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Expression of Interleukin-12-Related Cytokine Transcripts in Inflammatory Bowel Disease: Elevated Interleukin-23p19 and Interleukin-27p28 in Crohn's Disease But Not in Ulcerative Colitis

Expression of Interleukin-12-Related Cytokine Transcripts in Inflammatory Bowel Disease: Elevated... Abstract Background It has been suggested that Crohn's disease (CD) is associated with an exaggerated T-helper 1 cytokine response manifested by increased production of interleukin (IL)-12. IL-12 is a heterodimeric protein comprising 2 disulfide-linked subunits designated p35 and p40. Recently, IL-12-related cytokines, IL-23 and IL-27, were described. Biologically active IL-23 is a heterodimer whose p40 subunit is identical to IL-12p40 whereas its p19 subunit is distantly related to IL-12p35. IL-27 consists of EBI3, an IL-12p40-related protein, and p28, a newly discovered IL-12p35-related polypeptide. Aim We sought to determine whether mucosal expression of IL-23p19 and IL-27p28 transcripts correlate with the inflammatory activity in inflammatory bowel disease (IBD). Patients/Methods Messenger RNA expression in colonic mucosa from patients with Crohn's disease (CD; n = 37) and ulcerative colitis (UC; n = 19), and in non-IBD control subjects (specific colitis [SC]; n = 16) and normal, nondiseased control patients (n = 12) was measured by reverse-transcribed real-time polymerase chain reaction. Results IL-23p19 was significantly increased in inflamed mucosa in CD (P = 0.0377) and to a lesser extent also in UC patients but not in SC patients. Elevation of IL-23p19 transcript levels in CD correlated with the severity of endoscopic lesions. IL-27p28 transcripts and EBI3 transcripts were significantly elevated only in active CD. Discussion IL-23p19, IL-27p28, and EBI3 transcripts are strongly up-regulated in CD. The stimulatory effects of these cytokines on naive T cells in addition to a strongly synergistic action with IL-12 to trigger interferon-γ production may contribute to the perpetuation of the inflammatory process in patients with CD. Notably, increased expression of IL-23 and IL-27 transcripts in CD suggests a T helper 1-dominated immunologic function in this disease. Crohn's disease, interleukin-23, interleukin-27, realtime polymerase chain reaction, ulcerative colitis Despite intensive scientific research, the pathogenic mechanisms of inflammatory bowel disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC), remain unclear. However, distinct cell subsets and cell products have been identified that contribute to the inflammatory and destructive process. It has been shown that an uncontrolled or inadequately up-regulated cellular immune response of the intestinal mucosa toward an unknown pathogen, probably derived from the luminal content, plays a pivotal role in initiation and perpetuation of the disease.1,2 As a result of this mucosal injury, a breakdown of the epithelial barrier occurs, leading to an increased influx of intestinal content to the lamina propria, which further exaggerates the uncontrolled immune response.3,4 Recently, it has been suggested that CDis associated with an elevated T-helper 1 (TH1) response as manifested by increased production of interleukin (IL)-2, IL-12, IL-18, and interferon (IFN)-γ.4,–8 These cytokines are thought to contribute to disease development by orchestrating the inflammatory process. IL-12 is a 75-kDa heterodimeric protein that is produced by macrophages and dendritic cells. It comprises 2 disulfide-linked subunits designated p35 and p40 (according to their approximate molecular weights), forming the biologically active heterodimeric molecule (p70). IL-12 is secreted in mucosal tissue of patients with CD, contributing to the characteristic predominance of TH1 cytokine response of the mucosal immune system.9,–15 However, messenger (m) RNA transcripts for both subunits of IL-12 are only rarely detectable in situ and especially IL-12p40 expression is heterogeneous in CD patients.16 Very recently, a new p19 protein has been described that engages the IL-12 subunit p40 to form a new cytokine, named IL-23, which shows some biological activities similar to but others distinct from IL-12.17 Macrophages, dendritic cells and hemopoietic cells such as TH1 T cells have been identified as a source of IL-23. Although p19 shows no biologic activity by itself, combined with IL-12p40 it binds to IL-12 receptor β1 (IL-12Rβ1) but fails to engage IL-12Rβ2. Additionally, a new subunit of the receptor for IL-12 has been identified, termed IL-23R, that pairs with IL-12Rβ1 to confer IL-23 responsiveness.18 Similar to IL-12, human IL-23 stimulates IFN-γ production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.17 In respect of autoimmune inflammation Cua and coworkers19 demonstrated in a study that IL-23 rather than IL-12 is the critical cytokine for autoimmune inflammation of the brain. Another novel heterodimeric cytokine that has been termed IL-27 consists of a new discovered p28 protein, which is related to the p35-subunit of IL-12 and the Ebstein-Barr virus-induced gene 3 (EBI3), an IL-12p40-related protein. IL-27 is secreted by antigen-presenting cells, leading to rapid clonal expansion of naive CD4+ T cells and triggers IFN-γ secretion of these cells in synergism with IL-12.20 In this work, we determined whether the mucosal expression of IL-23p19, IL-27p28, and EBI3 was altered in patients with CD and UC by examining biopsy specimens from involved and normal mucosa, respectively, and comparing these results to patients with specific IBD as well as normal control patients. Materials and Methods Patients Mucosal biopsy specimens were obtained from ileocolonic mucosa from the following groups of patients. The first group represents 56 IBD patients with active disease (CD, 37 patients; UC, 19 patients; see Table 1). In all patients, paired samples from active and inactive mucosa were obtained. The diagnosis of these subjects was defined by standard clinical, radiologic, endoscopic, and histologic criteria.21 The second group (control subjects) included mucosal biopsy specimens of macroscopically and microscopically normal colonic mucosa from 12 patients without gastrointestinal disease (colonoscopy was performed to exclude colorectal neoplasia). The third group included 16 patients with specific IBD (ischemic colitis, bacterial colitis, i.e., specific colitis [SC]). Table 1. Patients Characteristics     View Large Table 1. Patients Characteristics     View Large Macroscopically, signs of inflammation in CD patients were assessed as described by Rutgeerts et al22: 0, no lesions; 1, less than 5 aphthous lesions; 2, more than 5 aphthous lesions with normal mucosa between the lesions; 3, diffuse aphthous lesions with diffusely inflamed mucosa; and 4, diffuse inflammation with larger ulcers, nodules or narrowing. In UC and SC patients, macroscopic signs of inflammation were assessed by a similar score ranging from 0 to 4 in adaptation to the score of Baron et al23: 0, no lesions; 1, granular mucosa; 2, contact bleeding/spontaneous bleeding; 3, erosions and small ulcers; 4, severe changes with ulcers, severely hemorrhagic mucosa and pus present. Biopsy samples were assessed blindly by the pathologist (I. M.