Defensins are small cationic peptides with a vast spectrum of antimicrobial activity (1–3). They comprise 2 major subfamilies termed α- and β-defensins with distinct structural characteristics (4–6). There are 6 human β-defensins (hBD1–hBD6), and 2 human α-defensins (hD5 and 6) known to be expressed by epithelial cells at various surfaces (5,6). In the gastrointestinal tract, defensins are regarded as primary effector molecules in host defense against invading microorganisms (7–10), thereby contributing to intestinal homeostasis (8,9).
Data about the expression and potential role of defensins in the healthy gastrointestinal tract and in various digestive diseases are derived mainly from adult populations or in vitro studies (4,7,11–13). However, the role of any of the α- or β-defensins in infectious gastritis in children has so far been investigated by a single study, which identified an upregulation of neutrophil α-defensins in Helicobacter pylori (Hp)–positive individuals (14).
Few studies in adult and pediatric populations have sought to characterize the potential role of defensins in the pathogenesis of celiac disease (CD), and contradictory findings have been published (15–19). We therefore aimed to further explore the expression patterns of defensins in gastric and duodenal biopsies of pediatric patients infected with Hp or experiencing active CD.
PATIENTS AND METHODS
Two adjacent biopsies from the duodenum, the corpus, and antrum mucosa were taken from 80 children (37 boys, 43 girls; 1.3–16.7 years of age) admitted to the Department of Pediatric Gastroenterology at the Children's University Hospital Bochum, Germany. All of the children underwent endoscopy of the upper gastrointestinal tract for medical reasons and were following no special diet at the time of endoscopy. One biopsy was used for routine pathology; the adjacent biopsy was stored for up to 2 weeks in RNAlater stabilization reagent (Ambion, Kassel, Germany). Biopsies were routinely stained with hematoxylin and eosin and evaluated by a professional pathologist based on the modified Sydney criteria (20). In 11 patients (3 boys, 8 girls; ages 4.2 to 16.2 years) the diagnosis of CD was reached based on European Society for Pediatric Gastroenterology, Hepatology and Nutrition criteria, which include characteristic histologic changes in small intestinal biopsies and unequivocal clinical improvement on a gluten-free diet (21). All of the children had newly diagnosed active CD and had not previously been receiving a gluten-free diet. Symptoms at the time of endoscopy were failure to thrive (n = 6), recurrent abdominal pain (n = 5), chronic diarrhea (n = 3), and family history for CD (n = 1). Antral biopsies of 18 (9 boys, 9 girls; ages 3.2–16.7 years) patients tested positive for Hp infection by urease test (Jatrox-H.p.-Test, C.H.R. Heim Arzneimittel, Darmstadt, Germany) (Hp group), and Hp-carrier status was confirmed by Giemsa stain. Symptoms present at the time of endoscopy were recurrent abdominal pain (n = 12), hoarseness (n = 3), recurrent vomiting (n = 4), and dyspepsia (n = 4). Twenty-one age-matched patients with dyspepsia (12 boys, 9 girls; ages 2.8–14.6 years) showed histologic normal mucosa of the antrum, corpus, and duodenum and were assigned to the control group. Nine patients had a mild esophagitis in the histologic workup; 12 children were diagnosed with functional abdominal pain. Thirty patients (14 boys, 16 girls; ages 3.6–15.7 years) whose biopsies were determined to be inflamed based on histopathologic evaluation, but in whom there was no detection of Hp by urease testing and histopathologic evaluation, were used for comparison with the Hp group (non-Hp). Symptoms present at the time of endoscopy were recurrent abdominal pain (n = 21), failure to thrive (n = 2), hoarseness (n = 2), recurrent vomiting (n = 4), and dyspepsia (n = 7). Nine patients were later diagnosed as experiencing bile reflux, 12 had simultaneous viral infections of the respiratory tract, and 4 had taken aspirin. No putative agent was identified in the remaining patients. The study was performed according to the Declaration of Helsinki and was approved by the ethics committee of the Ruhr University of Bochum.
RNA Preparation and Reverse Transcription
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Positive controls and external standards were generated from gastric biopsies of adults for hBD1, 2, and 4; from transbronchial biopsies for hBD3; from an operatively removed testicle for hBD5 and 6; and from duodenal biopsies for hD5 and 6. Eluation of RNA was followed by a second treatment with 0.2 U/μL DNase and 5.5 mmol/L MgCl in 10× reverse transcriptase (RT)-buffer solution (Roche, Mannheim, Germany) in the presence of RNasin (Promega, Madison, WI) for 20 minutes at 37°C followed by 8 minutes at 75°C. The obtained solution was cooled to −2°C and reverse transcribed adding 200 U of Moloney murine leukemia virus-RT, 10 mmol/L random-primer, and 5.5 mmol/L MgCl in the appropriate buffer (Carl Roth, Karlsruhe, Germany) in the presence of RNasin (Promega) for 60 minutes at 37°C, followed by 5 minutes at 95°C. Negative-RT controls were prepared for each biopsy adding RNase-free water (Qiagen) instead of reverse transcriptase to RNA solution.
