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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 3  |  Page : 418-421
Nuclear factor kappa-B and histopathology of chronic gastritis


Department of Pathology, Armed Forces Medical College, Pune, India

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Date of Web Publication22-Oct-2010
 

   Abstract 

Background: Studies suggest that nuclear factor kappa-B (NFκB) activation may be a critical event in the production of proinflammatory molecules in Helicobacter pylori-associated gastritis. Materials and Methods: This study examines the expression and activity of NFκB in situ in antral biopsies of 42 consecutive patients with immunohistochemical techniques. Results: NFκB was highly expressed in the gastric epithelial cells. The number of cells showing activated NFκB correlated with the activity of gastritis (P < 0.05), a measure of neutrophil influx, whereas no correlation was found with the chronicity of inflammation, a measure of the presence of mononuclear inflammatory cells. There was also a strong inverse association with the presence and grade of atrophy. Conclusion: This correlation is direct evidence of the importance of NFκB dependent signal transduction for neutrophil influx in gastritis. The role of NFκB appears to be only in the initial stages of gastritis, there is no role for the molecule in the development of chronic inflammation or atrophy.

Keywords: Gastritis, nuclear factor kappa B, antral biopsy

How to cite this article:
Moorchung N, Srivastava A N, Sharma A K, Achyut B R, Mittal B. Nuclear factor kappa-B and histopathology of chronic gastritis. Indian J Pathol Microbiol 2010;53:418-21

How to cite this URL:
Moorchung N, Srivastava A N, Sharma A K, Achyut B R, Mittal B. Nuclear factor kappa-B and histopathology of chronic gastritis. Indian J Pathol Microbiol [serial online] 2010 [cited 2020 Nov 25];53:418-21. Available from: https://www.ijpmonline.org/text.asp?2010/53/3/418/68255



   Introduction Top


Upon colonization by pathogenic bacteria, the host cells at the site of infection respond with an induction of the innate immune response. The host cells produce proinflammatory proteins such as cytokines, chemokines, and adhesive molecules, to counteract the external threat. 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 nuclear factor kappa B (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] The p65 subunit of NFκB is translocated to the nucleus after degradation of the inhibitory I-κB (inhibitory kappa B) in response to a wide variety of stimuli. [4],[5] It is then believed to regulate a wide variety of genes including those encoding cytokines, antimicrobial peptides and adhesion molecules. [2]

A classical model system to study the induction of the innate immune response by a single pathogenic bacterial species exists in the human stomach. Colonization by Helicobacter pylori results in an acute host response, mostly followed by persistence of the bacterium and chronic gastric inflammation. [6],[7] Activation of NFκB may cause gastritis via the induction of proinflammatory cytokines such as IL-1 (interleukin 1) and TNF-α (tumor necrosis factor a) and chemokines like IL-8 (interleukin 8). [8],[9] In this study, we examine the expression and activation of NF-κB in the antrum of the human stomach in gastritis. We also investigate the possible correlation of the activity of NF-κB with the severity of gastritis. The study aims to evaluate if the quantity of NFκB staining correlated with any of the inflammatory histological parameters.


   Materials and Methods Top


Patient Selection

Patients with non ulcer dyspepsia constituted the patient population. Exclusion criteria were a present or past history of gastric neoplasm and / or gastric surgery, long term therapy with nonsteroidal anti-inflammatory drugs, liver disease and previous treatment with antibiotics or bismuth salts. Endoscopic evidence of esophageal varices, pinpoint gastric ulcers and / or duodenal ulcers, the presence of mucosal scars and / or a suspicious mass in the upper digestive tract also constituted exclusion criteria. One hundred and twenty patients were biopsied of which 42 biopsies were immunostained for NFκB.