-M.). The hematoxylin and eosin-stained specimens were scored according to a global histologic disease activity score described previously by D'Haens et al.24 This score summarizes several different histologic characteristics, including architectural changes, epithelial damage, the presence of inflammation, and granulomas and erosions or ulcerations. Histology scores ranged from 0 (normal) to 12. Previously, it has been difficult to accurately and reproducibly quantify mRNA levels using real-time polymerase chain reaction (PCR). However, the recent development of the real time PCR methodology has eliminated much of this variability, and should allow for more a routine and reliable quantification of PCR products.25,26 Tissue Processing Mucosal biopsies were collected in 1 mL of RNA-later Ambion, Austin, TX) and shipped to the processing laboratory. Upon arrival, the samples were transferred into RiboLyser tubes “Green” (ThermoHYBAID, Heidelberg, Germany) containing 400 μL of lysis buffer from the MagnaPure mRNA Isolation Kit II (Roche Diagnostics, Mannheim, Germany). Tissue was disrupted by one run with the RiboLyser (ThermoHYBAID) for 20 seconds each. After each run, the RiboLyser tubes were centrifuged at 4 °C for 1 minute at 13,000 rpm, and 300 μL of the lysate was collected and stored at −80 °C until mRNA isolation. The samples were thawed at room temperature for 30 minutes with constant mixing. To each sample, 600 L of capture buffer containing oligo-dT was added. After centrifugation at 13,000 rpm for 5 minutes, 880 μL of this mix was transferred into a MagnaPure sample cartridge and the mRNA was isolated with the MagnaPure-LC device using the mRNA-II standard protocol. The elution volume was set to 50 μL. An aliquot of 8.2 μL of RNA was reverse transcribed using AMV-RT and oligo-(dT) as primer (First Strand cDNA synthesis kit, Roche) according to the manufacturer's protocol in a thermocycler. After termination of the cDNA synthesis, the reaction mix was diluted to a final volume of 500 μL and stored at −20 °C until PCR analysis. Real-time PCR Parameter specific primer sets optimized for the LightCycler were developed and purchased from Search-LC GmbH (Heidelberg, Germany). For further description of the used primers please visit www.search-lc.com. PCR was performed with the LightCycler FastStart DNA Sybr GreenI kit (RAS) according to the protocol provided in the parameter specific kits. To control for specificity of the amplification products, a melting curve analysis was performed. The copy number was calculated from a standard curve, which was obtained by plotting known input concentrations of 4 different plasmids at log dilutions to the PCR cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. Using 952 data points, the actual copy number per μl of cDNA was calculated as follows: X = e(−0.6585*CP + 20.972). The amplification efficiency of the PCR was determined by running log dilutions of standards. The slope of the standard curve was converted to the amplification efficiency E by the following algorithm: E = 10−1/slope. All used primer sets had an efficiency greater than 1.86. RNA input was normalized by the average expression of the 2 housekeeping genes β-actin and cyclophilin B. The data of 2 independent analyses for each sample and parameter were averaged and presented as adjusted transcripts/microliter cDNA. Multiplying this number by 3000 (because of dilution of the extracted cDNA within the process) gives an estimate of the transcripts in an average biopsy. Culture of Intestinal Mucosal Biopsies Whole biopsy samples were cultured in an humidified incubator (6% CO2) at 37 °C in RPMI 1640 medium (Gibco, Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco), 100 μg/mL penicillin, 100 μg/mL streptomycin, and 0.03% glutamine. At the beginning of the culture, phorbol 12-myristate 13-acetate (PMA; 5 μg/mL) and ionomycin (1 μg/mL) were added. After 2 hours of culture, biopsies were transferred into 1 mL of RNA-later (Ambion) and shipped to the processing laboratory. Statistical Analysis Results were described as medians and minimum/maximum values. Statistical differences were calculated using a Wilcoxon rank sum test and were considered to be significant at the P ≤ 0.05 level. Correlations were calculated by Spearman correlation coefficients using the StatView SE+Graphics program (SAS Institute, Cary, NC) on a Macintosh Computer. Ethical Considerations The study was approved by the local ethics committee. Each patient gave written informed consent to perform endoscopic procedures and subsequent scientific studies. Results Characterization of IL-23p19 Expression For characterization of the potential role of IL-23 and IL-27 in the pathogenesis of IBD we first investigated transcript levels of the 2 well-known IL-12 subunits p35 and p40. In accordance with former studies we could demonstrate low but detectable levels of IL-12p35 that have been described to be expressed ubiquitously. No significant differences could be observed between normal control subjects (median, 21 × 103 transcripts per biopsy [tpb]), patients with specific colitis (median, 24 × 103 in noninflamed mucosa and 18 × 103 tpb in inflamed mucosa), and patients with CD (median, 18–24 × 103 tpb in accordance with the macroscopic score of inflammation) or ulcerative colitis (15 to 24 × 103 tpb). In contrast to this IL-12 subunit, transcript levels of IL-12p40 were very low or even undetectable in normal control subjects (median, 3 × 103 tpb) as well as in IBD patients (median, 3 to 6 × 103 tpb in patients with CD and 0 to 3 × 103 tpb in UC patients) or in patients with specific colitis (median, 0 tpb). Again, no significant differences could be observed between these groups of patients. In initial experiments we determined IL-23p19 expression in control subjects. All mucosal samples revealed low but detectable transcripts of IL-23p19, ranging from 3 × 103 to 54 × 103 tpb with a median of 9 × 103 tpb (Table 2). In biopsy specimens taken from macroscopically normal mucosa from patients with CD or UC, respectively, IL-23p19 transcript levels showed no difference from those taken from normal control subjects. Compared with normal mucosa, biopsy samples taken from inflamed mucosa of CD patients revealed significantly elevated IL-23p19 transcript levels. Furthermore, a correlation between macroscopic signs of inflammation of the affected mucosal tissue (in terms of Rutgeert score) and IL-23p19 expression could be demonstrated. In parallel, in patients with active UC, elevated IL-23p19 transcript levels were observed. Otherwise, in patients with SC, elevated IL-23p19 levels have not been detected, whereas these patients' unaffected mucosa showed comparable IL-23p19 expression as in normal control subjects. The values of biopsy specimens from inflamed mucosa of these patients were summarized independent of the macroscopic score. Table 2. Number of IL-23 Transcript Copies With CD, UC, and Normal Control Patients Depending on the Macroscopical Signs of Inflammation     View Large Table 2. Number of IL-23 Transcript Copies With CD, UC, and Normal Control Patients Depending on the Macroscopical Signs of Inflammation     View Large It should be noted that in all patients paired samples obtained from inflamed and noninflamed-so-called normal-mucosa were analyzed. Intraindividual comparison supported the overall finding of clear differences in IL-23p19 transcript levels depending on disease activity. Successful Treatment of Active IBD Resulted in a Decrease of IL-23 Transcript Concentration Because mucosal IL-23p19 transcript levels were increased in patients with active CD and (to a lesser extent) with UC, we investigated the effects of steroid treatment on this parameter. Twenty-four patients (18 patients with CD and 6 patients with UC) underwent control ileocolonoscopy and determination of IL-23p19 transcript levels after a median follow-up of 8 weeks. We evaluated the effect of steroid treatment in patients with active disease, not having been treated with steroids at the time of the initial ileocolonoscopy and evaluation of transcript levels. None of these patients has received other concomitant therapy such as azathioprine. Asignificant decrease of initially elevated transcript levels in patients entering remission from a median of 63 × 103 transcripts per biopsy to 15 × 103 tpb was found. In these patients an improvement of clinical symptoms was observed, and patients were in a stable remission during the whole follow-up period of 6 months. Most patients showed a decrease of IL-23p19 transcripts, even if individual patients experienced an increase in transcript levels, with 3 patients still having elevated transcripts. However, 6 patients who did not enter complete remission or experienced early relapses of their IBD showed