Interleukin 6 , osteoprotegerin , sRANKL and bone metabolism in inflammatory bowel diseases

Results. The prevalence of osteoporosis and osteopenia was as follows: in I – CD, 18.92% and 32.43% in L2–L4; 13.51% and 35.13% in the neck, and in II – UC, 2.7% and 37.84% in L2–L4; 2.7%, and 29.73% in the femoral neck. The concentration of IL-6 correlated negatively with T-scores in the neck for the whole group, and in group I – CD, there was a significant positive correlation between serum OPG and IL-6.


Introduction
Inflammatory bowel diseases (Crohn's disease and ulcerative colitis) cause the development of bone pathologies, like osteopenia and osteoporosis, whose pathogenesis is not fully understood.There are several risk factors for osteoporosis in patients with inflammatory bowel diseases (IBD), including glucocorticoid therapy, reduced physical activity, bone resorption intensified by inflammation (elevated levels of proinflammatory cytokines such as interkeukin-6, interleukin 1 and tumor necrosis factor alpha), poor dietary calcium intake (related to lactose intolerance), magnesium deficiency in the diet, vitamin D deficiency, decreased albumin concentration, and impaired intestinal absorption. 1,2Patients with inflammatory bowel diseases, in whom inflammation is quenched, have higher bone mineral density (BMD) in remission.The overall risk of bone fractures in IBD patients, measured as 1 per 100 patientyears, is 40% higher than in the general population and this risk increases with age. 1,3Cytokines are a system that governs the functioning of many systems.They are mediators of inflammatory processes in the course of inflammatory bowel diseases and are involved in resorption and bone formation.The interrelation and regulation of the cytokine pathway may play a role in bone pathology in patients with IBD.Interleukin 6 (IL-6) is one of the pro-inflammatory cytokines that initiates and enhances inflammation, and also stimulates bone resorption by developing osteoclast progenitors.IL-6 is a glycosylated polypeptide of molecular weight 21-28 kDa, composed of 4 long α helices connected by loops.It is a typical secretory protein, which is produced together with the N-terminal signal peptide.The gene encoding IL-6 is located on chromosome 7 and contains 5 coding segments (exons).IL-6 is the main stimulus for the production of most acute phase proteins. 4Schulte et al. has demonstrated that the polymorphism of IL-6 does not affect bone loss in patients with IBD. 5 IL-6 induces a variety of factors, mainly interleukin-1 (IL-1), but also tumor necrosis factor alpha (TNF-α). 6The gene promoter has sequences that bind transcription factors such as nuclear factor kappa B (NF-κβ), which regulate gene transcription of IL-6 in a manner dependent on the cell type and the activating agent.It is worth noting that attempts have already been made to use antibodies against IL-6 in Crohn's disease. 7IL-6 is one of the proinflammatory cytokines that intensifies osteoclastogenesis, modulating activity of soluble receptor activator of nuclear factor kappa β ligand (sRANKL), which, contrary to osteoprotegerin, does not have osteogenic effects. 8IL-6 is produced by macrophages, monocytes, endothelial cells, and T and B lymphocytes.It shows biological activity only when combined with a specific receptor located on the membrane surface of the target cell and it is a potent stimulator of inflammatory processes.The receptor for IL-6 is composed of 2 subunits: glycoproteins of 80 kDa and 130 kDa.The importance of the osteoprotegerin-sRANKL system increases, as it can be modulated by cytokines, particularly interleukin-6, the concentration of which in serum correlates negatively with BMD. 7

Material and methods
The study group consisted of 37 patients with CD (I -CD) aged 31.7 ±8.0 years on average, including 15 women and 22 men; 37 patients with ulcerative colitis (II -UC) aged 40.6 ±15.1 years on average, including 21 women and 16 men; and 37 healthy volunteers aged 29.6 ±8.0 years on average, including 18 women and 19 men, who constituted the control group (III -CG).The inclusion criteria were as follows: age between 18 and 60 years, diagnosis of IBD based on cross-sectional imaging and/or endoscopy with histopathological confirmation, disease duration >1 year, lack of any other conditions (e.g., rheumatoid arthritis, chronic renal failure), lack of actually biological and steroids therapy which could affect the cytokines profile.
Densitometry of the lumbar spine with L2-L4 assessment and densitometry of the proximal epiphysis of the femur with the assessment of the femoral neck was carried out on all patients, using the Dual Energy X-ray Absorptiometry (DEXA-Lunar DPX-IQ; GE Healthcare Lunar, Boston, USA) technique.The analysis took into account the values of BMD as well as the T-score and Z-score indices.Each patient filled a specially designed questionnaire concerning the current progress and duration of the disease, number of exacerbations and hospitalizations, time and type of pharmacological treatment.
Serum samples for cytokin determinations were stored at -25°C for an average period of 2 months.The samples were not thawed until assay.Quantitative sandwich enzyme immunoassay method (enzyme-linked immunosorbent assay, ELISA) with monoclonal antibody specific for each cytokine/interleukin (IL) specified below was employed.The serum concentration of interleukin 6 was measured by ELISA kits (R&D Systems Inc., Minneapolis, USA) on microplater reader Sunrise TM (Tecan Group Ltd., Männedorf, Switzerland), with a sensitivity of 0.70 pg/mL.The following intra-assay and inter-assay coefficient of variations (CV) were calculated for IL-6: 1.8% and 3.4%.The serum concentrations of sRANKL and OPG were measured by ELISA kits by a sandwich immunoassay method including monoclonal antibodies (BioVendor -Laboratorni medicina a.s., Brno, Czech Republic) on microplater reader Sunrise TM (Tecan Group Ltd., Männedorf, Switzerland), with a sensitivity of 0.1 pmol/L, while intra-assay and inter-assay coefficient of variations were: 6.5% and 6.9% for sRANKL and 6.0% and 7.2% for OPG, respectively.Statistical analysis was carried out using the Kruskal-Wallis test with Dunn's post hoc test to distinguish homogeneous groups.The relationship between the analyzed parameters was assessed using Spearman's rank method.The osteopenia and osteoporosis prevalence in analyzed groups was compared with test for proportions.The analysis was carried out using STATISTICA PL v. 10 software (StatSoft, Tulsa, USA).All test were considered significant at p < 0.05.
Approval for the conduct of the study was obtained from the Bioethics Committee at the Poznan University of Medical Sciences (consent No. 92/09).Informed consent was obtained from every participant.

