Previous investigations have demonstrated a link between elevated levels of eosinophils, eosinophil activation, and adult IBD. However, there have been conflicting data regarding the individual contribution of the eosinophil-selective chemokines eotaxin-1 and eotaxin-2 in eosinophil recruitment in IBD. In the present study we demonstrate the following: 1) that eosinophil numbers are elevated in pediatric UC and that their level correlates with disease severity; 2) eotaxin-1 and not eotaxin-2 or eotaxin-3 is up-regulated in lesional colonic biopsy samples of pediatric UC patients; and 3) eotaxin-1 mRNA expression correlates with colonic eosinophil levels in pediatric UC. Using a chemical-induced colonic injury model, we define that eotaxin-1, and not eotaxin-2, is critical for eosinophil recruitment and that eotaxin-1 is predominantly derived from intestinal macrophages. Consistent with our experimental analysis, we show that eotaxin-1 is predominantly expressed by intestinal macrophages; however, we also identify intestinal epithelial cells as a source of eotaxin-1 in pediatric UC.
Previous clinical studies have demonstrated elevated levels of eosinophils in adult CD and UC and that the level of this leukocyte correlated with disease severity and GI dysfunction (2
). However, relatively few clinicopathologic studies have examined eosinophil involvement in pediatric UC. We now provide data demonstrating increased rectosigmoid eosinophil levels in the absence of significant increases in either the ascending or descending colon in pediatric UC. Increased eosinophils in adult UC have been shown to be localized to cecal and lesional large bowel biopsy samples (4
) and are considered to be a negative prognostic indicator for UC. Notably, while eosinophils are not a component of the established UCHIS (30
), we demonstrate a correlation between rectosigmoid eosinophil levels and disease severity. Interestingly, clinical severity of pediatric-onset UC is often greater in comparison to adult-onset disease (42
). Approximately 60% of pediatric patients have moderate-to-severe disease at diagnosis and almost 80 –90% have pancolitis whereas 24% have pancolitis or 33% extensive colitis in adult onset (42
). Furthermore, a higher rate of corticosteroid dependence (45%) in children as compared with adults as been observed (45
). The increased clinical severity and steroid dependency may be attributable to a number of factors, including differences in disease immunopathogenesis and intestinal location between children and adults (46
). However it is interesting to speculate that rectosigmoid eosinophils may contribute to corticosteroid dependency in pediatric UC. Notably, Lampinen and colleagues have previously demonstrated no reduction in eosinophil chemotactic activity in perfusion fluids from adult UC patients following corticosteroid therapy (47
The role of eosinophils in IBD, particularly UC, is not yet fully elucidated (48
). We demonstrate extensive eosinophil degranulation in pediatric UC, suggesting a link between eosinophil degranulation and the development of intestinal pathology. This is consistent with previous clinical investigations providing ultrastructural evidence of eosinophil degranulation in adult IBD (49
). Notably, we demonstrate the presence of extracellular EPO in colonic biopsies from UC patients. EPO catalyzes the oxidation of halides, pseudohalides, and nitrite to generate cytotoxic oxidants (3-bromotyrosine, 3-chlorotyrosine, and hypothiocyanite) and reactive nitrogen species (3-nitrotyrosine and peroxynitrite) that induce tissue damage and cell death (50
). Using a DSS-induced model of experimental colitis, we have previously demonstrated that ablation of EPO enzymatic activity by gene targeting or drug antagonism attenuated DSS-induced pathology (51
). We are currently assessing the contribution of EPO to the histopathology of pediatric UC.
Previous investigations have demonstrated elevated levels of serum eotaxin-1 in adult UC patients as compared with controls; however, an association between eotaxin-1 and colonic eosinophil levels has not yet been established (24
). In the present study, we demonstrate increased expression of eotaxin-1 in colonic biopsy samples from pediatric UC patients. In addition, we show that expression of this chemokine positively correlated with eosinophil recruitment, demonstrating a direct link between eotaxin-1 and eosinophils in IBD. Previous genetic analyses have also demonstrated a link between adult UC and eotaxin-2. Allelic frequency of +179T→C and +275C→T of eotaxin-2 and genotype differences correlated with UC susceptibility (27
). However, our analysis did not identify any difference in eotaxin-2 mRNA expression in pediatric UC, suggesting no role for eotaxin-2 in the recruitment of eosinophils in pediatric UC. Consistent with this observation, genetic deletion of eotaxin-2 had no effect on experimental DSS-induced colonic eosinophil inflammation. Collectively, these data and experimental analysis demonstrate an important role for eotaxin-1 in eosinophil recruitment into the colon.
We performed gene profiling analysis on pediatric UC patients at diagnosis and on normal patients and revealed a pediatric UC gene signature. The up-regulated genes possessed a broad range of functions primarily associated with cellular growth and proliferation, innate immune defense mechanisms, lipid and carbohydrate metabolism, and molecular transport. Innate immune genes up-regulated in UC consisted of an abundance of genes involved in leukocyte recruitment and activation (CXCL1
, eotaxin-1, and CXCL8/IL-8
). Elevated levels of CXCL8/IL-8, CXCL9/MIG, and CXL10/IP-10 mRNA and protein have previously been demonstrated in UC (52
). Furthermore, experimental analyses have demonstrated a key role for these chemokines in DSS-induced colonic intestinal inflammation and disease pathology (55
). IL-8, CXCL9, and CXCL10 are primarily involved in the regulation of macrophage, neutrophil, and T cell trafficking. Collectively, these network analyses reveal a link between macrophages, neutrophils, and T cells that is thought to play a pivotal role in the augmentation of the intestinal inflammatory response in IBD and the eotaxin-1/eosinophil pathway. Consistent with this notion, we demonstrate that eotaxin-1 is primarily secreted by macrophages.
Previous genome-wide array analysis in adult UC has demonstrated a significant increase in CXCL1, CXCL2, CXCL3, eotaxin-1, and IL-8, suggesting that there is up-regulation of common immune pathways between pediatric- and adult-onset UC gene signature (58
). However, the majority of up-regulated networks in adult UC were associated with biosynthesis, metabolism, and electrolyte transport (58
). In contrast, in our pediatric UC population a significant proportion of genes up-regulated were associated with immune response, immune and lymphatic system development, and function and immunological disease networks. One possible explanation for the predominance of immune networks in our studies is that our analysis was performed at diagnosis in the absence of therapy, whereas the previous study analyzed adult UC patients receiving therapy including aminosalicylates, antibiotics, steroids and immunomodulators (azathioprine, 6-mercaptopurine (6MP), or infliximab). Analysis of the UC gene signature in the absence of therapy will illuminate important gene networks involved in early stages of the immunopathophysiology of UC. Notably, we also observed altered expression of gene networks associated with cancer, cellular growth, and proliferation at diagnosis in our pediatric UC population (results not shown). UC is associated with an elevated risk for colorectal cancer (59
). This would suggest activation of proliferative pathways associated with tumorigenesis and proliferation early in disease onset.
Immunofluorescence analysis of colonic biopsy samples from UC patients demonstrated eotaxin-1 expression by intestinal epithelial cells and CD68+
macrophages. Notably, the eotaxin-1 signal from intestinal epithelial cells was restricted to the basolateral compartment. Consistent with this, we observed the accumulation of eosinophils between and beneath the intestinal epithelium. Previous studies with human intestinal epithelial cells have demonstrated Cryptosporidium parvum
- and Bacteroides fragilis
-induced basolateral expression of the CXC chemokines IL-8, and GROα
). In both human and mouse studies we also demonstrated that macrophages are a primary source of eotaxin-1. Intestinal macrophage levels have been shown to be elevated in IBD and activation correlated with disease severity (62
). Furthermore, previous immunohistochemistry analysis examining eotaxin-1 production in mucosal tissues derived from CD and UC identified a minor population of eotaxin-1-positive mononuclear inflammatory cells; however the cell type was not identified (26
). Experimental studies using macrophage-deficient op/op
mice have demonstrated a role for macrophages in Nocardia brasiliensis
infection-induced eosinophil recruitment (63
B-dependent signaling pathways are thought to be important in the orchestration of the intestinal inflammatory response associated with IBD. Furthermore, activated NF-κ
B has been localized to intestinal macrophages and epithelial cells from IBD patients (62
). Eotaxin-1 promoter possesses STAT-6 and NF-κ
B regulatory elements in the promoter region (64
). Furthermore, eotaxin-1 expression in airway smooth muscle cells and epithelial cells has been shown to be regulated by NF-κ
B- and STAT-6-dependent pathways (64
). It is tempting to speculate that NF-κ
B activation of intestinal epithelial cells and macrophages drives eotaxin-1 expression and subsequent eosinophil recruitment in experimental colitis and pediatric UC. Consistent with this hypothesis, blockade of NF-κ
B signaling by decoy oligonucleotides attenuates murine Th1- and Th2-mediated IBD (65
In conclusion, we demonstrate a direct link between eotaxin-1 and intestinal macrophages in eosinophilic inflammation of the colon in experimental models and pediatric UC. Further, we demonstrate an important role for eotaxin-1 in the recruitment of eosinophils into the colon during IBD and show that eosinophils may have a role in the disease pathogenesis. Because pediatric onset UC is often more extensive and severe as compared with adult UC and possesses a higher rate of steroid dependency, delineation of the inflammatory pathways involved in disease pathogenesis is critical as emerging therapeutic approaches are introduced into treatment. Our studies provide strong rationale for the usage of therapeutic approaches targeting eosinophils or the eotaxin-1 pathway (eotaxin-1/CCR3) for pediatric UC.