increasing IL-23p19 transcript levels from a median of 15 × 103 transcripts per biopsy to 63 × 103 tpb. In these patients, IL-23p19 transcripts decreased only marginally or even increased further. For further data on the development in every individual patient, see Figure 1a. Figure 1. View largeDownload slide Development of IL-23p19 transcripts (a) and IL-27p28 transcripts (b), respectively, after steroid treatment in patients with IBD (18 patients with CD and 6 patients with UC). The dotted line denotes the upper range of normal controls. White symbols characterize patients not entering complete remission or having early relapses of IBD, whereas black symbols characterize patients entering remission. Figure 1. View largeDownload slide Development of IL-23p19 transcripts (a) and IL-27p28 transcripts (b), respectively, after steroid treatment in patients with IBD (18 patients with CD and 6 patients with UC). The dotted line denotes the upper range of normal controls. White symbols characterize patients not entering complete remission or having early relapses of IBD, whereas black symbols characterize patients entering remission. Characterization of IL-27p28 Expression Low-but-detectable levels of IL-27p28 mRNA were found in all patients, in normal as well as in diseased mucosa. In normal control subjects a median concentration of 6 × 103 tpb was measured (minimum, 3 × 103 tpb; maximum, 12 × 103 tpb). Compared with these data, no significant elevation of IL-27p28 transcript levels in patients with SC could be demonstrated, neither in macroscopically normal nor in inflamed mucosal tissue samples. Patients with UC expressed the same number of transcripts in noninflamed and in inflamed tissue samples (meannon-inflamed 9 × 103 tpb (minimum-maximum [min-max], 3 × 103 tpb to 12 × 103 tpb) and meaninflamed 6−9 × 103 tpb (minimum-maximum, 3 × 103 tpb to 18 × 103 tpb), respectively). In contrast, patients with CD showed differing expression levels. Whereas the number of IL-27p28 transcripts in macroscopically normal or only moderately inflamed mucosa (grade 0 or 1 in terms of Rutgeert score) did not differ significantly from that in control subjects, more severe inflamed mucosal tissue (Rutgeert score, grade 2–4) exhibited significant differences in comparison with normal control subjects. The former mucosal biopsies revealed a median of 6 × 103 tpb to 9 × 103 tpb (min-max, 3 × 103 tpb to 36 × 103 tpb) and the latter samples exhibited a median of 18 × 103 tpb to 24 × 103 tpb (min-max, 6 × 103 tpb to 306 × 103 tpb; P = 0.0044 to 0.0348). Characterization of EBI3 Expression All mucosal samples examined revealed detectable levels of EBI3 transcripts. A median concentration of 117 × 103 tpb was measured (minimum 63 × 103 tpb, maximum 204 × 103 tpb) in normal control subjects. EBI3 expression in patients with SC did not differ significantly from the control group, neither in macroscopically normal nor in inflamed mucosal tissue. In patients with CD, we found a significant decrease in EBI3 transcript levels in macroscopically normal or only moderately inflamed mucosa (grade 0 or 1 in terms of Rutgeert score); transcript levels ranged from 12 × 103 tpb to 390 × 103 tpb (min-max median, 93 × 103 tpb). In contrast to this, in more severe inflamed mucosal tissue (Rutgeert score, grade 2 to 4) EBI3 transcripts were significantly elevated (median, 129 × 103 tpb; min-max, 15 × 103 tpb to 972 × 103 tpb; P = 0.001). Interestingly, and in contrast to the data published by Omata et al,16 we did not find significant differences in EBI3 expression in patients with UC, neither in tissue samples from normal (median, 93 × 103 tpb; min-max, 0 × 103 tpb to 303 × 10 3 tpb; P = 0.065 to 0.515), nor from inflamed colonic areas (median, 102 × 103 tpb; min-max, 0 × 103 tpb to 372 × 103 tpb; P = 0.068 to 0.686). IL-27 Transcript Concentration After Treatment of Active IBD Because mucosal IL-27p28 transcripts were increased in a subset of patients with active CD, we investigated the effects of steroid treatment on this parameter in both groups of patients with IBD. Again, 24 patients (18 patients with CD and 6 patients with UC) underwent control ileocolonoscopy and determination of IL-27p28 transcripts after a median follow-up of 8 weeks. The 2 UC patients not entering remission had transcript levels within the reference range. Two patients with UC who entered remission after 8 weeks of treatment had normal transcript levels before and after steroid treatment, and only 2 patients (of 6 CU patients examined) showed slightly elevated IL-27p28 levels (15 × 103 tpb) that became normal 8 weeks after treatment (3 and 6 × 103 tpb, respectively). In 9 CD patients entering remission, IL-27p28 levels were elevated in the first biopsy (median, 21 × 103 tpb; min-max, 15 × 103 tpb to 360 × 103 tpb), and 8 of those patients showed normal transcript levels after treatment (median, 6 × 103 tpb; min-max, 0 to 9 × 103 tpb). However, 6 patients entering remission showed normal transcript levels in the initial examination (≤ 12 × 103 tpb). Three patients with CD did not enter remission, 2 with initially elevated transcript levels that normalized in 1 case and 1 patient with a normal IL-27p28 mRNA levels at the initial examination. Cytokine Expression in Mucosal Biopsies After Activation of Immunocompetent Cells in Vitro In 7 patients with UC and 14 patients with CD, mucosal biopsy specimens were examined regarding their IL-23p19-expression after stimulation with an activator of T cells PMA and ionomycin [Iono]. These data show an up-regulation of IL-23p19 responsiveness in intestinal tissue samples of CD patients compared with UC patients (P = 0.046; Fig. 2). Interestingly, the responsiveness in inflamed areas of either disease was not as high as that in noninflamed specimens, possibly because of higher transcript levels in these areas before stimulation with PMA/Iono. No significant differences in cytokine responsiveness upon stimulation with PMA/ionomycin could be observed concerning the expression of IL-12p35 (P = 0.753), IL-12p40 (P = 0.463), IL-27p28 (P = 0.917), or EB13 (P = 0.080). Moreover, the trend toward increased EB13 responsiveness was in favor of a higher expression in patients with CD. Figure 2. View largeDownload slide Increase of IL-23 transcript levels after stimulation of intestinal biopsies by PMA and ionomycin as described in patients and methods. The figure demonstrates an up-regulated responsiveness of IL-23p19 transcript levels on nonspecific stimulation of LPMC in patients with CD in comparison with patients with UC (P = 0.046). Figure 2. View largeDownload slide Increase of IL-23 transcript levels after stimulation of intestinal biopsies by PMA and ionomycin as described in patients and methods. The figure demonstrates an up-regulated responsiveness of IL-23p19 transcript levels on nonspecific stimulation of LPMC in patients with CD in comparison with patients with UC (P = 0.046). Discussion CD and UC are chronic inflammatory disorders of the colon associated with an exaggerated immune response, leading to destruction of the mucosal tissue. Several reports have shown that elevated TH1 cytokines such as IL-12 or IFN-γ are associated with CD, whereas up-regulated TH2 cytokines such as IL-4 and IL-5 characterize the inflammatory process in UC. Recently, in animal models it has been shown that anti-IL-12 strategies such as anti-IL-12-antibodies or antagonizing IL-12p40 homodimers may become useful therapeutic options in CD by leading to a specific abrogation of the inflammatory process. The newly described novel p19 protein, engaging IL-12p40 to form the new termed cytokine IL-23, is distantly related to the p35 subunit of IL-12.17 Hence, the question has been raised of whether the previously mentioned anti-IL-12 strategies may be directed against IL-23 rather than against IL-12 itself. Moreover, very recently IL-27, a novel heterodimeric cytokine that consists of p28, a newly discovered IL-12p35-related polypeptide, and EB13, has been described. EB13 is an IL-12p40-related