Real-time Reverse Transcriptase-Polymerase Chain Reaction
Following the manufacturer's protocol, 1 μL LightCycler-DNA Master SYBR Green I (Roche) and 1 μL of reversely transcribed RNA solution were added to 550 nmol/L forward and reverse primers (TIB Molbiol, Berlin, Germany) and sterile water to produce a total volume of 9 μL. Sequences of primer pairs were designed or obtained from the literature as indicated in Table 1 (22–24).
RT-polymerase chain reaction (PCR) profile for hD5 and 6 was as follows: 95°C for 10 minutes; 45 cycles at 94°C for 15 seconds, 55°C for 5 seconds, and 72°C for 15 seconds. RT-PCR profile for hBD1 to -6 was as follows: 95°C for 10 minutes; 45 cycles at 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 15 seconds. For melting curve analysis both profiles were completed with warming to 95°C for 2 seconds, 65°C for 15 seconds, and a heating of 0.1°C/second up to 97°C. Real-time measurement of fluorescence was monitored by the LightCycler System. Calibrator-normalized relative quantification using glyceraldehyde-3-phosphate dehydrogenase as internal standard and isolated mRNA of the above-mentioned positive controls as external standard was carried out according to the manufacturer's instructions.
The Mann-Whitney U test was used to compare the relative amounts of mRNA between groups and carry out subgroup analysis. A P value <0.05 was considered significant.
Levels of defensin expression did not show a significant correlation with age in any of the groups, and there was no difference in defensin expression based on sex in any of the defensins tested. HBD6 was not found in any of the biopsies tested, whereas hBD5 was found in 1 of the 3 biopsies taken from each patient in 7 cases (2 antrum, 2 corpus, 3 duodenum). Patients belonged to all of the groups, and subgroup analysis did not reveal any concise differences between patients with and without hBD5 expression. Because of the inconsistent expression, no comparison between groups was carried out for either hBD5 or hBD6.
Based on the amount of polymorphonuclear cell infiltration, antral biopsies of all but 2 patients of the Hp group who had severe inflammation were graded as mildly to moderately inflamed. Infiltration of mononuclear cells was severe in 5, moderate in 8, and mild in 5 patients. In the non-Hp group, 4 patients showed moderate and 9 mild infiltration with polymorphonuclear cells, whereas mononuclear cell infiltration was severe in 7, moderate in 14, and mild in 9 cases. No correlation between defensin mRNA expression and histologic grading was found for either polymorphonuclear or mononuclear cell infiltration at any of the biopsy sites of the Hp and non-Hp groups.
In children carrying Hp, relative amounts of hBD2-mRNA were significantly higher in biopsies from the antrum and corpus than in healthy individuals (Fig. 1). Biopsies that showed mucosal inflammation without proof of Hp infection (non-Hp) showed significantly lower levels of hBD2-mRNA when compared with the Hp group in antral biopsies, whereas the level of significance was not reached comparing hBD2 levels of healthy individuals and those of the non-Hp group (Fig. 1). There were no differences observed in the expression level of defensins in the duodenum or of the α-defensins and hBD1, 3, and 4.
Expression in CD
Five patients of the CD group displayed mild lymphocytic gastritis without polymorphonuclear cell infiltrates in the histologic workup of antral specimen. Villous atrophy in patients with CD was severe in 2 cases and moderate in 9 children. None of the patients with CD had a positive urease test.
The expression level of hBD2 in antral biopsies of patients experiencing active CD was markedly elevated when compared with healthy controls (Fig. 2). Careful reevaluation of the adjacent routine biopsies revealed that antral lymphocytic gastritis was present in 5 of the 11 patients with CD. However, subgroup analysis did not reach a statistically significant level concerning the expression of hBD2 between patients having CD with and without lymphocytic gastritis. Although there were no changes in defensin-mRNA noted in biopsies taken from the corpus, mRNA expression of hBD1 was significantly decreased in duodenal specimen of patients with CD when compared with healthy controls (Fig. 2). There was no detection of hBD4-mRNA in the duodenum of patients with CD, whereas controls expressed this defensin at low levels. No differences between the groups were observed in the expression levels of hBD3 and the α-defensins tested.
Based on the previous studies using in vitro assays or adult tissue, hBD1 expression has been described mostly as constitutive in epithelial cells of the intestinal mucosa (22,25–27). On the contrary, hBD2 expression has been characterized as inducible by Hp and inflammatory mediators in vitro (25,27–29) and in vivo (22,25,27,30,31). In the present study, we were able to confirm these results by demonstrating constitutive hBD1 and increased hBD2 expression in Hp-positive children.
