Nancy Wyzga

Relationship of functional and antigenic interleukin 6 to disease activity in inflammatory bowel disease

BACKGROUND Intestinal and peripheral blood mononuclear cell interleukin 6 (IL-6) production in inflammatory bowel disease might present an increased quantity of IL-6 into the systemic circulation. The aim of the present study was to examine the relationship of circulatory IL-6 to the clinical and laboratory expression of inflammatory bowel disease in children. METHODS Sera were obtained from 26 children with ulcerative colitis, 49 with Crohns disease, and 29 control patients. Serum functional IL-6 was measured by a bioassay and antigenic IL-6 by enzyme linked immunosorbent assay. RESULTS Functional and antigenic serum IL-6 levels were higher in Crohns disease than in ulcerative colitis or controls (P < 0.0001) and higher in ulcerative colitis than controls (P < 0.04). In Crohns disease affecting the colon, functional and antigenic serum IL-6 levels were greater than in disease limited to the small bowel (P < 0.002). Increasing disease severity was reflected by increasing antigenic but not functional IL-6 levels in both Crohns disease (P < 0.001) and ulcerative colitis (P < 0.02). Serum antigenic IL-6 levels were related to acute phase reactants in both diseases (P < 0.001) whereas functional levels were not. CONCLUSIONS Our results underscore the importance of using both functional and antigenic methodologies in examining the relationship of circulating cytokines to the clinical manifestations of inflammatory bowel disease.

Natural history of bone metabolism and bone mineral density in children with inflammatory bowel disease.

Background: In children with inflammatory bowel disease (IBD) it is not known whether reductions in bone mineral density (BMD) are a consequence of bone turnover alterations and if BMD improves with treatment. Methods: In a cohort of children with IBD, we prospectively measured indicators of bone remodeling, body mass index (BMI), disease activity, intact parathyroid hormone, serum IL‐6, and insulin‐like growth factor‐I at diagnosis and then every 6 months for 2 years. BMD was determined annually using dual x‐ray absorptiometry (DXA). BMD Z‐scores were calculated using height/age. Baseline measurements and calcium intake were compared with a group of age‐ and sex‐matched healthy children. Results: We observed that at diagnosis total body BMD Z‐score (mean ± SD) was −0.78 ± 1.02 for Crohns disease (CD, n = 58), −0.46 ± 1.14 for ulcerative colitis (UC, n = 18), and −0.17 ± 0.95 for control (CL, n = 49) (P < 0.01, CD versus CL). In CD, a BMD Z‐score <−1.0 was associated with lower BMI and higher serum IL‐6. Patients with CD and UC had low bone turnover. Activation of bone formation paralleled clinical improvement, but BMC gain was less than expected over the 2‐year study period, especially in CD. Prednisone use did not correlate with low BMD. Conclusions: Decreased bone turnover occurs in children newly diagnosed with IBD. Although indicators of osteoblast activity increase with clinical improvement, bone mineral accrual does not accelerate. Children with low BMI may be considered for BMD screening, since they are at risk for low bone mass.

Alterations in bone metabolism in children with inflammatory bowel disease: an in vitro study.

BACKGROUND In patients with inflammatory bowel disease (IBD), accelerated bone loss and osteopenia have been found. Potential etiologies of these bone abnormalities have included malnutrition, poor calcium intake or absorption, and the use of corticosteroids. Recent studies have suggested that circulating pro-inflammatory cytokines, which are produced in inflamed bowel, can have a profound effect on bone metabolism, particularly bone resorption. Our aim was to characterize the effects of serum from subjects with IBD on bone metabolism in an in vitro bone culture system. METHODS Organ cultures of fetal rat parietal bones were treated with sera from 9 subjects with Crohns disease, 7 with ulcerative colitis, and 10 controls with functional bowel disease (age range of all subjects 7-16 years). Patients were also classified by disease activity, serum albumin level, erythrocyte sedimentation rate (ESR), and serum interleukin (IL) 6 levels. The effects of sera on bone formation and resorption were quantified. RESULTS Compared with control serum, serum from patients with Crohns disease significantly decreased bone dry weight (p < 0.01) and calcium content (p < 0.001) during 96 h of culture, while serum from ulcerative colitis patients had no effect. While no difference in collagen synthesis was noted between any of the three experimental groups, noncollagen protein synthesis was lower in the ulcerative colitis group than in the control group or those with Crohns disease (p < 0.05). DNA content was similar in all groups. There was no significant effect of serum from any experimental group on bone resorption. There was no demonstrable relationship between clinical disease activity, ESR, or serum IL-6 levels and measures of bone metabolism. Histologic evaluation of cultured bone showed marked differences between control subjects and Crohns disease patients, with the latter being characterized by disorganization of mineral and osteoid and morphologically abnormal osteoblasts. CONCLUSIONS Serum from children with IBD has a significantly different effect than control serum on an in vitro model of bone metabolism. Our data suggest that circulating factors may affect osteoblasts and bone formation, leading to bone loss. Further work will be required to further characterize the nature of these factors and develop treatment strategies to minimize their effects.