protein and is much more widely expressed than p28. It is able to form a homodimer (with another EB13 protein) as well as heterodimers with IL-12p35 itself, its homolog p28,20 or maybe other yet-unidentified partners. In animal models EB13 seems to play a critical role in the induction of TH2-mediated tissue inflammation in vivo. It has been suggested to mediate this function through the control of invariant natural killer T cell function.27 However, IL-27 has been shown to drive rapid clonal expansion of naive but not memory CD4+ T cells and to synergize with IL-12 to trigger IFN-γ production of naive CD4+ T cells, thus promoting TH1-mediated immune response.20 In the presented study we therefore examined IL-23p19, IL-27p28, and EB13 transcript levels in mucosal biopsy specimens derived from inflamed and normal tissue from IBD patients, as well as from normal control subjects and patients with SC (infectious colitis and ischemic colitis). The data presented show that the expression of the IL-23p19 gene is strongly up-regulated in CD, whereas only a lower elevation of these transcripts could be observed in patients with UC. There are only small, but detectable amounts of IL-23p19 transcripts in normal mucosa as well as in mucosa from patients with specific colitis. Especially in CD, we could demonstrate a positive correlation between elevated IL-23p19 transcript levels and macroscopic signs of inflammation in terms of Rutgeerts score. Moreover, the elevated IL-23p19 responsiveness in stimulated mucosal tissue samples of patients with CD compared with control subjects or patients with UC supports the view of a pathogenetic role of this cytokine. Nevertheless, the source of IL-23p19 within the mucosal tissue needs further evaluation. In addition to an increased expression of IL-23p19 transcripts, we could demonstrate that increased IL-27p28 transcripts were detected only in inflamed mucosal biopsies from patients suffering from CD that is associated with an exaggerated TH1-mediated immune response. Interestingly, we could observe an increased expression of EB13 transcripts in these patients, as well, whereas Christ et al28 did not observe increased EB13 levels in CD patients at all and Omata et al16 found an increased expression in EB13 only in a subset of patients with CD. In our study in biopsies from patients with SC as well as UC, considered as a TH2-mediated disease, no significant increases in IL-27p28 or EB13 transcripts have been observed. Thus, our data confirm the view of a predominantly TH1-promoting role of IL-27p28. The role of EB13 seems to be less clear, as we found an increased expression in CD patients with active disease, whereas Omata et al16 found this subunit predominantly elevated in patients with active as well as inactive UC. In contrast to our data, Christ et al28 observed increased EB13-levels only in active UC on mRNAas well as on protein level (but not in active CD patients). Moreover, EB13 seems to play an important role in animal models of TH2-mediated diseases, as it has been shown by the use of EB13−/− mice.27 Maybe, even if highly speculative, EB13 is able to play a role in different types of mucosal inflammation, depending on the type of dimerization of this subunit, for instance, as a homodimer or in association with other subunits, such as IL-12p35 or other, yet unknown proteins. Undoubtedly, these inconsistent results require further investigation. From a critical point of view, it should be pointed out that Desreumaux et al29 demonstrated that early ileal lesions of patients with CDwere associated with a significant increase of IL-4 mRNA and a decrease of IFN-γ mRNA compared with the normal mucosa of patients with CD or control subjects. The typical TH1-type pattern was observed in this study only in chronic ileal lesions.29 Therefore, concerning the in vivo data and experimental models, including transgenic mice, the TH1/TH2 paradigm seems to be an oversimplification of the intricacies of the immune system; however, it offers an useful concept for understanding of the chronic inflammation. In patients with CD who were successfully treated with corticosteroids, we found a remarkable decrease of IL-23p19 transcript levels. In addition, 8 of 9 CD patients entering remission after steroid treatment showed a normalization of initially elevated IL-27p28 levels. In contrast to this, the average IL-23p19 transcript levels in patients not entering remission or experiencing an early relapse were increasing. The sometimes variable expression levels in an individual patient, who enters remission in the absence of a normalized IL-23p19 level or who experiences a disease relapse while having normal IL-23p19 transcripts may be explained by the fact that the stage or chronicity of the disease influences cytokine production in patients with IBD. The immunomodulary effects of the mentioned anti-IL-12 strategies in animal models of IBD may act (at least in part) via modulating IL-23 action as a proinflammatory cytokine rather than acting via direct IL-12 antagonism. Even if a study published very recently could demonstrate that IL-23 rather than IL-12 is the critical cytokine for inflammation in experimental autoimmune encephalomyelitis,19 further studies have to evaluate the potential role of IL-23 and IL-12 and their respective receptors16,30 in IBDs. In summary, our data on mucosal expression of IL-23p19, IL-27p28, and EB13 transcripts suggest that these recently discovered cytokines play a pivotal role in the network of (pro-) inflammatory cytokines in IBD (and particularly in CD) in contrast to infectious diseases or ischemic lesions. The differential stimulatory effects of IL-23 and IL-27 on naive and memory T cells together with a strongly synergistic action of IL-12 to trigger IFN-γ production may contribute to the perpetuation of the inflammatory process in these patients. Future studies should address the question, whether our results can be confirmed on protein level, and have to evaluate whether these cytokines might become targets of influencing the inflammatory mucosal process. Acknowledgment The authors thank the nursing staff of the Endoscopy Department for their help in specimen collection. References 1. Duchmann R, Schmitt E, Knolle P, et al. 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D'Haens GR, Geboes K, Peeters M, et al. Early lesions of recurrent Crohn's disease caused by infusion of intestinal contents in excluded ileum. Gastroenterology.  1998; 114: 262– 267. CrossRef Search ADS PubMed  25. Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res.  1996; 6: 995– 1001. CrossRef Search ADS PubMed  26. Heid CA, Stevens J, Livak KJ, et al. Real time quantitative PCR. Genome Res.  1996; 6: 986– 994. CrossRef Search ADS PubMed  27. Nieuwenhuis EES, Neurath MF, Corraza N, et al. Disruption of T helper 2-immune responses in Epstein-Barr virus-induced gene 3-deficient mice. Proc Natl Acad Sci USA.  2002; 26: 16951– 16956. CrossRef Search ADS   28. Christ AD, Stevens AC, Koeppen H, et al. An interleukin 12-related cytokine is up-regulated in ulcerative colitis but not in Crohn's disease. Gastroenterology.  1998; 115: 307– 313. CrossRef Search ADS PubMed  29. Desreumaux P, Brandt E, Gambiez L, et al. Distinct cytokine patterns in early and chronic ileal lesions of Crohn's disease. Gastroenterology.  1997; 113: 118– 126. CrossRef Search ADS PubMed  30. Parham C, Chirica M, Timans J, et al. A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rβ1 and a novel cytokine receptor subunit, IL.23R. J Immunol.  2002; 168: 5699– 5708. CrossRef Search ADS PubMed  Copyright © 2004 by Lippincott Williams & Wilkins. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Inflammatory Bowel Diseases Oxford University Press

Expression of Interleukin-12-Related Cytokine Transcripts in Inflammatory Bowel Disease: Elevated Interleukin-23p19 and Interleukin-27p28 in Crohn's Disease But Not in Ulcerative Colitis

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Copyright © 2004 by Lippincott Williams & Wilkins.
