The incidence of childhood-onset Crohn disease (CD) and ulcerative colitis (UC) has dramatically increased in Western countries in the last several decades (1). The etiology of inflammatory bowel disease (IBD) is unknown, but evidence suggests that it results from a combination of genetic predisposition and environmental factors (2). The latter is clearly important because IBD is almost exclusively a disease of developed countries. The data have shown a rapidly increasing incidence in Asia, which seems to occur in parallel with the rapid socioeconomic development taking place (3,4). A change to a more Westernized diet may underlie this epidemiological change in the Asian population (4). Indeed, diet has been the subject of much discussion and speculation. Most studies dealing with this subject have, however, only provided indirect evidence of a possible cause-and-effect relation between specific dietary factors and IBD (2). Upon light microscopy, Shepherd et al (5) have found that the base of human Peyer patches of the terminal ileum contains black granular pigment deposits. Further analysis has demonstrated that these pigment deposits consist of titanium dioxide and aluminosilicate (5,6). There are 2 hypotheses concerning the route these microparticles may take to reach the Peyer patches. The first hypothesis assumes that these metals and minerals in the bowel are derived from ingested materials, such as food additives, pharmaceuticals, and toothpaste (7,8). The second hypothesis assumes that materials from atmospheric dust are inhaled and that macrophages in the lung bearing these materials are expectorated and swallowed (8). Following digestion, these materials are reingested by macrophages in the bowel. A variation of this hypothesis is that the macrophages in the lung are deposited in the bowel via the bloodstream or lymphatic vessels (8). Based on the anatomical distribution of these microparticles in the Peyer patches of the terminal ileum, it has been suggested that these particles may play a role in the etiology of CD (9). Until now little has been known about the presence of exogenous pigment in the gastrointestinal tract of children. Pigment deposits have been reported in only a few children, all older than 6 years (5). It has been suggested that extremely young children, while being exposed to these exogenous materials, may not have accumulated enough pigment in the Peyer patches to be detected by light microscopy (5). Therefore, the aims of the present study were as follows: to map the distribution of exogenous pigment throughout the gastrointestinal tract of children suspected as having IBD, to assess the correlation between age and the presence and amount of exogenous pigment in the Peyer patches of the terminal ileum, and to determine its relation to pediatric IBD.
All children suspected as having IBD visiting our Department of Pediatric Gastroenterology and Nutrition between January 2003 and December 2008 were selected for the present study. As part of the clinical workup, all patients underwent both ileocolonoscopy and upper gastrointestinal tract endoscopy. Only children in whom the terminal ileum was intubated were included. All of the patients had at least 1, but mostly 2 or more biopsy specimens taken from each part of the colon (cecum, ascending colon, transverse colon, descending colon, and rectum), terminal ileum, duodenum, stomach (antrum and corpus), and esophagus. Biopsies were taken from macroscopically normal mucosa and from inflamed areas. The tissue was formalin fixed, paraffin embedded, and routinely processed and stained with hematoxylin and eosin. Each biopsy specimen was cut in 2 or more levels to increase the chance to detect pigment. Biopsies from all sites were reassessed, using light microscopy, by an expert pathologist, who was blinded to the clinical condition. The amount of pigment was scored using a semiquantitative scale, range 0 to (+++). In the (+) group, only some spots of pigment were seen. The biopsies that were full of pigment were scored as (+++). All biopsies that contained pigment less than (+++) and more than (+) were grouped into (++). Patients were divided into 3 groups on the basis of their diagnosis: CD, UC, and no IBD (non-IBD). The diagnosis was made based on the reference standard procedure, which consisted of endoscopic findings and histopathological interpretation, imaging studies, and on clinical follow-up data and/or repeated endoscopy. Presenting symptoms, duration of symptoms, and clinical follow-up data were extracted from medical charts. A statement of no objection was released by our institutional review board.
The Statistical Package for Social Sciences version 20 (IBM SPSS Statistics, Armonk, NY) was used for all analyses. Fisher exact tests were used comparing categorical variables between groups and nonparametric tests (Kruskal-Wallis test) comparing continuous variables, such as age and duration of disease. Spearman correlation coefficient was used to assess the relation between the amount of pigment and age at endoscopy. The criterion for statistical significance was defined as a P value of <0.05.
A total of 172 children suspected of IBD were selected. The terminal ileum was successfully intubated in 151 of 172. Of these 151 children, 54% were boys, with a mean age of 12.2 years (age range 1.6–18.1 years). Based on our reference standard diagnostic procedures, 62 children fulfilled the criteria for CD, 26 children for UC, and 63 children did not have IBD. No differences were found between the 3 main groups with regard to age at endoscopy, sex, and duration of symptoms. The follow-up period was significantly different between groups (P < 0.0001). These data are shown in Table 1. None of the patients with non-IBD developed IBD, or other gastrointestinal disease, during the follow-up period.