Effect of Crohn's Disease on Bone Metabolism In Vitro: A Role for Interleukin-6†

Circulating proinflammatory cytokines may be involved in osteopenia associated with Crohns disease (CD). Therefore, the effect of interleukin (IL)‐6, IL‐1β, and tumor necrosis factor (TNF) α contained in Crohns serum on bone formation was examined in a bone organ culture system. Initially, serum levels of IL‐6, IL‐1β, and TNF‐α were determined by ELISA in newly diagnosed, untreated children with CD and healthy age‐matched controls. Serum IL‐6 levels were significantly higher in patients with CD than in controls (23.9 ± 2.8 pg/ml vs. 0.7 pg/ml ± 0.2; p < 0.001), whereas IL‐1β and TNF‐α serum levels were not. In the organ culture studies, 20‐day‐old fetal rat parietal bones were incubated for 96 h with CD or control serum, serum preincubated with a neutralizing antibody to each cytokine or a nonimmune immunoglobulin control, and with IL‐6. Bone formation measured by assaying calcium content and dry weight was significantly decreased in bones exposed to Crohns serum. Light microscopy of the bones treated with CD serum revealed a discontinuous, uneven mineralized bone matrix and disorganized osteoblasts with altered morphology. Incubation with an antibody that neutralized IL‐6 activity prevented the change in osteoblast and bone morphology. TNF‐α and IL‐1β antibodies had no apparent effects. Collagen synthesis and DNA content were not affected by CD serum. Also, addition of IL‐6 to the culture medium decreased mineralization. These results suggest that IL‐6 is a mediator of the effects of Crohns serum on in vitro mineralization and may be a contributing factor to the osteopenia associated with CD.

Tumor necrosis factor-α is not elevated in children with inflammatory bowel disease

Chronic undernutrition and high-dose daily corticosteroid therapy are well-accepted causes of growth failure in children with inflammatory bowel disease. Occasionally, children are seen with minimal gastrointestinal symptoms but in whom severe anorexia and profound growth impairment are evident. Recent observations that elevated serum levels of tumor necrosis factor-alpha (TNF) in cachexia associated with a number of disease states have suggested a similar possible role in inflammatory bowel disease. Accordingly, we determined TNF levels in 45 children and adolescents with inflammatory bowel disease (18 ulcerative colitis, 27 Crohns disease) at varying times during their clinical course and compared them to values obtained from a group of 25 children with functional bowel disease. No differences were noted in serum TNF levels between the children with inflammatory bowel disease and the control population. Values were generally within the range of the lower limit of detection of the assay. In the children with inflammatory bowel disease, there was no significant correlation between TNF levels and disease activity or growth parameters. Our observations suggest that elevated TNF levels are not associated with inflammatory bowel disease in children.

Effects of serum from children with newly diagnosed Crohn disease on primary cultures of rat osteoblasts

Objectives We propose that Crohn disease (CD) decreases bone formation via circulating inflammatory mediators. We therefore examined the effects of serum from newly diagnosed, untreated children with CD on osteoblasts in culture and the role of interleukin-6 (IL-6), a cytokine present in excess in active CD that also has direct effects on bone. Methods Bone mineral density was measured by dual x-ray absorptiometry. Primary cultures of rat osteoblasts were treated with serum from patients with CD and healthy controls. We measured expression of osteoblast proliferation, viability, differentiation markers, and mineralized nodule formation. Neutralizing antibodies were used to inhibit the effects of IL-6 present in serum. Results We studied 24 children with CD (14 male) and 31 controls (15 male). Spine bone mineral density was lower in patients with CD (Z score, −0.8 ± 0.9 vs. 0.0 ± 1.0 for controls;P < 0.05). Nodule formation was markedly decreased in osteoblasts treated with CD serum. However, CD serum did not affect osteoblast proliferation or viability. Expression of proteins characteristic of mature osteoblasts—osteocalcin and alkaline phosphatase—was reduced. Unlike our results in a model of intact bone, neutralization of IL-6 did not inhibit the effects of CD serum. Addition of IL-6 to control serum to match serum concentrations in CD had no effect either. Conclusions CD serum affects osteoblast function and probably differentiation in vitro, suggesting a mechanism by which CD may affect bone formation. IL-6 by itself is not sufficient to cause these effects and probably needs a cofactor present in intact bone.