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1078-0998
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1536-4844
DOI
10.1097/00054725-200501000-00003
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Abstract

Abstract Background It has been suggested that Crohn's disease (CD) is associated with an exaggerated T-helper 1 cytokine response manifested by increased production of interleukin (IL)-12. IL-12 is a heterodimeric protein comprising 2 disulfide-linked subunits designated p35 and p40. Recently, IL-12-related cytokines, IL-23 and IL-27, were described. Biologically active IL-23 is a heterodimer whose p40 subunit is identical to IL-12p40 whereas its p19 subunit is distantly related to IL-12p35. IL-27 consists of EBI3, an IL-12p40-related protein, and p28, a newly discovered IL-12p35-related polypeptide. Aim We sought to determine whether mucosal expression of IL-23p19 and IL-27p28 transcripts correlate with the inflammatory activity in inflammatory bowel disease (IBD). Patients/Methods Messenger RNA expression in colonic mucosa from patients with Crohn's disease (CD; n = 37) and ulcerative colitis (UC; n = 19), and in non-IBD control subjects (specific colitis [SC]; n = 16) and normal, nondiseased control patients (n = 12) was measured by reverse-transcribed real-time polymerase chain reaction. Results IL-23p19 was significantly increased in inflamed mucosa in CD (P = 0.0377) and to a lesser extent also in UC patients but not in SC patients. Elevation of IL-23p19 transcript levels in CD correlated with the severity of endoscopic lesions. IL-27p28 transcripts and EBI3 transcripts were significantly elevated only in active CD. Discussion IL-23p19, IL-27p28, and EBI3 transcripts are strongly up-regulated in CD. The stimulatory effects of these cytokines on naive T cells in addition to a strongly synergistic action with IL-12 to trigger interferon-γ production may contribute to the perpetuation of the inflammatory process in patients with CD. Notably, increased expression of IL-23 and IL-27 transcripts in CD suggests a T helper 1-dominated immunologic function in this disease. Crohn's disease, interleukin-23, interleukin-27, realtime polymerase chain reaction, ulcerative colitis Despite intensive scientific research, the pathogenic mechanisms of inflammatory bowel disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC), remain unclear. However, distinct cell subsets and cell products have been identified that contribute to the inflammatory and destructive process. It has been shown that an uncontrolled or inadequately up-regulated cellular immune response of the intestinal mucosa toward an unknown pathogen, probably derived from the luminal content, plays a pivotal role in initiation and perpetuation of the disease.1,2 As a result of this mucosal injury, a breakdown of the epithelial barrier occurs, leading to an increased influx of intestinal content to the lamina propria, which further exaggerates the uncontrolled immune response.3,4 Recently, it has been suggested that CDis associated with an elevated T-helper 1 (TH1) response as manifested by increased production of interleukin (IL)-2, IL-12, IL-18, and interferon (IFN)-γ.4,–8 These cytokines are thought to contribute to disease development by orchestrating the inflammatory process. IL-12 is a 75-kDa heterodimeric protein that is produced by macrophages and dendritic cells. It comprises 2 disulfide-linked subunits designated p35 and p40 (according to their approximate molecular weights), forming the biologically active heterodimeric molecule (p70). IL-12 is secreted in mucosal tissue of patients with CD, contributing to the characteristic predominance of TH1 cytokine response of the mucosal immune system.9,–15 However, messenger (m) RNA transcripts for both subunits of IL-12 are only rarely detectable in situ and especially IL-12p40 expression is heterogeneous in CD patients.16 Very recently, a new p19 protein has been described that engages the IL-12 subunit p40 to form a new cytokine, named IL-23, which shows some biological activities similar to but others distinct from IL-12.17 Macrophages, dendritic cells and hemopoietic cells such as TH1 T cells have been identified as a source of IL-23. Although p19 shows no biologic activity by itself, combined with IL-12p40 it binds to IL-12 receptor β1 (IL-12Rβ1) but fails to engage IL-12Rβ2. Additionally, a new subunit of the receptor for IL-12 has been identified, termed IL-23R, that pairs with IL-12Rβ1 to confer IL-23 responsiveness.18 Similar to IL-12, human IL-23 stimulates IFN-γ production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.17 In respect of autoimmune inflammation Cua and coworkers19 demonstrated in a study that IL-23 rather than IL-12 is the critical cytokine for autoimmune inflammation of the brain. Another novel heterodimeric cytokine that has been termed IL-27 consists of a new discovered p28 protein, which is related to the p35-subunit of IL-12 and the Ebstein-Barr virus-induced gene 3 (EBI3), an IL-12p40-related protein. IL-27 is secreted by antigen-presenting cells, leading to rapid clonal expansion of naive CD4+ T cells and triggers IFN-γ secretion of these cells in synergism with IL-12.20 In this work, we determined whether the mucosal expression of IL-23p19, IL-27p28, and EBI3 was altered in patients with CD and UC by examining biopsy specimens from involved and normal mucosa, respectively, and comparing these results to patients with specific IBD as well as normal control patients. Materials and Methods Patients Mucosal biopsy specimens were obtained from ileocolonic mucosa from the following groups of patients. The first group represents 56 IBD patients with active disease (CD, 37 patients; UC, 19 patients; see Table 1). In all patients, paired samples from active and inactive mucosa were obtained. The diagnosis of these subjects was defined by standard clinical, radiologic, endoscopic, and histologic criteria.21 The second group (control subjects) included mucosal biopsy specimens of macroscopically and microscopically normal colonic mucosa from 12 patients without gastrointestinal disease (colonoscopy was performed to exclude colorectal neoplasia). The third group included 16 patients with specific IBD (ischemic colitis, bacterial colitis, i.e., specific colitis [SC]). Table 1. Patients Characteristics     View Large Table 1. Patients Characteristics     View Large Macroscopically, signs of inflammation in CD patients were assessed as described by Rutgeerts et al22: 0, no lesions; 1, less than 5 aphthous lesions; 2, more than 5 aphthous lesions with normal mucosa between the lesions; 3, diffuse aphthous lesions with diffusely inflamed mucosa; and 4, diffuse inflammation with larger ulcers, nodules or narrowing. In UC and SC patients, macroscopic signs of inflammation were assessed by a similar score ranging from 0 to 4 in adaptation to the score of Baron et al23: 0, no lesions; 1, granular mucosa; 2, contact bleeding/spontaneous bleeding; 3, erosions and small ulcers; 4, severe changes with ulcers, severely hemorrhagic mucosa and pus present. Biopsy samples were assessed blindly by the pathologist (I. M.