Distribution of Pigment
In 63 children (42%), aggregates of fine black pigment were found in biopsies from the terminal ileum (Fig. 1A and B). The pigment was located in the Peyer patches, just within the lymphoid follicle, outside the germinal center. Pigment cells were not observed in lymphoid follicles in the duodenum, colon, or elsewhere in the gut.
Pigment in Relation to Age
The age of children with pigment in the Peyer patches ranged from 3.7 to 18.1 years (mean age 12.1 years, standard deviation 3.8). There was a significant correlation between age at endoscopy and the amount of pigment (P = 0.004, Fig. 2). The amount of pigment became denser with increasing age.
Pigment in Relation to Diagnosis
Pigment was found in a significantly lower number of patients with CD compared with that in patients with UC and those with non-IBD (26% vs, respectively, 62% and 49%, P = 0.002, Fig. 3). Terminal ileitis was found in 11 of 16 patients with CD (69%) with pigment in the terminal ileum biopsies and in 36 of 46 patients with CD (78%) with no pigment in the terminal ileum (P = 0.5). Lymphoid tissue was found in terminal ileum biopsies of 50% of patients with CD, 70% of patients with UC, and 72% of patients with non-IBD (P = 0.4). In this group of children with lymphoid tissue present in the terminal ileum biopsies, pigment was still found significantly less often in patients with CD compared with that in patients with UC and those with non-IBD (P = 0.02).
The present study shows that pigment deposits were present in 42% of all of the children undergoing endoscopy because of complaints of suspected IBD located only in the Peyer patches in the terminal ileum biopsies. Furthermore, a significant correlation was found between the amount of pigment in the Peyer patches and age, with the amount of pigment becoming denser with increasing age. Surprisingly, these pigment deposits were found significantly less often in patients with CD compared with that in children with UC and those with non-IBD.
Shepherd et al (5) were the first to describe their findings of “pigment cells” at the base of Peyer patches in 1987 in samples of human small intestine. Since then, these pigment cells have been described several times, but never in a cohort of children (6,8,9). In accordance with the report by Shepherd et al (5), in the present study, pigment deposits were only observed in Peyer patches, not in gut-associated lymphoid tissue in other parts of the gastrointestinal tract. Powell et al (6) have shown pigment also in lymphoid aggregates in the colon/ileocecal region in a few adult patients. Pigment deposits have also been seen in the endothelial cells of blood vessels (n = 2) (8), around dilated lymphatics in the submucosa of the ileum (n = 2), in mesenteric lymph nodes, and in 1 patient with ileal CD in transmural inflammatory aggregates (5). All of these studies, however, examined intestinal resection preparates, which can explain the difference in these studies where only biopsies were available.
Pigment cells are either mature or maturing macrophages containing lysosomes that are full of dense, small microparticles that do not allow transmission of light and, thus, appear black upon regular light microscopy (6,9). These microparticles appear to be principally aluminosilicates, titanium dioxide, and a small proportion of nonaluminum-containing silicates (6). Titanium dioxide is a common whitening and brightening agent used in the food and pharmaceutical industry. Aluminosilicates are widely used in the food industry as anticaking and thickening agents, as pharmaceutical excipients, and in toothpaste (6,10). Intakes of aluminosilicates are greater than intakes of titanium dioxide, respectively, 35 and 2.5 mg per person per day in the United Kingdom (10). Microparticles can enter the body by crossing the intestinal epithelium via endocytic M cells overlying intestinal lymphoid aggregates before reaching macrophages. M cells are specialized, differentiated epithelial cells, which function as an entry and have an exceptional capacity in the uptake of exogenous particles (11,12). The exogenous particles are resistant to enzymatic or chemical degradation and are accumulated in the pigment cells, which are of low metabolic and immunological activity (9). Our finding that the density of pigment deposits increases with age confirms that pigment cells are inert storage cells of exogenous particles, taken up by the gut, which become saturated in the course of time. Shepherd et al (5) have shown that pigment in Peyer patches is present in adults and children >6 years of age; however, we have shown in our study that pigment in Peyer patches can be found in even younger children. In our cohort, 6 children with pigment deposits in Peyer patches were younger than 6 years (2 CD, 1 UC, and 3 non-IBD). Apparently, in these young children, exogenous microparticles may have accumulated enough to be detected by light microscopy.
Based upon the site of uptake of these particles in the Peyer patches in the terminal ileum, the “classical” site of inflammation in CD, they may play a role in the pathogenesis of CD. It is well known that similar particles can cause granulomatous diseases in other organs (13,14). Our study shows that pigment in Peyer patches was found in a significantly lower number of patients with CD compared with that in patients with UC and those with non-IBD. We hypothesize that in patients with CD, the pigment cells have turned into an immunological active state and have become involved in the inflammatory process. Former studies have found that exogenous microparticles can act as adjuvants in the presence of lipopolysaccharide, significantly enhancing interleukin-1β (IL-1β) (titanium dioxide, in the presence of additional calcium cations) (15,16), IL-10, IL-8, and tumor necrosis factor-α responses in vitro and can impair macrophage phagocytic capacity (titanium dioxide and aluminosilicate) (17).