Relationship of interleukin-1 receptor antagonist to mucosal inflammation in inflammatory bowel disease

Summary: Previous work has suggested that the interleu-kin-1 (IL-1) receptor antagonist, IL-Ira, may regulate mucosal inflammation in inflammatory bowel disease. The present study assessed the relationship of mucosal IL-Ira levels to histologic severity of inflammation and the related proinflammatory cytokines IL-1β and IL-6 in children with inflammatory bowel disease. Colonic biopsy specimens from 29 patients with ulcerative colitis, 27 with Crohns disease, and 24 noninflammatory control subjects were assayed for IL-Ira, IL-1β, and IL-6 by enzyme-linked immunosorbent assay. Histologic activity was graded as none, mild, moderate, or severe. Mucosal IL-1β levels, but not IL-lra levels, were significantly elevated in moderate/severely inflamed biopsies from patients with either ulcerative colitis (p < 0.01) or Crohns disease (p < 0.001) compared with those with none/mild inflammation. The mucosal molar ratio of IL-lra/IL-1β was significantly lower for moderate/severe inflammation compared with none/mild inflammation for patients with ulcerative colitis (p < 0.05) and Crohns disease (p < 0.01). The mucosal IL-lra/IL-1β ratio was similar in controls to none/mild inflamed biopsies from subjects with either ulcerative colitis or Crohns disease. Our observations suggest that increasing mucosal inflammation in inflammatory bowel disease in children is associated with a decrease in the “normal” effective IL-lra/IL-1β ratio in which IL-lra predominates. The importance of this abnormality to the pathogencsis of inflammatory bowel disease awaits further study.

Characterization of Circulating Interleukin-1 Receptor Antagonist Expression in Children with Inflammatory Bowel Disease

The cytokines IL-1β and IL-6 appear to be important in the pathogenesis of inflammatory bowel disease (IBD). Recently, a naturally occurring interleukin-1 receptor antagonist, designated IL-1ra, which inhibits IL-1β activityin vitro andin vivo has been described. The purpose of the present study was to assess the circulating levels and relative relationships of IL-1ra, IL-1β, and IL-6 in children with IBD of varying severity. Serum/plasma samples were obtained from 32 children with ulcerative colitis, 45 with Crohns disease, and 24 control patients. Cytokine assays were performed by enzymelinked immunoassay. IL-1ra levels were significantly elevated in children with ulcerative colitis or Crohns disease of moderate/severe activity compared to patients with inactive/mild IBD or control subjects (P<0.001). IL-1β was only detectable in the circulation of two subjects with severe colitis (one ulcerative colitis, one Crohns disease), and both had extremely elevated IL-1ra levels. IL-1ra levels were significantly related to IL-6 levels for patients with IBD (P<0.00001). Our results suggest that circulating IL-1ra appears in increasing concentrations in children with mounting degrees of disease severity as determined by clinical scoring methods as well as by the level of IL-6. Future work will need to address the clinical and prognostic value of measuring circulating IL-1ra in individuals with inflammatory bowel disease.

S1225 Fecal Osteoprotegerin: A Marker for Pediatric Ulcerative Colitis At Diagnosis - a Pilot Study

SMO levels in involved UC rectal tissues versus uninvolved right colon in a group of patients with left-sided colitis. There was an increase in SMO levels from 0.75 ± 0.21 to 2.63 ± 0.94 x 1019 copies, which represented a 3.5-fold increase (n = 10, p = 0.006 by paired t test). Conclusions: In general, SMO mRNA expression is increased in UC tissues. The increase was more marked in tissues with quiescent or mild colitis than in tissues from moderate or severe colitis. This pattern suggests that like ODC, SMO expression and polyamine metabolism also vary depending on disease activity. Assessment of SMO levels is important in understanding dysregulated polyamine homeostasis in UC and may prove useful as a marker of early or mild UC.