-M.). The hematoxylin and eosin-stained specimens were scored according to a global histologic disease activity score described previously by D'Haens et al.24 This score summarizes several different histologic characteristics, including architectural changes, epithelial damage, the presence of inflammation, and granulomas and erosions or ulcerations. Histology scores ranged from 0 (normal) to 12. Previously, it has been difficult to accurately and reproducibly quantify mRNA levels using real-time polymerase chain reaction (PCR). However, the recent development of the real time PCR methodology has eliminated much of this variability, and should allow for more a routine and reliable quantification of PCR products.25,26 Tissue Processing Mucosal biopsies were collected in 1 mL of RNA-later Ambion, Austin, TX) and shipped to the processing laboratory. Upon arrival, the samples were transferred into RiboLyser tubes “Green” (ThermoHYBAID, Heidelberg, Germany) containing 400 μL of lysis buffer from the MagnaPure mRNA Isolation Kit II (Roche Diagnostics, Mannheim, Germany). Tissue was disrupted by one run with the RiboLyser (ThermoHYBAID) for 20 seconds each. After each run, the RiboLyser tubes were centrifuged at 4 °C for 1 minute at 13,000 rpm, and 300 μL of the lysate was collected and stored at −80 °C until mRNA isolation. The samples were thawed at room temperature for 30 minutes with constant mixing. To each sample, 600 L of capture buffer containing oligo-dT was added. After centrifugation at 13,000 rpm for 5 minutes, 880 μL of this mix was transferred into a MagnaPure sample cartridge and the mRNA was isolated with the MagnaPure-LC device using the mRNA-II standard protocol. The elution volume was set to 50 μL. An aliquot of 8.2 μL of RNA was reverse transcribed using AMV-RT and oligo-(dT) as primer (First Strand cDNA synthesis kit, Roche) according to the manufacturer's protocol in a thermocycler. After termination of the cDNA synthesis, the reaction mix was diluted to a final volume of 500 μL and stored at −20 °C until PCR analysis. Real-time PCR Parameter specific primer sets optimized for the LightCycler were developed and purchased from Search-LC GmbH (Heidelberg, Germany). For further description of the used primers please visit www.search-lc.com. PCR was performed with the LightCycler FastStart DNA Sybr GreenI kit (RAS) according to the protocol provided in the parameter specific kits. To control for specificity of the amplification products, a melting curve analysis was performed. The copy number was calculated from a standard curve, which was obtained by plotting known input concentrations of 4 different plasmids at log dilutions to the PCR cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. Using 952 data points, the actual copy number per μl of cDNA was calculated as follows: X = e(−0.6585*CP + 20.972). The amplification efficiency of the PCR was determined by running log dilutions of standards. The slope of the standard curve was converted to the amplification efficiency E by the following algorithm: E = 10−1/slope. All used primer sets had an efficiency greater than 1.86. RNA input was normalized by the average expression of the 2 housekeeping genes β-actin and cyclophilin B. The data of 2 independent analyses for each sample and parameter were averaged and presented as adjusted transcripts/microliter cDNA. Multiplying this number by 3000 (because of dilution of the extracted cDNA within the process) gives an estimate of the transcripts in an average biopsy. Culture of Intestinal Mucosal Biopsies Whole biopsy samples were cultured in an humidified incubator (6% CO2) at 37 °C in RPMI 1640 medium (Gibco, Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco), 100 μg/mL penicillin, 100 μg/mL streptomycin, and 0.03% glutamine. At the beginning of the culture, phorbol 12-myristate 13-acetate (PMA; 5 μg/mL) and ionomycin (1 μg/mL) were added. After 2 hours of culture, biopsies were transferred into 1 mL of RNA-later (Ambion) and shipped to the processing laboratory. Statistical Analysis Results were described as medians and minimum/maximum values. Statistical differences were calculated using a Wilcoxon rank sum test and were considered to be significant at the P ≤ 0.05 level. Correlations were calculated by Spearman correlation coefficients using the StatView SE+Graphics program (SAS Institute, Cary, NC) on a Macintosh Computer. Ethical Considerations The study was approved by the local ethics committee. Each patient gave written informed consent to perform endoscopic procedures and subsequent scientific studies. Results Characterization of IL-23p19 Expression For characterization of the potential role of IL-23 and IL-27 in the pathogenesis of IBD we first investigated transcript levels of the 2 well-known IL-12 subunits p35 and p40. In accordance with former studies we could demonstrate low but detectable levels of IL-12p35 that have been described to be expressed ubiquitously. No significant differences could be observed between normal control subjects (median, 21 × 103 transcripts per biopsy [tpb]), patients with specific colitis (median, 24 × 103 in noninflamed mucosa and 18 × 103 tpb in inflamed mucosa), and patients with CD (median, 18–24 × 103 tpb in accordance with the macroscopic score of inflammation) or ulcerative colitis (15 to 24 × 103 tpb). In contrast to this IL-12 subunit, transcript levels of IL-12p40 were very low or even undetectable in normal control subjects (median, 3 × 103 tpb) as well as in IBD patients (median, 3 to 6 × 103 tpb in patients with CD and 0 to 3 × 103 tpb in UC patients) or in patients with specific colitis (median, 0 tpb). Again, no significant differences could be observed between these groups of patients. In initial experiments we determined IL-23p19 expression in control subjects. All mucosal samples revealed low but detectable transcripts of IL-23p19, ranging from 3 × 103 to 54 × 103 tpb with a median of 9 × 103 tpb (Table 2). In biopsy specimens taken from macroscopically normal mucosa from patients with CD or UC, respectively, IL-23p19 transcript levels showed no difference from those taken from normal control subjects. Compared with normal mucosa, biopsy samples taken from inflamed mucosa of CD patients revealed significantly elevated IL-23p19 transcript levels. Furthermore, a correlation between macroscopic signs of inflammation of the affected mucosal tissue (in terms of Rutgeert score) and IL-23p19 expression could be demonstrated. In parallel, in patients with active UC, elevated IL-23p19 transcript levels were observed. Otherwise, in patients with SC, elevated IL-23p19 levels have not been detected, whereas these patients' unaffected mucosa showed comparable IL-23p19 expression as in normal control subjects. The values of biopsy specimens from inflamed mucosa of these patients were summarized independent of the macroscopic score. Table 2. Number of IL-23 Transcript Copies With CD, UC, and Normal Control Patients Depending on the Macroscopical Signs of Inflammation     View Large Table 2. Number of IL-23 Transcript Copies With CD, UC, and Normal Control Patients Depending on the Macroscopical Signs of Inflammation     View Large It should be noted that in all patients paired samples obtained from inflamed and noninflamed-so-called normal-mucosa were analyzed. Intraindividual comparison supported the overall finding of clear differences in IL-23p19 transcript levels depending on disease activity. Successful Treatment of Active IBD Resulted in a Decrease of IL-23 Transcript Concentration Because mucosal IL-23p19 transcript levels were increased in patients with active CD and (to a lesser extent) with UC, we investigated the effects of steroid treatment on this parameter. Twenty-four patients (18 patients with CD and 6 patients with UC) underwent control ileocolonoscopy and determination of IL-23p19 transcript levels after a median follow-up of 8 weeks. We evaluated the effect of steroid treatment in patients with active disease, not having been treated with steroids at the time of the initial ileocolonoscopy