Based on genomewide association studies, it has become clear that a defective autophagy plays an important role in the pathogenesis of CD. NOD2 gene variants have been implemented in muramyl dipeptide–induced autophagy and bacterial targeting toward lysosomes. Dendritic cells that express the disease-associated variants have been shown to be defective in both these functions (18). The disease-associated variant in the ATG16L1 gene is involved in the same pathway and has been implicated in antimicrobial peptide release and negative regulation of proinflammatory cytokine production (19). Thus, the role of autophagy in the immune response is highly pleiotropic because it is involved in both innate and adaptive immune response and dependent on cell type and interactions with specific microbial factors (20).
Our observation that pigment is found significantly less often in the lysosomes of patients with CD raises the question of whether this observation is associated with autophagy-related disease variants and prompts future research in this direction.
An alternative explanation for our observation could be that the biopsies just represent a fraction of the total terminal ileum, which may lead to sampling bias in patients with CD if biopsies have been taken from chiefly inflamed mucosa. The terminal ileum biopsies of patients with CD contained significantly less lymphoid tissue compared with that of others; however, analyzing only the group of children with lymphoid tissue present in the terminal ileum biopsies, pigment was still found significantly less often in patients with CD compared with that in patients with UC and those with non-IBD, which makes sampling bias less likely.
In summary, in children, pigment cells are only present in Peyer patches in the terminal ileum, not in gut-associated lymphoid tissue in other parts of the gastrointestinal tract. Pigment in Peyer patches can be found in children >3.7 years of age and the density increases with age. The pigment in Peyer patches was, however, found in a significantly lower number of patients with CD compared with that in children with UC and those with non-IBD. Knowing that the etiology of IBD is still unclear, the absence of pigment in Peyer patches in patients with CD strengthens the evidence that microparticles may play a role in the inflammatory process of CD, which may be the result of a defective autophagy pathway. This hypothesis, however, remains to be established.
1. Benchimol EI, Fortinsky KJ, Gozdyra P, et al. Epidemiology of pediatric inflammatory bowel disease
: a systematic review of international trends. Inflamm Bowel Dis
2. Rogler G. Interaction between susceptibility and environment: examples from the digestive tract. Dig Dis
3. Ahuja V, Tandon RK. Inflammatory bowel disease
in the Asia-Pacific area: a comparison with developed countries and regional differences. J Dig Dis
4. Goh K, Xiao SD. Inflammatory bowel disease
: a survey of the epidemiology in Asia. J Dig Dis
5. Shepherd NA, Crocker PR, Smith AP, et al. Exogenous pigment in Peyer's patches. Hum Pathol
6. Powell JJ, Ainley CC, Harvey RS, et al. Characterisation of inorganic microparticles in pigment cells of human gut associated lymphoid tissue. Gut
7. Lomer MCE, Thompson RPH, Powell JJ. Fine and ultrafine particles of the diet: influence on the mucosal immune response and association with Crohn's disease. Proc Nutr Soc
8. Urbanski SJ, Arsenault AL, Green FHY, et al. Pigment resembling atmospheric dust in Peyer's patches. Mod Pathol
9. Thoree V, Skepper J, Deere H, et al. Phenotype of exogenous microparticle
-containing pigment cells of the human Peyer's patch in inflamed and normal ileum. Inflamm Res
10. Lomer MCE, Hutchinson C, Volkert S, et al. Dietary sources of inorganic microparticles and their intake in healthy subjects and patients with Crohn's disease. Br J Nutr
11. Powell JJ, Thoree V, Pele LC. Dietary microparticles and their impact on tolerance and immune responsiveness of the gastrointestinal tract. Br J Nutr
12. Powell JJ, Faria N, Thomas-McKay E, et al. Origin and fate of dietary nanoparticles and microparticles in the gastrointestinal tract. J Autoimmun
13. Redline S, Barna BP, Tomashefski JF Jr, et al. Granulomatous disease associated with pulmonary deposition of titanium. Br J Ind Med
14. Chen WJ, Monnat RJ Jr, Chen M, et al. Aluminum induced pulmonary granulomatosis. Hum Pathol
15. Powel JJ, Harvey RS, Ashwood P, et al. Immune potentiation of ultrafine dietary particles in normal subjects and patients with inflammatory bowel disease
. J Autoimmun
16. Butler M, Boyle JJ, Powell JJ, et al. Dietary microparticles implicated in Crohn's disease can impair macrophage phagocytic activity and act as adjutants in the presence of bacterial stimuli. Inflamm Res
17. Evans SM, Ashwood P, Wharley A, et al. The role of dietary microparticles and calcium in apoptosis and interleukin-1beta release of intestinal macrophages. Gastroenterology
18. Cooney R, Baker J, Brian O, et al. NOD2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation. Nat Med
19. Cadwell K, Liu JY, Brown SL, et al. A key role for autophagy and the autophagy gene ATG16L1 in mouse and human intestinal Paneth cells. Nature
20. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature