMC cultures were also stimulated with 10 ng/ml IFN-γ in the presence or the absence of anti-IFN-γ (1 μg/ml). In additional experiments LPMC were isolated from the inflamed colon of four patients with CD and cultured (2 × 10⁶ cells/ml) in the presence of a neutralizing IL-12 Ab or a nonrelevant control Ab (rabbit IgG; 1 μg/ml; both purchased from Sigma) for 24 h. At the end of the culture, LPMC were collected and used for RNA extraction.

Tissue homogenate preparation

Biopsy or surgical mucosal samples taken from all patients enrolled in this study were used for both RNA and protein analysis on freshly obtained whole tissue. Mucosal samples were separately placed in sterile tubes containing 1–2 ml of cold guanidine thiocyanate buffer (for RNA extraction) or 0.5 ml of lysis buffer (for protein extraction). The latter contained 0.0625 mol/L Tris (pH 6.8), 2% SDS, 3% 2-ME, 10% glycerol, 100 mmol/L sodium fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mmol/L PMSF (all from Sigma). Tissue samples were homogenized using a tissue homogenizer (Ystral, Dottingen, Germany).

RNA and cDNA preparation and semiquantitative RT-PCR

Total RNA extracted from freshly obtained mucosal samples and (both unstimulated and stimulated) LPMC, CD3⁺ LPL, and CD3⁺-depleted LPMC were used to prepare cDNA as previously described (2, 5). In preliminary experiments we established the optimal number of cycles for obtaining a PCR product within the linear portion of the curve. For this purpose an equivalent amount of cDNA per sample (2 μl/reaction) was amplified using specific primers for β-actin (for 16, 18, 22, 24, and 26 cycles) or for each IL-12R subunit, IFN-γ, IL-4, or IL-5 (for 18, 20, 23, 26, and 28 cycles). PCR were performed in a total volume of 50 μl as previously described (2, 26). For Southern blotting experiments cDNA samples were incubated for 18 cycles with β-actin primers or for 23 cycles with primers for each IL-12R subunit, IL-12/p40, IFN-γ, IL-4, and IL-5 and were detected using specific cDNA probes (26). The cDNA probes were DNA fragments encoding the full-length PCR product. RT-PCR products were used as probes only after each product was cloned and its sequence verified. PCR primers (Genosys, Cambridge, U.K.) were as follows: IL-12Rβ1, 5′-CTTCCAGAAGGCTGTCAAG-3′ and 3′-CTGTGATTCAATGCAATACG-5′; IL-12Rβ2, 5′-GGATGCTCATTGGCATTTAT-3′ and 3′-CAGGCCAGTTTGCAGACAA-5′; IL-12/p40, 5′-CATTCGCTCCTGCTGCTTCAC-3′ and 3′-TACTCCTTGTTGTCCCCTCTG-5′; IFN-γ, 5′-AATGCAGGTCATTCAGATG-3′ and 3′-TTGGACATTCAAGTCAGTT-5′; IL-4, 5′-GCTAGCATGTGCCGGCAACTT-3′ and 3′-CAACGTACTCTGGTTGGCTTC-5′; IL-5, 5′-GAGGATTCCTGTTCCTGT-3′ and 3′-GCGCAACAAACCAGTTTAG-5′; and β-actin, 5′-CGAGGCCCAGAGCAAGAGA-3′ and 3′-CGTGACATTAAGGAGAAGCTGTG-5′. The level of RNA transcripts was measured by laser densitometry and expressed as arbitrary units.

Determination of phosphorylated STAT4 level

Total proteins were extracted from freshly obtained mucosal samples by using the above-mentioned lysis buffer. After cell lysis the supernatant was collected, run at 4000 × g for 40 min (4°C), and stored at −80°C until assay. Total proteins (500 μg/sample) were incubated with anti-STAT4 (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 2 h. Immune complexes were collected by incubation with protein A/G agarose (Santa Cruz Biotechnology), washed three times with lysis buffer, and heated for 5 min in a boiling water bath in sample buffer for SDS-PAGE. Immunoprecipitates from extracts containing the same amount of protein were analyzed by Western blotting with Ab against phosphotyrosine (p-Tyr; Santa Cruz Biotechnology) and subsequent incubation with HRP-conjugated goat anti-mouse IgG mAb (Santa Cruz Biotechnology). The Ab reaction was detected with a chemiluminescence detection kit (Amersham International, Arlington Heights, IL). After p-Tyr analysis, blots were stripped by incubation for 30 min at 50°C in stripping medium (2% SDS, 0.05 M Tris, and 0.1 M 2-ME) and then incubated with Ab against STAT4 (H-119; Santa Cruz Biotechnology).