and evaluation of transcript levels. None of these patients has received other concomitant therapy such as azathioprine. Asignificant decrease of initially elevated transcript levels in patients entering remission from a median of 63 × 103 transcripts per biopsy to 15 × 103 tpb was found. In these patients an improvement of clinical symptoms was observed, and patients were in a stable remission during the whole follow-up period of 6 months. Most patients showed a decrease of IL-23p19 transcripts, even if individual patients experienced an increase in transcript levels, with 3 patients still having elevated transcripts. However, 6 patients who did not enter complete remission or experienced early relapses of their IBD showed increasing IL-23p19 transcript levels from a median of 15 × 103 transcripts per biopsy to 63 × 103 tpb. In these patients, IL-23p19 transcripts decreased only marginally or even increased further. For further data on the development in every individual patient, see Figure 1a. Figure 1. View largeDownload slide Development of IL-23p19 transcripts (a) and IL-27p28 transcripts (b), respectively, after steroid treatment in patients with IBD (18 patients with CD and 6 patients with UC). The dotted line denotes the upper range of normal controls. White symbols characterize patients not entering complete remission or having early relapses of IBD, whereas black symbols characterize patients entering remission. Figure 1. View largeDownload slide Development of IL-23p19 transcripts (a) and IL-27p28 transcripts (b), respectively, after steroid treatment in patients with IBD (18 patients with CD and 6 patients with UC). The dotted line denotes the upper range of normal controls. White symbols characterize patients not entering complete remission or having early relapses of IBD, whereas black symbols characterize patients entering remission. Characterization of IL-27p28 Expression Low-but-detectable levels of IL-27p28 mRNA were found in all patients, in normal as well as in diseased mucosa. In normal control subjects a median concentration of 6 × 103 tpb was measured (minimum, 3 × 103 tpb; maximum, 12 × 103 tpb). Compared with these data, no significant elevation of IL-27p28 transcript levels in patients with SC could be demonstrated, neither in macroscopically normal nor in inflamed mucosal tissue samples. Patients with UC expressed the same number of transcripts in noninflamed and in inflamed tissue samples (meannon-inflamed 9 × 103 tpb (minimum-maximum [min-max], 3 × 103 tpb to 12 × 103 tpb) and meaninflamed 6−9 × 103 tpb (minimum-maximum, 3 × 103 tpb to 18 × 103 tpb), respectively). In contrast, patients with CD showed differing expression levels. Whereas the number of IL-27p28 transcripts in macroscopically normal or only moderately inflamed mucosa (grade 0 or 1 in terms of Rutgeert score) did not differ significantly from that in control subjects, more severe inflamed mucosal tissue (Rutgeert score, grade 2–4) exhibited significant differences in comparison with normal control subjects. The former mucosal biopsies revealed a median of 6 × 103 tpb to 9 × 103 tpb (min-max, 3 × 103 tpb to 36 × 103 tpb) and the latter samples exhibited a median of 18 × 103 tpb to 24 × 103 tpb (min-max, 6 × 103 tpb to 306 × 103 tpb; P = 0.0044 to 0.0348). Characterization of EBI3 Expression All mucosal samples examined revealed detectable levels of EBI3 transcripts. A median concentration of 117 × 103 tpb was measured (minimum 63 × 103 tpb, maximum 204 × 103 tpb) in normal control subjects. EBI3 expression in patients with SC did not differ significantly from the control group, neither in macroscopically normal nor in inflamed mucosal tissue. In patients with CD, we found a significant decrease in EBI3 transcript levels in macroscopically normal or only moderately inflamed mucosa (grade 0 or 1 in terms of Rutgeert score); transcript levels ranged from 12 × 103 tpb to 390 × 103 tpb (min-max median, 93 × 103 tpb). In contrast to this, in more severe inflamed mucosal tissue (Rutgeert score, grade 2 to 4) EBI3 transcripts were significantly elevated (median, 129 × 103 tpb; min-max, 15 × 103 tpb to 972 × 103 tpb; P = 0.001). Interestingly, and in contrast to the data published by Omata et al,16 we did not find significant differences in EBI3 expression in patients with UC, neither in tissue samples from normal (median, 93 × 103 tpb; min-max, 0 × 103 tpb to 303 × 10 3 tpb; P = 0.065 to 0.515), nor from inflamed colonic areas (median, 102 × 103 tpb; min-max, 0 × 103 tpb to 372 × 103 tpb; P = 0.068 to 0.686). IL-27 Transcript Concentration After Treatment of Active IBD Because mucosal IL-27p28 transcripts were increased in a subset of patients with active CD, we investigated the effects of steroid treatment on this parameter in both groups of patients with IBD. Again, 24 patients (18 patients with CD and 6 patients with UC) underwent control ileocolonoscopy and determination of IL-27p28 transcripts after a median follow-up of 8 weeks. The 2 UC patients not entering remission had transcript levels within the reference range. Two patients with UC who entered remission after 8 weeks of treatment had normal transcript levels before and after steroid treatment, and only 2 patients (of 6 CU patients examined) showed slightly elevated IL-27p28 levels (15 × 103 tpb) that became normal 8 weeks after treatment (3 and 6 × 103 tpb, respectively). In 9 CD patients entering remission, IL-27p28 levels were elevated in the first biopsy (median, 21 × 103 tpb; min-max, 15 × 103 tpb to 360 × 103 tpb), and 8 of those patients showed normal transcript levels after treatment (median, 6 × 103 tpb; min-max, 0 to 9 × 103 tpb). However, 6 patients entering remission showed normal transcript levels in the initial examination (≤ 12 × 103 tpb). Three patients with CD did not enter remission, 2 with initially elevated transcript levels that normalized in 1 case and 1 patient with a normal IL-27p28 mRNA levels at the initial examination. Cytokine Expression in Mucosal Biopsies After Activation of Immunocompetent Cells in Vitro In 7 patients with UC and 14 patients with CD, mucosal biopsy specimens were examined regarding their IL-23p19-expression after stimulation with an activator of T cells PMA and ionomycin [Iono]. These data show an up-regulation of IL-23p19 responsiveness in intestinal tissue samples of CD patients compared with UC patients (P = 0.046; Fig. 2). Interestingly, the responsiveness in inflamed areas of either disease was not as high as that in noninflamed specimens, possibly because of higher transcript levels in these areas before stimulation with PMA/Iono. No significant differences in cytokine responsiveness upon stimulation with PMA/ionomycin could be observed concerning the expression of IL-12p35 (P = 0.753), IL-12p40 (P = 0.463), IL-27p28 (P = 0.917), or EB13 (P = 0.080). Moreover, the trend toward increased EB13 responsiveness was in favor of a higher expression in patients with CD. Figure 2. View largeDownload slide Increase of IL-23 transcript levels after stimulation of intestinal biopsies by PMA and ionomycin as described in patients and methods. The figure demonstrates an up-regulated responsiveness of IL-23p19 transcript levels on nonspecific stimulation of LPMC in patients with CD in comparison with patients with UC (P = 0.046). Figure 2. View largeDownload slide Increase of IL-23 transcript levels after stimulation of intestinal biopsies by PMA and ionomycin as described in patients and methods. The figure demonstrates an up-regulated responsiveness of IL-23p19 transcript levels on nonspecific stimulation of LPMC in patients with CD in comparison with patients with UC (P = 0.046). Discussion CD and UC are chronic inflammatory disorders of the colon associated with an exaggerated immune response, leading to destruction of the mucosal tissue. Several reports have shown that elevated TH1 cytokines such as IL-12 or IFN-γ are associated with CD, whereas up-regulated TH2 cytokines such as IL-4 and IL-5 characterize the inflammatory process in UC. Recently, in animal models it has been shown that anti-IL-12 strategies such as anti-IL-12-antibodies or antagonizing IL-12p40 homodimers may become useful therapeutic options in CD by leading to a specific abrogation of the inflammatory process. The newly described novel p19 protein, engaging IL-12p40 to form the new termed cytokine IL-23, is distantly related to the p35 subunit of IL-12.17 Hence, the question has been raised of whether the previously mentioned anti-IL-12 strategies may be directed against IL-23 rather than against IL-12 itself. Moreover, very recently IL-27, a novel heterodimeric cytokine that consists of p28, a newly discovered IL-12p35-related polypeptide, and EB13, has been described. EB13 is an IL-12p40-related protein and is much more widely expressed than p28. It is able to form a homodimer (with another EB13 protein) as well as heterodimers with IL-12p35 itself, its homolog p28,20 or maybe other yet-unidentified partners. In animal models EB13 seems to play a critical role in the induction of TH2-mediated tissue inflammation in vivo. It has been suggested to mediate this function through the control of invariant natural killer T cell function.27 However, IL-27 has been shown to drive rapid clonal expansion of naive but not memory CD4+ T cells and to synergize with IL-12 to trigger IFN-γ production of naive CD4+ T cells, thus promoting TH1-mediated immune response.20 In the presented study we therefore examined IL-23p19, IL-27p28, and EB13 transcript levels in mucosal biopsy specimens derived from inflamed and normal tissue from IBD patients, as well as from normal control subjects and patients with SC (infectious colitis and ischemic colitis). The data presented show that the expression of the IL-23p19 gene is strongly up-regulated in CD, whereas only a lower elevation of these transcripts could be observed in patients with UC. There are only small, but detectable amounts of IL-23p19 transcripts in normal mucosa as well as in mucosa from patients with specific colitis. Especially in CD, we could demonstrate a positive correlation between elevated IL-23p19 transcript levels and macroscopic signs of inflammation in terms of Rutgeerts score. Moreover, the elevated IL-23p19 responsiveness in stimulated mucosal tissue samples of patients with CD compared with control subjects or patients with UC supports the view of a pathogenetic role of this cytokine. Nevertheless, the source of IL-23p19 within the mucosal tissue needs further evaluation. In addition to an increased expression of IL-23p19 transcripts, we could demonstrate that increased IL-27p28 transcripts were detected only in inflamed mucosal biopsies from patients suffering from CD that is associated with an exaggerated TH1-mediated immune response. Interestingly, we could observe an increased expression of EB13 transcripts in these patients, as well, whereas Christ et al28 did not observe increased EB13 levels in CD patients at all and Omata et al16 found an increased expression in EB13 only in a subset of patients with CD. In our study in biopsies from patients with SC as well as UC, considered as a TH2-mediated disease, no significant increases in IL-27p28 or EB13 transcripts have been observed. Thus, our data confirm the view of a predominantly TH1-promoting role of IL-27p28. The role of EB13 seems to be less clear, as we found an increased expression in CD patients with active disease, whereas Omata et al16 found this subunit predominantly elevated in patients with active as well as inactive UC. In contrast to our data, Christ et al28 observed increased EB13-levels only in active UC on mRNAas well as on protein level (but not in active CD patients). Moreover, EB13 seems to play an important role in animal models of TH2-mediated diseases, as it has been shown by the use of EB13−/− mice.27 Maybe, even if highly speculative, EB13 is able to play a role in different types of mucosal inflammation, depending on the type of dimerization of this subunit, for instance, as a homodimer or in association with other subunits, such as IL-12p35 or other, yet unknown proteins. Undoubtedly, these inconsistent results require further investigation. From a critical point of view, it should be pointed out that Desreumaux et al29 demonstrated that early ileal lesions of patients with CDwere associated with a significant increase of IL-4 mRNA and a decrease of IFN-γ mRNA compared with the normal mucosa of patients with CD or control subjects. The typical TH1-type pattern was observed in this study only in chronic ileal lesions.29 Therefore, concerning the in vivo data and experimental models, including transgenic mice, the TH1/TH2 paradigm seems to be an oversimplification of the intricacies of the immune system; however, it offers an useful concept for understanding of the chronic inflammation. In patients with CD who were successfully treated with corticosteroids, we found a remarkable decrease of IL-23p19 transcript levels. In addition, 8 of 9 CD patients entering remission after steroid treatment showed a normalization of initially elevated IL-27p28 levels. In contrast to this, the average IL-23p19 transcript levels in patients not entering remission or experiencing an early relapse were increasing. The sometimes variable expression levels in an individual patient, who enters remission in the absence of a normalized IL-23p19 level or who experiences a disease relapse while having normal IL-23p19 transcripts may be explained by the fact that the stage or chronicity of the disease influences cytokine production in patients with IBD. The immunomodulary effects of the mentioned anti-IL-12 strategies in animal models of IBD may act (at least in part) via modulating IL-23 action as a proinflammatory cytokine rather than acting via direct IL-12 antagonism. Even if a study published very recently could demonstrate that IL-23 rather than IL-12 is the critical cytokine for inflammation in experimental autoimmune encephalomyelitis,19 further studies have to evaluate the potential role of IL-23 and IL-12 and their respective receptors16,30 in IBDs. In summary, our data on mucosal expression of IL-23p19, IL-27p28, and EB13 transcripts suggest that these recently discovered cytokines play a pivotal role in the network of (pro-) inflammatory cytokines in IBD (and particularly in CD) in contrast to infectious diseases or ischemic lesions. The differential stimulatory effects of IL-23 and IL-27 on naive and memory T cells together with a strongly synergistic action of IL-12 to trigger IFN-γ production may contribute to the perpetuation of the inflammatory process in these patients. Future studies should address the question, whether our results can be confirmed on protein level, and have to evaluate whether these cytokines might become targets of influencing the inflammatory mucosal process. Acknowledgment The authors thank the nursing staff of the Endoscopy Department for their help in specimen collection. References 1. Duchmann R, Schmitt E, Knolle P, et al. 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Journal

Inflammatory Bowel Diseases – Oxford University Press

Published: Jan 1, 2005

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