Abstract
Both in vitro studies and experiments in mice suggest a key role for transcription factor NF-κB as a mediator of mucosal inflammation. Experiments in vitro show that NF-κB activation may be a critical event in the production of proinflammatory molecules in Helicobacter pylori-associated gastritis. This study examines the expression and activity of NF-κB in situ in antral biopsies of 69 consecutive patients with immunohistochemical techniques. In the uninflamed stomach, NF-κB was highly expressed and active in a subset of epithelial cells, which were identified as predominantly G cells. In accordance with this activity, G cells were shown to express high levels of the NF-κB target cytokine TNF-α, a well-documented stimulator of gastrin production. In patients with H. pylori-associated gastritis, NF-κB activity was markedly enhanced. Activation occurred preferentially in the epithelial cells. The number of cells showing activated NF-κB correlated with the activity of gastritis, a measure of neutrophil influx, whereas no correlation was found with the chronicity of inflammation, a measure of the presence of mononuclear inflammatory cells. This correlation is direct evidence of the importance of NF-κB-dependent signal transduction for neutrophil influx in H. pylori-associated gastritis.
Upon colonization by pathogenic bacteria, the host cells at the site of infection respond with an induction of the innate immune response. This response is elicited after a signal from the invader triggers an alteration in host cell gene transcription. Subsequently, the host cells will produce proinflammatory proteins, such as cytokines, chemokines, and adhesive molecules, to counteract the external threat. Moreover, at the site of colonization, both the rate of proliferation and apoptosis of the epithelial cells are often markedly affected. The molecular details of the interaction between the host epithelium and pathogenic bacterial invaders are now slowly emerging (1).
One of the sentinel transcriptional modulators in the host response to bacterial invasion is the NF-κB family of proteins (2, 3, 4, 5). This transcription factor mediates both acute and chronic inflammation through the regulation of many proinflammatory proteins (2). NF-κB is present in the cell as a hetero- or homodimer and remains inactive by binding to an inhibitory protein, I-κB, within the cytoplasm (3). Subunits that can form these dimers are NF-κB1 and NF-κB2 (expressed as precursors p105 and p100, processed to p50 and p52, respectively), RelA (p65), RelB, and c-Rel (4, 5). The dimer, typically composed of a p50 and a p65 subunit, is translocated to the nucleus after degradation of the inhibitory I-κB in response to a wide variety of stimuli (4, 5). Although it is generally assumed that NF-κB is ubiquitously expressed by all cell types, exact expression patterns remain to be studied for most human tissues.
A unique model system to study the induction of the innate immune response by a single pathogenic bacterial species exists in the human stomach. Here, colonization of the mucous layer overlaying the gastric epithelial cells by the Gram-negative bacterium Helicobacter pylori results in an acute host response, mostly followed by persistence of the bacterium and chronic gastric inflammation (6, 7). The presence of H. pylori is associated with peptic ulcer disease (8), atrophic gastritis, gastric adenocarcinoma (9, 10), and gastric mucosa-associated lymphoid tissue lymphoma (11). NF-κB plays an important role in the inflammatory response in the intestine (12), and accordingly, H. pylori activates this transcription factor (13, 14, 15). This activation may in turn cause gastritis via the induction of proinflammatory cytokines such as IL-1 and TNF-α and chemokines like IL-8 (15, 16). We examined in this study the expression and activation of NF-κB in the antrum of the human stomach in both the histologically uninflamed mucosa and H. pylori-associated gastritis. We also investigated a possible correlation of the activity of NF-κB with the commonly used histopathological classification of the severity of gastritis, the Sidney score. Our findings show that in the antrum of the stomach, activation of NF-κB correlated with the activity of gastritis, a measure of neutrophil influx, whereas this did not correlate with the chronicity of gastritis, a measure of the presence of mononuclear inflammatory cells. Furthermore, we found that expression and activation of NF-κB are associated with the epithelial cells, especially G cells.
Materials and Methods
Patients
Gastroduodenoscopy was performed on 69 consecutive patients referred to the endoscopy unit of the Academic Medical Center in Amsterdam for upper abdominal complaints. These patients were enrolled in a prospective study investigating the prevalence of H. pylori in patients with upper abdominal complaints in the general practitioner’s setting; the study was approved by the Ethics Committee of the Academic Medical Center. Biopsies were taken from the antrum of the stomach as part of these investigations. Specimens were fixed in PBS-buffered paraformaldehyde for 30 min and embedded in paraffin with the use of standard methods. H. pylori status was determined by serology, by culturing the bacteria, and by routine histology (hematoxylin and eosin stain) by an independent pathologist. A patient was considered H. pylori positive if the bacterium was cultured or if both serology and pathology were positive. The histological grade of gastritis was scored according to the Sydney classification (17), by a pathologist blinded to the NF-κB activation score (see below).
Antibodies
Antisera to different NF-κB subunits, anti-p65 rabbit polyclonal IgG Ab C-20, anti-p65 mouse monoclonal IgG1 Ab F-6, anti-p50 rabbit polyclonal IgG Ab H-119, anti-p52 rabbit polyclonal IgG Ab K-27, anti-RelB rabbit polyclonal IgG Ab C-19, and anti-TRAF2 rabbit polyclonal IgG Ab M-19, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal IgG3 Ab (Boehringer Mannheim, Mannheim, Germany), raised against the p65 nuclear localization signal, was used to detect activated NF-κB. This Ab binds to the p65 unit only after release from the inhibitory I-κB subunit and thus specifically recognizes activated p65, allowing assessment of NF-κB activation in situ (14, 18, 32). For detection of chromogranin A and gastrin, we used anti-chromogranin A mouse mAb clone DAK-A3 and anti-gastrin rabbit polyclonal Ab A568 (DAKO, Glostrup, Denmark). A mouse monoclonal IgM Ab against TNF-α, clone 4C6-H6, was purchased from Instruchemie (Hilversum, The Netherlands). For H. pylori visualization, the rabbit polyclonal Ab B0471 (DAKO) was used.
Immunohistochemistry
Paraffin sections (4 μm) were dewaxed and rehydrated in graded alcohols. Endogenous peroxidase activity was quenched with 1.5% H2O2 in PBS for 30 min at room temperature. Nonspecific staining was blocked with 10 mM Tris, 5 mM EDTA, 0.15 M NaCl, 0.25% gelatin, 0.05% (v/v) Tween 20, pH 8.0, for 30 min at room temperature. After a washing with PBS, one of the following primary Abs was applied: anti-p65 polyclonal (1:500); anti-p65 monoclonal (1:50); anti-p50 (1:50); anti-p52 (1:10); anti-RelB (1:20); anti-TRAF2 (1:50); anti-active p65 (1:500 of 1 μg/ml stock); anti-chromogranin A (1:50); anti-gastrin (1:200) anti-TNFα (1:50) in PBS containing 1% BSA. For double staining, combinations of monoclonal and polyclonal Abs were used. Sections were stored overnight at 4°C. The following day, for single staining, sections were washed in PBS and incubated with a secondary biotinylated goat anti-rabbit Ig (DAKO, 1:500) or anti-mouse Ig Ab (DAKO, 1:200) for 1 h at room temperature and washed with PBS. Hereafter, sections were incubated with AB complex (DAKO) as described in the manufacturer’s instructions for 1 h at room temperature. Peroxidase activity was detected with diaminobenzidine (DAB,2 Sigma, St. Louis, MO) (5 mg DAB and 10 μl H2O2 in 10 ml 0.05 M Tris, pH 7.8), resulting in the formation of a brown reaction product. Sections were briefly counterstained with hematoxylin, dehydrated in graded alcohols, and mounted. For double staining experiments, secondary Abs used were combinations of alkaline phosphatase-conjugated goat anti-rabbit (1:100) with goat anti-mouse biotin, or alkaline phosphatase-conjugated goat anti-mouse (1:100) with goat anti-rabbit biotin. Sections were then incubated with streptavidin β-galactosidase (1:40, Boehringer Mannheim) at room temperature for 30 min. The streptavidin β-gal was detected with 1% X-gal (DAKO) in iron phosphate buffer (0.02% MgCl2·6H2O, 0.099% potassium ferricyanide, 0.127% potassium ferrocyanide) at 37°C for 15 min, resulting in a blue color. After washing in Tris-buffered saline, the alkaline phosphatase was detected in purple by the Fast Red detection method (DAKO). Double-stained sections were mounted in Ultramount (DAKO), an aqueous mounting medium. Controls consisted of omitting the primary and secondary Ab and use of an appropriate Ig control. Single staining in adjacent sections always preceded double staining experiments.
NF-κB activation score
To assess the activity of NF-κB in situ in the human stomach, specimens from all patients were stained for active NF-κB. Two pictures of each section were taken at ×200 magnification (0.0325 mm2/picture), and positive cells were counted, blind to the clinical diagnosis, in each microscope field with the use of an image analysis program (EFM Software, Rotterdam, The Netherlands). The mean of the two fields was taken as a relative measure of NF-κB activity. To be able to compare the results between patients, it was ensured that all sections visualized the entire axis from the superficial epithelium to the muscularis mucosa.
Statistical analysis
Data are presented as mean ± SEM. Comparisons between groups of data were made using a one-way ANOVA followed by a Tukey post hoc test. p values < 0.05 were considered statistically significant.
Results
Patients
To investigate the expression of NF-κB in situ in human tissue and to assess the site and extent of activation of this transcription factor, in response to colonization by a pathogenic bacterium, we collected biopsies of 69 consecutive patients with upper abdominal complaints (Table I⇓). Of these, 26 patients were H. pylori positive, 6 after an unsuccessful attempt at eradication. Of the 43 H. pylori-negative patients, 8 were recently successfully eradicated. The histological grade of gastritis was scored according to the Sydney classification (Table II⇓).
Patient characteristics
Severity of inflammation according to the Sydney classification, number of patients per group
Expression of NF-κB in the H. pylori-negative stomach
It is now clear that H. pylori activates the transcription factor NF-κB and that this event plays a central role in the induction of the inflammatory reaction often associated with colonization with this bacterium. However, the expression of NF-κB in the antral mucosa, the main site of colonization, has thus far not been studied. To study the expression of different NF-κB subunits in the normal gastric mucosa, we examined their expression in histologically normal gastric biopsy specimens (no activity and no chronicity of inflammation as assessed by the Sidney classification (Table II⇑)) with the use of immunohistochemical techniques. In the antrum of the normal stomach (n = 26 patients), low but detectable expression of the p65 NF-κB subunit was found in the cytoplasm of the superficial gastric pit cells (Fig. 1⇓A). Deeper in the gastric glands, however, many cells were found with a very high p65 content (Fig. 1⇓, A, B, and H). In some of these cells, NF-κB was detected not only in large amounts in the cytoplasm but also in the nucleus, suggesting activation of NF-κB in these cells in the absence of overt signs of inflammation. The expression pattern of the p50 subunit (n = 15 patients; not shown), and RelB subunit (n = 15 patients; not shown) was similar to that of the p65 protein. The expression of the p52 subunit (n = 15 patients) was clearly different; it showed a staining pattern reminiscent of the p65, p50, and RelB subunits in gastric epithelial cells, but in addition and in contrast to the other subunits a relatively high expression in clusters of inflammatory cells (Fig. 1⇓C). We concluded that NF-κB subunits are highly expressed in a specific subpopulation of cells in the uninflamed stomach.
Immunohistochemical staining of the antrum of the uninflamed human stomach. A–C, DAB stain (brown precipitate), nuclear counterstain with hematoxylin; arrows show high NF-κB expression. A, Total p65 NF-κB. p65 NF-κB is differentially expressed in the antrum of the stomach. B, Higher magnification of the boxed area in A, showing cells with high p65 content. C, Adjacent section, p52 NF-κB subunit; in addition to the epithelial cells, some clusters of inflammatory cells (large asterisk) show intense staining. D–F, Double stainings for p65 and enteroendocrine cells as described in Materials and Methods, no nuclear counterstain. D, Total p65 NF-κB (red) chromogranin A (blue); arrows show examples of double staining cells. E, p65 NF-κB (red) and gastrin (blue). F, Blowup of boxed area in E; almost all high p65-expressing cells were G cells (arrows denote examples of double staining cells). G–I, Immunohistochemical staining with DAB, no hematoxylin in G. Three adjacent sections, of noninflamed mucosa, showing constitutive activity of p65 NF-κB. G, Active p65 NF-κB; note the nuclear staining (arrows). H, Adjacent section, total p65 NF-κB, stained the same cells (arrows); I, Adjacent section, TRAF2, a transcriptional target of NF-κB; many of the cells with high NF-κB expression also showed high TRAF2 expression. Original magnifications: A, ×33; B and C, ×660; D, ×650; E, ×200; F, ×1000; G, ×660; H, ×1220; A–C, ×1600).
High NF-κB expression in the endocrine cell region
The immunohistochemical determination of NF-κB protein expression showed that high levels of NF-κB subunits are expressed in a subset of epithelial cells deeper in the gastric glands. The morphology and localization of the NF-κB-expressing cells suggested that NF-κB expression in the stomach corresponded to the endocrine cell population. To investigate this, sections were double-stained with a monoclonal anti-chromogranin A Ab, a marker for enteroendocrine cells, and a polyclonal α-NF-κB p65 Ab. We observed that the NF-κB-expressing region in the stomach indeed corresponded to the chromogranin A-positive cells and those cells immediately adjacent to the chromogranin A-positive cells (n = 69 patients; Fig. 1⇑D). An almost complete correlation was observed in double staining experiments in which sections were stained with a polyclonal Ab against gastrin and an anti-NF-κB p65 mAb (n = 10 patients; Fig. 1⇑, E and F). Apparently, many of the highly NF-κB-expressing cells are G cells. These results were further confirmed by single staining experiments of adjacent sections (not shown). We concluded that expression of NF-κB in the uninflamed stomach is associated with the G cells.
Activity of NF-κB in the uninflamed stomach
The nuclear localization of NF-κB found staining with an Ab that recognizes total p65 protein suggested that this transcription factor may be active in the uninflamed mucosa. The possible activation of NF-κB was further investigated using an Ab directed against the nuclear localization sequence of the p65 subunit (14, 18, 32). Because this epitope is exposed only after degradation of the inhibitory protein I-κB, this Ab recognizes activated p65. In adjacent sections, this Ab (Fig. 1⇑H) identified the same cells that displayed nuclear localization of the protein when stained with the polyclonal anti-total p65 Ab (Fig. 1⇑, G and H). For further proof of activation, we also examined whether these cells showed expression of TRAF2, a transcriptional target of NF-κB (28). Staining for TRAF2 revealed high expression of this protein in the same cells that contain activated NF-κB (n = 12 patients; Fig. 1⇑, H and I). Furthermore, double staining for activated p65 and gastrin in the uninflamed, H. pylori-negative stomach (n = 26 patients) revealed that activation of NF-κB occurs preferentially in the endocrine cell region of the stomach, mostly in the G cells (Fig. 3⇓, B, E, and F). We concluded that the uninflamed stomach shows constitutive activation of NF-κB, in the same cells that maintain high NF-κB expression, and that these cells are mainly endocrine G cells.
Expression of TNF-α in the uninflamed mucosa
Because active NF-κB was found predominantly in the G cells, we were interested to see whether these cells would express the cytokine TNF-α, because the production of this cytokine is stimulated by NF-κB. Therefore, we stained for TNF-α and gastrin in adjacent sections (n = 20 patients; Fig. 2⇓, A and B) and double staining experiments (n = 10 patients; Fig. 2⇓, C and D). As shown in Fig. 2⇓, these stainings showed that the G cells are the main site of production of TNF-α in the histologically uninflamed stomach. Thus, in accordance with the observed activity of NF-κB, G cells produce the proinflammatory cytokine TNF-α.
H. pylori-negative, uninflamed mucosa. A and B, Immunohistochemical stain. DAB detection, with nuclear counterstain. A, Detection of gastrin staining the G cells. B, Adjacent section, detection of TNF-α, expressed in the same cell region. C and D, Double stain for gastrin and TNF-α; D is a blowup of the boxed area in C. The G cells are the main site of production of TNF-α in the uninflamed stomach (arrows denote double staining cells). Original magnifications: A–C, ×660; D, ×800.
NF-κB activation in the H. pylori-infected stomach
The activation of NF-κB in the gastric mucosa in response to colonization by a pathogenic bacterium was studied in patients with H. pylori infection. To this end, a double stain of active NF-κB and gastrin was performed for all 69 patients studied. The number of cells with activity of NF-κB in the H. pylori-colonized stomach was markedly enhanced, as compared with uninflamed tissue (Fig. 3⇓, A and B). Whereas p65 NF-κB was found in all cell types (Fig. 3⇓C), the active NF-κB was mainly detected in the epithelial cells. Remarkably, whereas some isolated inflammatory cells scattered throughout the mucosa often also stained with the anti-active p65 Ab, the large infiltrates of neutrophils did not (Fig. 3⇓D). Double staining of active NF-κB and H. pylori showed that many of the NF-κB-positive cells were not in direct contact with the bacteria (Fig. 3⇓B). Active NF-κB was primarily detected in the cells deeper in the gastric glands, many of them G cells (Fig. 3⇓, E--H). Thus, we found markedly enhanced activity of NF-κB in H. pylori-infected patients, predominantly in the epithelial cells deeper in the gastric glands.
Immunohistochemical double staining, performed as described in Materials and Methods. A, The mucosa of the uninflamed antrum of the stomach, gastrin (red), and active p65 NF-κB (blue). B, H. pylori (red), active p65 NF-κB (blue). The bacteria resided mostly in the superficial mucous layer (denoted by #), and although some of the epithelial cells that contained active p65 NF-κB were in direct contact with the H. pylori (asterisk), most of them were not (arrows). C and D, Adjacent sections, H. pylori infected patient. C, Total p65 NF-κB (blue), chromogranin A (red); all cells expressed p65 NF-κB. d, Active p65 NF-κB (blue), gastrin (red), p65 NF-κB was predominantly activated in the epithelial cells (arrows), whereas no active p65 NF-κB was detected in the neutrophil infiltrate (asterisk). E–F, H. pylori-infected patient, active p65 NF-κB (blue), gastrin (red). p65 NF-κB activation in the epithelial cells was highest deeper in the epithelium. F, Blowup of boxed area in E; in many patients NF-κB was preferentially activated in the G cells. H and I, Double stain of gastrin and active p65 NF-κB, clearly showing nuclear staining with the anti-active p65 NF-κB Ab (blue) and cytoplasmic staining with the anti-gastrin Ab (red) in the G cells. Original magnifications: A, ×660; B–D, ×500; E, ×500; F, ×1000; G and H, ×1000.
Active NF-κB and the histopathological severity of gastritis
Because NF-κB seems to play a pivotal role in the induction of inflammation in the gastric mucosa, we compared a measure of the activity of this transcription factor with the severity of gastritis scored according to the Sidney criteria. To obtain a quantitative measure of NF-κB activity in the stomach biopsies, the active NF-κB-positive cells were counted, blind to the clinical diagnosis. As depicted in Fig. 4⇓A, H. pylori infection results in a marked increase in active NF-κB-positive cells (uninflamed (870 cells/mm2) vs inflamed (2430 cells/mm2); p ≪ 0.01). Separate analysis of the patients who received treatment (Fig. 4⇓B) revealed that in patients successful eradication of the bacterium restored the mean active NF-κB score to normal whereas in those patients in whom treatment was unsuccessful this score remained high. To investigate the relation of the number of active NF-κB-positive cells to the severity of gastritis, we examined a possible correlation between the active NF-κB score and the histopathological score based on the Sydney classification system. The number of active NF-κB-positive cells correlated well with the activity of gastritis, a measure of neutrophil influx (Fig. 5⇓A). Interestingly, however, no such correlation was found between the activity of NF-κB and the chronicity of inflammation, a score of the number of mononuclear inflammatory cells (Fig. 5⇓B). As shown in Fig. 5⇓B, H. pylori-negative patients with chronic gastritis (n = 17 patients, with moderate to severe chronicity of gastritis (Table II⇑)) did not show enhanced activation of NF-κB compared with patients without chronic gastritis (n = 26). The significantly higher NF-κB activity in patients with severely chronic gastritis is most likely explained by the fact that 9 of 10 of these patients have a marked to severe activity of disease (see Table II⇑). We concluded that activation of NF-κB correlates the activity of gastritis and thus with neutrophil influx in the gastric mucosa in response to colonization with H. pylori.
NF-κB activation score. Number of active p65 NF-κB positive (pos) cells/mm2. A, The normal stomach (884 cells/mm2; n = 43) vs H. pylori-infected patients (2492 cells/mm2; n = 26). Data are means ± SEM; ∗∗∗, p ≪ 0.001. B, Whereas successful eradication of H. pylori restores NF-κB activity to normal (n = 8 patients), the number of active p65 NF-κB-positive cells remains high after treatment failure (n = 6 patients). Data are means ± SEM, ∗, p < 0,02.
NF-κB activation score vs the severity of gastritis scored according to the Sydney classification system. A, NF-κB activation score vs the activity of gastritis, a measure of neutrophil influx. This shows a clear correlation between the extent of NF-κB activation and amount of neutrophil influx. Activity: none, 893; mild, 1558; moderate (mod), 2963; severe (sev), 4078 cells/mm2. B, NF-κB activation score vs the chronicity of gastritis, a measure of the presence of mononuclear inflammatory cells (lymphocytes and macrophages). A considerable number of H. pylori-negative patients (n = 17) showed chronic gastritis (moderate to severe (Table II⇑)), the NF-κB activation score in these patients was comparable with that for patients without inflammation (n = 26), suggesting that enhanced activity of NF-κB is not necessary for the development of chronic gastritis. Although the NF-κB activation score was higher in the H. pylori-positive (pos) patients, this is probably because all of these patients also have active gastritis. Data are means ± SEM; n.p., no patients; n.s., not significant; ∗∗∗, p ≪ 0.001 vs no activity, after Tukey post hoc test.
Discussion
This study investigated the expression and activation of NF-κB in the uninflamed and inflamed stomach. NF-κB was differentially expressed in the antrum of the human stomach. Although most cell types expressed some NF-κB, the G cells displayed particularly high levels of this transcription factor. We found activity of p65 NF-κB in the G cells in the histologically uninflamed gastric mucosa. Activation was judged by four different criteria: nuclear localization of the protein; staining with an Ab against active NF-κB; and high expression of the NF-κB target genes, TRAF2 and TNF-α. The activity was markedly enhanced activation in H. pylori-infected patients in these cells.
NF-κB is a key regulator of the innate immune response and its high expression and activity in the G cells, which are not known to be involved in the immune response, is a surprising finding. However, G cells stimulate release of acid into the stomach via the production of gastrin (19, 20), and therefore these cells may help combat bacterial infection of the stomach by increasing their gastrin secretion and subsequent bactericidal acidification of the stomach. Indeed, the production of gastric acid provides an essential nonimmunological first line of defense against colonization by enteropathogenic bacteria (21). Exogenous bacteria are usually rapidly destroyed at a pH up to 4.0, and impaired gastric acid production caused by use of antacids, stomach resections, and especially chronic autoimmune gastritis with parietal cell destruction and pernicious anemia is associated with colonization of the stomach and small intestine with fecal type bacteria and increased risk of disease during cholera epidemics (21). The notion that G cells are capable of reacting to bacterial infection is further supported by the hypergastrinemia observed in H. pylori-infected patients (22). Indeed, three reports describe enhanced production of gastrin after exposure of isolated antral G cells to live H. pylori or H. pylori extracts (23, 24, 25). Thus, the expression and activity of NF-κB in the G cells may be a bona fide reflection of a role of these cells in innate immunity.
In accordance, we show that these cells are the main site of production of the NF-κB-regulated cytokine TNF-α (4) in the uninflamed stomach. Addition of exogenous TNF-α to isolated G cells stimulates the production of gastrin in vitro (23, 25, 26, 27). Our finding that G cells produce TNF-α and respond to H. pylori with enhanced NF-κB activation may suggest that colonization by this bacterium increases expression of TNF-α in the G cells through activation of NF-κB and causes increased gastrin production in an autocrine manner. Thus, the high levels of NF-κB and TNF-α expression in the G cells may couple the innate immune response to the production of bactericidal gastric acid.
Additionally, NF-κB may play a specific role in the cell fate and/or differentiation of these cells. Because NF-κB plays an important role in the protection of cells from apoptosis via stimulation of the production of anti-apoptotic proteins (4, 28), such as TRAF2 (28, 29), the activity of this transcription factor is likely to play a role herein. Indeed, whereas the superficial gastric epithelial cells undergo enhanced apoptosis in H. pylori-infected patients, G cells may be protected from this process by their high expression of NF-κB, and increased G cell mass has been reported in these patients (30, 31).
H. pylori infection greatly increased the number of cells containing active NF-κB, activation occurred predominantly in the epithelial cells, and little activation was detected in neutrophils. This is in agreement with findings by Rogler et al. (32) in patients with inflammatory bowel disease, who showed that NF-κB was mainly activated in epithelial cells and macrophages. These authors also demonstrated a correlation between the activity of NF-κB and an endoscopic score of inflammation. In our study, the increase in NF-κB activity correlated well with the activity of gastritis, a measure of neutrophil influx, as scored according to the Sydney classification system. Together, these studies support an important role for epithelial NF-κB activation in the production of neutrophil chemoattractants like IL-8.
No correlation was found with the chronicity of inflammation, a measure of the number of mononuclear inflammatory cells in the mucosa, given that H. pylori-negative patients with chronic inflammation did not have a higher NF-κB activity score than those without. This lack of correlation is further supported by our finding that the NF-κB activity score was restored to normal values in patients recently treated successfully whereas it is well known that the chronic component of gastritis may last for up to 1 year after successful treatment.
Double staining for H. pylori and active p65 NF-κB showed that most of the epithelial cells exhibiting activation of NF-κB were not in direct contact with the bacteria. The activation occurred preferentially deeper in the gastric glands, in the endocrine cell region, where no H. pylori are found because the bacterium resides in the superficial mucus layer. Therefore, NF-κB activation in the stomach does not seem to require direct contact with bacteria and may thus be dependent on a secreted factor. Accordingly, H. pylori virulence and induction of epithelial IL-8 expression are strongly associated with a group of 31 bacterial genes, the pathogenicity island (33, 34), which encodes a type IV toxin secretion system (35).
In conclusion, our findings show that in the uninflamed antrum of the stomach high NF-κB expression and activity are associated with the G cells, which produce TNF-α, a well-documented stimulator of gastrin release. This suggests an autocrine mechanism of gastrin production by TNF-α that may be enhanced on colonization by pathogenic bacteria. Our data also show a strong correlation between epithelial NF-κB activation and neutrophil influx in H. pylori-associated gastritis, a clear illustration of the importance of NF-κB activation in chemokine production.
Acknowledgments
We thank Dr. W. P. van den Brink for expert statistical advice.
Footnotes
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↵1 Address correspondence and reprint requests to Dr. Gijs van den Brink, Laboratory of Experimental Internal Medicine, Room G2-130, Academic Medical Center, Meibergdreef 9, 1005 AZ, Amsterdam, The Netherlands. E-mail address: g.r.vandenbrink{at}amc.uva.nl
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↵2 Abbreviation used in this paper: DAB, diaminobenzidine.
- Received October 12, 1999.
- Accepted January 4, 2000.
- Copyright © 2000 by The American Association of Immunologists
References
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