Results
The aim of the study was to evaluate bone mineral density (BMD) and the prevalence of osteopenia and osteoporosis and to determine the concentration of interleukin-6, osteoprotegerin (OPG) and sRANKL protein (sRANKL) in patients with inflammatory bowel diseases in relation to the control group, and to assess the relationship between IL-6 and OPG, RANKL and s-BMD.The research objective was also to assess the impact of disease duration and number of hospitalizations on BMD.Characteristics of Crohn's disease patients (A) ulcerative colitis (UC) and the control group (B) is included in the table (Table 1).
The analyzed groups were not homogeneous with respect of age.The oldest was group of patients with UC (41 years).This group significantly differs from CD (32 years; p < 0.0001) and control group (30 years; p = 0.0002).No significant difference was observed between CD and control group with respect of age.
The characteristics of patients and control groups are presented in Table 1.
The prevalence of osteoporosis and osteopenia in group I -CD and II -UC in L2-L4 and the femoral neck are presented in Table 2.
I -CD patients group is characterized by a significantly higher rate of osteoporosis compared to II -UC patients group 18.92% vs 2.7%, p = 0.0247.Neck BMD and T-score in I -CD group differs significantly from III -CG group (p < 0.05 = 0.0007), but is not significantly different from II -UC group.The mean concentrations of IL-6 (pg/mL), OPG (pmol/L) and sRANKL (pmol/L) of patients and control groups are presented in Table 3.
The level of OPG (pmol/L) differed significantly between all analyzed groups.The highest level was observed in the I -CD patients group.For both I -CD and II -UC group the IL-6 (pg/mL) level was significantly higher compared to III -CG (I -CD vs III -CG group p < 0.0001; II -UC vs III -CG p < 0.0001).No significant differences in IL-6 (pg/mL) level were observed between the I -CD and the II -UC group (Table 3).
The concentration of IL-6 correlated negatively with neck T-scores for the whole group (r = -0.33;p = 0.0004), and there was a significant positive correlation (r = 0.51; p = 0.0017) between OPG and IL-6 in group I -CD.In patients with CD and UC, the mean concentrations of IL-6 were higher than in CG.This difference was statistically significant (p < 0.0001).There was no significant difference in disease duration between analyzed groups 8.05 ±5.29 in group I -CD, and 8.03 ±7.92 in group II -UC.Disease duration correlated with neck T-and Z-scores (r = -0.40;p < 0.0001 and r = -0.24;p = 0.0120).A similar correlation  15 According to other authors, the concentrations of IL-6 in patients with IBD and osteoporosis are increased, and it is thus believed that IL-6 plays a role in the pathogenesis of bone loss, but the mechanism is not fully understood and requires further studies. 16The findings suggest that the concentration of IL-6 in the serum of patients with IBD in the Croatian population is a clinically important parameter and correlates with the activity of inflammatory disease. 17IL-6, as a pro-inflammatory cytokine, adversely affects bone turnover by influencing the activity of osteoclasts.The negative effect of IL-6 on bone tissue has been shown in patients with rheumatoid arthritis. 18n increased activity of IL-6 accelerates the process of bone resorption through the activity of osteoclasts.Other authors indicate that IL-6 concentrations were higher in postmenopausal women with osteoporosis than in the control group.This allows for the conclusion that this cytokine has destructive effects on bone tissue. 19,20Our results are similar to the study conducted by Polinska et al. 21The study demonstrated that the serum concentrations of IL-6 in UC was 3 times higher than in healthy group.Clinical studies of tocilizumab, a humanized monoclonal antibody against IL-6 receptors, have shown its high efficiency in the treatment of rheumatoid arthritis. 22,23Therefore, these studies may contribute to the development and implementation of new secondary osteoporosis therapy in the course of IBD.

Conclusions
The incidence of osteopenia and osteoporosis in patients with inflammatory bowel diseases is high and increases with the duration of the disease and the number of hospitalizations.Patients with Crohn's disease are at a higher risk of skeletal pathology than patients with ulcerative colitis.Interleukin 6, as a proinflammatory cytokine, can modulate bone mineral density in the femoral neck, which can cause a loss of bone mass, especially in the course of Crohn's disease.The effect of interleukin 6, which modulates the OPG/ sRANKL system, on bone mass density in the course of inflammatory bowel diseases requires further study.

Table 3 .
Mean serum concentrations of tested cytokines, Crohn's disease, ulcerative colitis and control group (comparison of the studied groups) a, b groups followed by the same letter do not differ statistically significantly; SD -standard deviation; OPG -osteoprotegerin; sRANKL -soluble receptor activator of nuclear factor kappa B ligand.

Table 2 .
Prevalence of osteoporosis and osteopenia in IBD (CD, UC)

Table 1 .
Characteristics of Crohn's disease patients, ulcerative colitis patients and control group parameters values presented as means and standard deviations