The IGF system in patients with inflammatory bowel disease treated with prednisolone or infliximab: potential role of the stanniocalcin-2 / PAPP-A / IGFBP-4 axis

Details of the study participants and procedures have previously been described [27]. A total of 38 consecutive patients with active IBD were enrolled, of which 20 patients presented with ulcerative colitis and 18 with Crohn’s disease. The patients had been diagnosed according to the standard clinical, biochemical, endoscopic, and histopathological criteria. Inclusion criteria were age above 18 years and moderate-to-severe active ulcerative colitis or Crohn’s disease that required systemic anti-inflammatory treatment with prednisolone or infliximab. Thus, all patients exhibited clinically active, inflammatory disease on the basis of standard criteria, including elevated C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), orosomucoid, and fecal calprotectin, and disease activity was furthermore scored according to the Harvey Bradshaw Index (HBI) [28] in patients with Crohn’s disease and the Simple Clinical Colitis Activity Index (SCCAI) [29] in patients with ulcerative colitis. All patients were bio-naïve, but had previously been treated with azathioprine (n = 8), 5-aminosalicylic acid (n = 13), both azathioprine and 5-aminosalicylic acid (n = 2) or budesonide (n = 2). Patients treated with budesonide were not assigned to the prednisolone treatment group. Thirteen patients were newly diagnosed or treatment naïve. Exclusion criteria were the use of either prednisolone or infliximab within 8 weeks prior to enrolment. Furthermore, patients with active infections, malignancies, or other catabolic or chronic inflammatory diseases were excluded. Of the included patients, 17 were treated with prednisolone (13 ulcerative colitis and 4 Crohn’s disease patients), and 21 were treated with infliximab (7 ulcerative colitis and 14 Crohn’s disease patients). The department’s gastroenterologists made the treatment decision according to clinical criteria and before informed consent was obtained from the participant. Intravenous treatment was either with methylprednisolone (40 mg twice daily) (Solu-Medrol, Pfizer, NY, USA) or infliximab (5 mg/kg body weight, one dose) (Remicade; Janssen Biologics B.V., Leiden, Netherlands). Patients receiving infliximab treatment had previously experienced steroid resistance or had severe adverse effects in response to steroid treatment. The two treatment groups were balanced with regard to age, sex, height, and weight. All investigations were performed after an overnight fast. Blood samples were collected from all patients at baseline (day 0) and on day 7 and stored at − 80 °C until analysis.

To specifically characterize the STC2/PAPP-A/IGFBP-4-axis in IBD patients, protein measurements on day 0 and day 7 were compared to healthy control subjects. Control EDTA-plasma for IGFBP-4 and STC2 measurements was collected from 50 randomly selected Danish registered blood donors at Aarhus University Hospital (25 women and 25 men, age range 18–50 years). Control serum for PAPP-A measurements was collected from 150 adults (75 women and 75 men, age 44 ± 15 years) [30]. All donors were healthy and received no medication.

The study was approved by The Local Ethics Committee (Journal no. 20060197) and registered at ClinicalTrials.​gov (NCT00955123). Written informed consent was obtained from all participants, and the study conformed to the Declaration of Helsinki.

CRP, ESR, orosomucoid, and creatinine were assayed using routine laboratory methods. Estimated glomerular filtration rate (eGFR) was calculated using the MDRD formula [31]. Cortisol and fecal calprotectin were measured by specific ELISAs (DRG Diagnostics, Marburg, Germany and Bühlmann Laboratories, Schönenbuch, Switzerland, respectively), and TNF-α and IL-6 by highly sensitive immunoassays (RnD Systems, Minneapolis, MN, USA).

Total serum IGF-I was assayed after acid-ethanol extraction using a validated in-house sandwich assay as previously described [32] with minor modifications: the secondary detection antibody was replaced by a biotinylated IGF-I antibody (Cat# I-8773, Sigma-Aldrich, St. Louis, MO, USA). Serum IGF-II concentrations were measured by a validated in-house, time-resolved immunofluorometric assay (TR-IFMA) [33]. IGFBP-3 was determined using the IDS-iSYS Multi-Discipline Automated Analyzer (Immunodiagnostic Systems, Copenhagen, Denmark), as previously published [22].

The ability of serum IGFs to activate the IGF-IR in vitro (bioactive IGF) was determined by an in-house kinase receptor activation assay (KIRA) as originally described [34] with slight modifications. The assay measures the ability of IGF to phosphorylate the IGF-IR in an in vitro-based model employing IGF-IR gene-transfected human embryonic renal cells. In brief, transfected cells were stimulated with diluted serum for 15 min at 37 °C. A serial dilution of rhIGF-I (WHO 02/254) served as calibrator. Following cell lysis, the concentration of phosphorylated IGF-I receptor was determined in the crude cell lysates using a phospho-IGF-IR ELISA (Cat# DYC 1770E, RnD Systems, Abingdon, UK). The KIRA assay signal primarily reflects binding of IGF-I to the IGF-IR, but also binding of IGF-II and pro-IGF-II (cross-reactivity 12 and 2%, respectively). The cross-reactivity of pro-insulin and insulin is negligible (< 1%). Hence, the assay signal is referred to as IGF bioactivity. The KIRA assay has a detection limit < 0.08 μg/L and intra- and inter-assay CVs of 12 and 20%, respectively.