In antral biopsies, expression levels of hBD2-mRNA in the Hp group were significantly increased in comparison to both healthy controls and the non-Hp group, which did not show a statistically upregulated hBD2-expression when compared with the control group. Although the extent of polymorphonuclear cell infiltration was tendentially higher in the Hp group, no correlation of hBD2 to the histologic grading of inflammatory changes was observed. Hence, it may be speculated that hBD2 gene expression is specifically upregulated by Hp in children as well as in adults, with the latter having been reported previously (22,25,27,30,31) and that this phenomenon may not be solely because of the inflammatory process. The inhomogeneous composition of the non-Hp group regarding underlying pathologies can be regarded as a limitation to this conclusion, however.
hBD3 was shown to be inducible by Hp in vitro (28). This effect could not be confirmed by the present in vivo study, suggesting an overall low level of hBD3 expression and a rather constitutive expression pattern.
α-Defensin gene expression in the course of an Hp infection has been studied in mostly adults. In accordance with the present results, hD5 showed a rather constitutive pattern in adult antral biopsies (22,31), whereas hD6 was previously shown to be upregulated in the fundus but not in the corpus of Hp-positive individuals (22). The present results suggest a rather constitutive pattern of both hD5 and -6 during Hp infection in children. In a recently published immunohistochemical study on Hp-infected children, the amount of inflammation correlated to the rate of α-defensin-positive individuals (14). The antibody used, however, was designed to detect the neutrophil α-defensins hD1 to -3 (human neutrophil peptides), although we detected mRNA of hD5 and -6 that are predominantly expressed in Paneth cells of the small intestine rather than in leukocytes (32,33).
Studies regarding defensin expression in patients with CD have reported contradictory results in the past: hBD1 expression has been reported to be unchanged (15,18) or diminished (17,19), whereas hBD2 has been shown to be upregulated (15) or diminished (17,19) in duodenal biopsies. Similarly, significantly increased amounts of hD5 and hD6 have been found in duodenal biopsies from patients with CD when compared with healthy controls (17–19), whereas others reported comparable expression levels in the duodenum of healthy and affected individuals (16,19). Different study designs and patient numbers or backgrounds may explain these contradictory results. In accordance with Forsberg et al (17) and Taha et al (19), we detected diminished amounts of hBD1-mRNA in patients with CD, whereas hBD2-mRNA expression levels remained unchanged when compared with healthy controls. However, we did not observe any change in the levels of α-defensin-mRNA. Interestingly, hBD4 was completely absent in all of the duodenal biopsies of patients with CD in our study and this has previously not been recognized. Because CD leads to a villous atrophy in the small intestine, and hBD1 and hBD4 are preferentially expressed by epithelial cells (34,35), the diminished amounts of these defensins in our patients may be because of villous atrophy, and therefore a different cellular composition of the tissue sample rather than real changes in the gene expression of epithelial cells in response to the illness. However, this effect should consequently hold true for hBD2 and -3, which are also preferentially expressed by epithelial cells in the gastrointestinal tract (22,25–27,30,34,36) but whose expression in the present study was not affected by CD when compared with the control group. Thus, the downregulation of specific defensins may well be a consequence of CD, possibly leading to a change in hBD1 and -4 mRNA expression. Whether the downregulation of defensins can lead to a deficit of the local immune defense in CD, as previously suggested (17), remains to be evaluated. Surprisingly, antral biopsies of patients with active CD showed an increased amount of hBD2-mRNA. Lymphocytic antral gastritis in association with CD has been previously recognized (37–41) and it is present in approximately 50% of patients with CD (23,42). However, although 5 of the 11 patients enrolled had concomitant lymphocytic gastritis, this finding alone did not provide a sufficient explanation because a subgroup analysis did not reveal statistically different results. Together with the finding of changed amounts of defensin-mRNA in the stomach, this finding further supports the notion of a modulation of defensin gene expression in the upper gastrointestinal tract during CD, the mechanism and impact of which have yet to be recognized. Further research in this interesting field is clearly required.
hBD5 and -6 were believed to be exclusively detectable in epididymidis tissue (43). We have previously reported on the presence of hBD5- and -6-mRNA in duodenal biopsies obtained from healthy children (32). In the present study, expression of hBD5-mRNA was again detectable in individual patients, confirming our earlier results. Because there were no differences between patients with and without expression, the potential meaning of this finding remains unclear.
In addition to an earlier description of the defensin-expression pattern in the upper gastrointestinal tract of healthy children (32), we have identified modulations of defensin gene expression of children infected by Hp or experiencing active CD. To our knowledge, this is the first study to evaluate β-defensin expression in Hp-infected children and parallel different pathologies of the upper gastrointestinal tract concerning defensin-expression patterns in a pediatric population. Further studies are needed to support the pathophysiologic relevance of these findings. The present results strongly support a key role for defensins in host defense in gastrointestinal disease of children.
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