IL-12Rβ2 expression is enhanced at the site of disease in CD

Transcripts for IL-12Rβ2 were detected in tissue homogenates from both affected and unaffected intestinal mucosal areas of CD patients. Similarly, transcripts for IL-12Rβ2 were found in mucosal samples taken from patients with UC and non-IBD controls. In addition, IL-12Rβ2 was expressed in freshly isolated LPMC from CD and UC patients as well as in those from non-IBD controls. When the IL-12Rβ2 content was examined by a semiquantitative RT-PCR, an increased accumulation was seen in whole mucosal samples from CD compared with UC and non-IBD controls (Fig. 1⇓). In CD, IL-12Rβ2 expression was more abundant in mucosal samples taken from affected areas compared with that in spared mucosa (Fig. 1⇓). No difference in terms of IL-12Rβ2 RNA transcripts was observed between spared mucosa of CD patients and UC or normal controls. IL-12Rβ2 RNA level in active UC did not differ from that in non-IBD controls (Fig. 1⇓). To establish which cells express IL-12Rβ2, total RNA was extracted from both CD3⁺ LPL and CD3⁺-depleted LPMC and analyzed by Southern blotting after PCR amplification. As shown in Fig. 2⇓, both CD3⁺ LPL and CD3⁺-depleted LPMC contained transcripts for IL-12Rβ2. However, an enhanced accumulation of IL-12Rβ2 RNA transcripts was observed only in CD3⁺ LPL from CD compared with controls (Fig. 2⇓).

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FIGURE 1.

The expression of IL-12Rβ2, but not IL-12Rβ1, is increased in the affected mucosal areas from CD compared with that in ulcerative colitis and non-IBD controls. Upper panel, RNA transcripts for IL-12Rβ2, IL-12Rβ1, and β-actin in mucosal tissue homogenates from three CD (lanes 1–6) and two UC (lanes 7–10) patients and two non-IBD controls (lanes 11 and 12). In patients with CD and those with UC, mucosal samples were taken from involved and uninvolved tissue. Southern blot analysis of RT-PCR products was performed as indicated in Materials and Methods. One representative experiment of four independent experiments is shown. Lower panel, Mean ± SEM of the ratio between IL-12Rβ2 and β-actin or between IL-12Rβ1 and β-actin in mucosal samples taken from involved areas of eight CD patients, eight UC patients, and eight normal controls. The level of RNA transcripts was measured by laser densitometry, and the relative values are expressed in arbitrary units.

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FIGURE 2.

RNA transcripts for IL-12Rβ2 and β-actin in CD3⁺ LPL (lanes 2, 4, 6, and 8) and CD3⁺-depleted LPMC (lanes 1, 3, 5, and 7) from two patients with CD (lanes 1–4) and two non-IBD controls (lanes 5–8). cDNA (1.5 and 3 μl for β-actin and IL-12Rβ2, respectively) was incubated with specific primers for β-actin and IL-12Rβ2 for 18 and 23 cycles. Southern blot analysis of RT-PCR products was performed as indicated in Materials and Methods. In CD mucosa, IL-12Rβ2 is expressed at a high level in CD3⁺ LPL. One of two representative experiments is shown.

Transcripts for IL-12Rβ1 were expressed at comparable levels in mucosal samples from CD and UC patients and non-IBD controls (Fig. 1⇑). Both IL-12Rβ1 and IL-12Rβ2 were barely detectable in PBMC (data not shown).

In CD, IL-12Rβ2 expression strictly correlates with tyrosine phosphorylation of STAT4 and IFN-γ expression

STAT4 is an essential component of the IL-12-dependent pathway for IFN-γ production (7, 8). There is good evidence that the phosphorylation and activation of STAT4 in response to IL-12 are dependent on the presence of IL-12Rβ2 (14, 16, 27, 28). To examine whether up-regulation of IL-12Rβ2 was associated with a pronounced expression of active STAT4, protein extracts from whole mucosal samples were immunoprecipitated with anti-STAT4 Ab and immunoblotted with anti-phosphotyrosine Ab. The level of STAT4 phosphorylation in affected mucosal areas of CD was greater than that in UC and non-IBD controls, although all these groups expressed comparable amounts of the STAT4 proteins (Fig. 3⇓). In paired mucosal biopsy specimens available from three CD patients, three UC patients, and three non-IBD controls, the profile of regulatory cytokines was analyzed by Southern blotting. As shown in Fig. 4⇓, a strong accumulation of transcripts for IFN-γ was detected in CD mucosal samples. Transcripts for IL-4 were expressed at very low levels in both CD and UC as well as non-IBD control mucosa (Fig. 4⇓). IL-5 RNA transcripts were detected only in UC (Fig. 4⇓).

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FIGURE 3.

Expression of both active (phosphorylated) and inactive STAT4 proteins in mucosal samples from two CD (lanes 1–4) and two UC (lanes 5–8) patients and two non-IBD controls (lanes 9 and 10). In both patients with CD and UC, mucosal samples were taken from involved and uninvolved tissue. Total proteins were immunoprecipitated (IP) with STAT4 Ab, run on 10% SDS-PAGE under reducing conditions, and immunoblotted with anti-phosphotyrosine Ab (p-Tyr). After detection of p-Tyr-STAT4 proteins, the membrane was stripped and reblotted with another STAT4 Ab to ascertain equivalent loading of the lanes. A marked phosphorylation of STAT4 is seen in affected mucosal samples from CD compared with UC or non-IBD controls. The example is representative of three separate experiments.

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FIGURE 4.

Transcripts for IFN-γ, IL-4, IL-5, and β-actin in mucosal tissue homogenates from CD and UC patients and non-IBD controls. Southern blot analysis of RT-PCR products was performed as indicated in Materials and Methods. A strong accumulation of transcripts for IFN-γ is documented in CD. IL-4 is expressed at a low level in all samples tested, whereas IL-5 is detectable only in UC. One representative experiment of three independent experiments is shown.

IL-12Rβ2 is enhanced in IL-12-associated Th1 gastrointestinal diseases

To investigate whether up-regulation of IL-12Rβ2 is a feature of Crohn’s disease or occurs in other Th1-mediated gastrointestinal diseases, the content of IL-12Rβ2 RNA was measured in mucosal samples taken from the distal duodenum of patients with celiac disease and the gastric antrum of patients with HP-associated gastritis. In addition, IL-12Rβ2 RNA transcripts were analyzed in mucosal samples of patients with infectious colitis. For all these groups of patients appropriate controls were included. As shown in Fig. 5⇓, the amount of transcripts for IL-12Rβ2 in the gastric mucosa of HP-infected patients was greater than that detected in the mucosa of HP-negative subjects. Similarly, the level of IL-12Rβ2 RNA was greater in patients with infectious colitis than in normal controls (Fig. 5⇓). In contrast, the IL-12Rβ2 RNA level in celiac disease duodenum did not differ from that in normal duodenal mucosa (Fig. 5⇓).

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FIGURE 5.

Southern blot analysis of RT-PCR products for IL-12Rβ2 and β-actin in mucosal tissue homogenates from an HP-infected patient (lane 1), an HP-negative control (lane 2), an infectious colitis patient (lane 3), a non-IBD control (lane 4), a celiac disease patient (lane 5), and a normal subject (lane 6). Southern blot analysis of RT-PCR products was performed as indicated in Materials and Methods. The amount of transcripts for IL-12Rβ2 in patients with HP infection or infectious colitis is greater than that in their respective controls. In contrast, the level of IL-12Rβ2 in the duodenal mucosa of patients with celiac disease is not different from that in normal duodenum. The example is representative of three separate experiments.

Transcripts for IL-12/p40 were detected in mucosal samples of patients with CD, HP-associated gastritis, and infectious colitis, but not in celiac disease (Fig. 6⇓). In addition, transcripts for IL-12/p40 were barely detectable in patients with UC (Fig. 6⇓) and non-IBD controls, confirming previous observations (2, 3, 4). Together, these data indicate that up-regulation of the IL-12Rβ2 chain occurs only in IL-12-associated Th1 gastrointestinal diseases.

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FIGURE 6.

Transcripts for IL-12/p40 and β-actin in mucosal tissue homogenates from patients with CD (lanes 1 and 2), infectious colitis (lanes 3 and 4), HP infection (lanes 5 and 6), UC (lanes 7 and 8), and celiac disease (lanes 9 and 10). Southern blot analysis of RT-PCR products was performed as indicated in Materials and Methods. Transcripts for IL-12/p40 are expressed at a high level in the mucosa of patients with CD, infectious colitis, and HP infection. The example is representative of two separate experiments.

IL-12 enhances IL-12Rβ2 expression in normal LPMC

Recent reports have demonstrated that IL-12 can regulate the expression of IL-12Rβ2 on T cells (15, 29). These data together with the documented observation that IL-12Rβ2 is enhanced in IL-12-associated Th1 gastrointestinal diseases prompted us to investigate whether IL-12 can enhance IL-12Rβ2 expression in human gastrointestinal mucosa. To address this issue, nonadherent LPMC isolated from normal controls were cultured with exogenous IL-12, and the amount of IL-12Rβ2 RNA transcripts was examined by Southern blotting after PCR amplification. As shown in Fig. 7⇓ (upper panel), IL-12 enhanced LPMC IL-12Rβ2, and this effect was inhibited by the addition of a neutralizing anti-IL-12 Ab. As IL-12 is a potent inducer of IFN-γ, and IFN-γ has the ability to enhance IL-12Rβ2, we next examined whether the effect of IL-12 on IL-12Rβ2 was dependent by the induction of IFN-γ. For this purpose, in parallel experiments nonadherent LPMC were cultured with exogenous IL-12 in the presence of a neutralizing anti-IFN-γ Ab, and the amount of IL-12Rβ2 RNA transcripts was examined by Southern blotting after PCR amplification. Interestingly, the anti-IFN-γ reduced, but did not completely abrogate, the induction of IL-12Rβ2 RNA by IL-12 (Fig. 7⇓, upper panel). Finally, we showed that addition of anti-IL-12 Ab to the CD LPMC cultures resulted in a marked inhibition of IL-12Rβ2 (Fig. 7⇓, lower panel).

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FIGURE 7.

IL-12 enhances IL-12Rβ2 in the human intestinal mucosa. Upper panel, Effect of rIL-12 on LPMC IL-12Rβ2 expression. LPMC, isolated from normal colonic mucosa, were cultured as described in Materials and Methods in the presence of medium alone (lane 1) or with IL-12 (10 ng/ml; lane 2), IL-12 plus anti-IL-12 Ab (1 μg/ml; lane 3), or IL-12 plus anti-IFN-γ Ab (1 μg/ml; lane 4). At the end of culture, total RNA was extracted from the LPMC and tested for IL-12Rβ2 expression by Southern blotting. The addition of IL-12 to the LPMC cultures results in an increase in IL-12RB2. This effect is inhibited by the addition of a neutralizing anti-IL-12 Ab. Induction of IL-12Rβ2 by IL-12 is reduced, but is not completely abrogated, by coincubation with a neutralizing anti-IFN-γ Ab. One of two representative experiments is shown. Lower panel, Effect of a neutralizing anti-IL-12 Ab on the expression of IL-12Rβ2 in CD LPMC. LPMC, isolated from the inflamed colon of two patients with CD, were cultured in the presence of medium alone (lanes 1 and 4) or a neutralizing IL-12 Ab (1 μg/ml; lanes 2 and 5) or a nonrelevant control Ab (1 μg/ml; lanes 3 and 6) for 24 h. At the end of culture, total RNA was extracted from the LPMC and tested for IL-12Rβ2 expression by Southern blotting. Addition of a neutralizing anti-IL-12, but not a control Ab (rabbit IgG), inhibits CD LPMC IL-12Rβ2. One of two representative experiments is shown.