EDTA-plasma levels of intact IGFBP-4 and the two PAPP-A generated fragments, C-terminal (CT)-IGFBP-4 and N-terminal (NT)-IGFBP-4, were measured in duplicate by in-house TR-IFMAs using monoclonal antibodies (mAb) and recombinant human (rh) calibrators generously provided by HyTest Ltd. (Turku, Finland). The assays were performed as recently described [14, 21‐23]. In each fragment assay, one of the antibodies specifically recognized the proteolytic neoepitope generated upon cleavage by PAPP-A. Detection limits were 0.5 μg/L for IGFBP-4, 0.4 μg/L for CT-IGFBP-4 and 0.9 μg/L for NT-IGFBP-4. Intra- and inter-assay CVs were < 10 and < 15%, respectively.

PAPP-A and STC2 levels were determined by commercial sandwich ELISAs (PAPP-A; Cat# AL-101 and STC2; Cat# AL-143) from AnshLabs (Webster, TX, USA). Assay procedures were as described by the manufacturer, and both assays behaved linearly within the analytical range.

Baseline data have previously been described and analyzed [27]. The assumption of normality was checked using quantile plots, and non-normally distributed variables were transformed using the natural logarithm prior to statistical analyses. Unpaired continuous observations were analyzed at baseline using Student’s t-test, and paired observations on day 0 and day 7 were analyzed using paired t-test. Variables that remained non-normally distributed upon transformation were analyzed using the nonparametric Wilcoxon matched-pairs signed-ranks test or Mann Whitney U-statistics as appropriate. Categorical variables were compared using χ²-test. Differences in protein concentrations between IBD patients and controls were assessed by unpaired t-test. Correlations were examined using Pearson’s correlation analysis. To investigate the overall change from day 0 to day 7 in IGF system parameters and inflammatory and metabolic markers, we calculated delta values in each subject. Correlations between delta values were investigated using Pearson or Spearman correlation coefficient as appropriate. The proteins investigated in the present study have previously shown to provide ample statistical power in cohorts of 9–24 subjects [10, 35, 36], supporting that differences in proteins levels would be detectable in a cohort of 38 patients. Data are reported as means ± SD for normally distributed variables and medians with interquartile range for non-normally distributed variables. Level of significance was p < 0.05. Statistical analyses were performed using STATA 13 (StataCorp, College Station, TX, USA).

Measurement details of healthy subjects and IBD patients before and after treatment are summarized in Table 1. At baseline, disease scores, CRP, ESR, orosomucoid, fecal calprotectin, cortisol, TNF-α and IL-6 were higher compared with levels in healthy subjects (reference levels not shown), but similar in the two treatment groups. The eGFR was slightly higher in the infliximab group. Irrespective of anti-inflammatory treatment, patient SCCAI and HBI disease scores as well as CRP, ESR, orosomucoid, and fecal calprotectin improved during the course. Both treatments resulted in reduced IL-6 levels, whereas infliximab triggered a possibly compensatory increment in TNF-α levels. These data have previously been published [27].

Paired individual levels of IGF system proteins are shown in Fig. 2. At baseline, IGF-I levels were higher in the infliximab group, whereas IGF-II, IGF bioactivity, and IGFBP-3 levels were similar in the two treatment groups (Table 1). Following seven days of prednisolone treatment, both total IGF-I concentration and bioactive IGF, as measured by in vitro IGF-IR activation, were significantly increased (p < 0.001 and p = 0.048, respectively). In contrast, concentrations of IGF-II and IGFBP-3 remained unchanged. When looking at the individual changes, six patients demonstrated numerically lower levels of bioactive IGF after prednisolone treatment, whereas 11 patients showed numerically higher levels following prednisolone. By contrast, the concentration of IGF-I was only reduced in two patients. Upon infliximab treatment, levels of IGF-I, IGF-II, and IGFBP-3 were slightly increased (p = 0.025, p = 0.049, and p = 0.035, respectively), whereas IGF bioactivity remained unaltered.

Total IGF-I and IGF bioactivity were not correlated on day 0 (r = 0.22, p = 0.19), but were strongly associated on day 7 (r = 0.72, p < 0.001). At baseline, intact IGFBP-4 was negatively correlated with PAPP-A (r = − 0.31, p < 0.05) and positively correlated with CT- and NT-IGFBP-4 (r = 0.45, p < 0.005 and r = 0.48, p < 0.005, respectively). In addition, a strong positive correlation was observed between concentrations of CT- and NT-IGFBP-4 at both time points (day 0: r = 0.84, p < 0.001, and day 7: r = 0.93, p < 0.001). CT- and NT-IGFBP-4 were positively associated with TNF-α at baseline (r = 0.34, p < 0.05 and r = 0.44, p < 0.01, respectively), and negatively associated with eGFR (day 0: r = − 0.48, p < 0.005 and r = − 0.62, p < 0.001, respectively, and day 7: r = − 0.34, p < 0.05 and r = − 0.43, p < 0.01, respectively). IGF system proteins did not correlate with CRP, IL-6 or the functional hepatic nitrogen clearance at either time point.

To investigate the individual change from baseline in IGF system parameters within each subject, we calculated the delta values from day 0 to day 7. We observed a positive association between the delta value of IGF-I and IGF bioactivity from day 0 to day 7 (r = 0.47, p < 0.005). Correlations were also observed between the change in CT- and NT-IGFBP-4 and the change in PAPP-A (r = 0.32, p < 0.05 and r = 0.37, p < 0.05, respectively). Thus, the patients that experienced the most substantial increase in fragmented IGFBP-4 also demonstrated the largest increase in PAPP-A. The shift in intact IGFBP-4 from day 0 to day 7 was positively associated with the change in STC2 (r = 0.48, p < 0.005). Accordingly, patients with the most massive reductions in intact IGFBP-4 also showed the most pronounced declines in levels of the PAPP-A inhibitor STC2.