Histopathology

Three antral biopsies were taken for histological examination from the distal lesser and greater curvature, 2 to 3 cm from the pylorus. The biopsies were immediately fixed in formalin for histopathological examination. Sections were stained with the standard hematoxylin and eosin (H and E) stain and the Giemsa stain for H. pylori. The Giemsa stain was carried out using standard histological laboratory methods. Positive H. pylori showed deep blue-stained bacillary structures in epithelium of mucosa or in glandular recess. Histopathological analysis of the biopsies was done based on a method proposed by the authors. [10] The grading of the presence of lymphoid follicles was done as per the histological scoring index as proposed by Wotherspoon et al.[11]

Immunohistochemistry

Serial 4 mm thick sections were made and mounted on poly L lysine coated slides. Paraffin sections were immersed in xylene for five minutes and hydrated using a gradient series of alcohol. Antigen retrieval was routinely performed by immersing the sections in citric acid buffer (pH 6.0), in a microwave oven for 15 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes and then incubated with a primary antibody in a humidified chamber at 4 overnight. Primary antibody was monoclonal mouse anti-NF-κB/p65 antibody (Santa Cruze) at 1:200 dilution. Expressions of NFκB were evaluated according to the ratio of positive cells and graded as a percentage.

Statistical Analysis

The statistical package for social sciences (SPSS) for Windows (version 13) was used for statistical analysis. Association between the different parameters was evaluated using the Kruskal Wallis test for non parametric data. Differences were considered to be statistically significant at P < 0.05.


   Results Top


Neutrophils

Sixteen patients did not show the presence of neutrophils on the biopsy in the mucosa. Seventeen patients showed mild neutrophilic infiltrate, seven - moderate and two - marked inflammatory infiltrate. When no neutrophils were seen, the mean NFκB positivity was 21.56% [Figure 1]. With a mild, moderate and marked neutrophilic infiltrate, the percentage of NFκB staining was 16.68%, 29.21% and 35% respectively. There was a marked correlation between the quantity of NFκB staining and the grade of the neutrophilic infiltrate [Figure 2].
Figure 1 :Sections showing a comparison of the NFêB staining. The section on the left shows a strong perinuclear and nuclear staining for NFêB in the gastric epithelial cells. In comparison, the secti on on the right shows a complete absence of NFêB staining

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Figure 2 :Boxplots showing the marked association between the percentage of NFƒÈB positi ve cells and the neutrophilic infi ltrate. The figure also shows the lack of correlati on of the NFƒÈB positi ve cells and the plasma cell density

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Lymphocytes and Plasma Cells

Thirteen patients showed a mild lymphocytic infiltrate, 11 a moderate and 18 a marked inflammatory infiltrate. The mean NFκB positivity with a mild, moderate and a marked lymphocytic infiltrate was 22.62%, 18.91% and 2.28% respectively. Twelve patients showed a mild plasmacytic infiltrate, 16- moderate and 14 - marked plasmacytic infiltrate. The mean NFκB positivity with a mild, moderate and marked plasma cell infiltrate was 23.92%, 16.44% and 25.21% respectively. There was no correlation of the lymphocytic or plasma cell infiltrate with the mean NFκB positivity [Figure 2].

Eosinophils

Nineteen patients showed no eosinophils, 19 showed a mild eosinophillic infiltrate and four patients showed a moderate inflammatory infiltrate. The mean NFκB positivity for an absent, mild and moderate eosinophillic infiltrate was 21.95%, 20.16% and 25.75% respectively [Figure 3]. There was no correlation between the mean NFκB positivity and the presence or the grade of the eosinophillic infiltrate.
Figure 3 :Boxplots showing the lack of associati on between the percentage of NFƒÈB positi ve cells and the presence and grade of lymphoid follicles. The figure also shows the negati ve correlati on of the NFƒÈB positi ve cells and the presence and grade of atrophy

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Lymphoid Follicles

Twenty nine patients did not show the presence of lymphoid follicles, six patients showed Grade 2 lymphoid follicles and seven patients showed Grade 3 lymphoid follicles. The mean NFκB positivity for an absent, Grade 2 and Grade 3 lymphoid follicles was 20.62%, 23.83% and 23.14% respectively. There was no correlation between the grade of the lymphoid follicles and the mean NFκB positivity [Figure 3].

Atrophy

Fifteen patients showed no evidence of atrophy, eight patients showed mild, eleven showed moderate and eight patients showed marked atrophy on histopathology. There was a negative correlation between the mean NFκB positivity and the presence and grade of atrophy. The mean NFκB positivity for absent, mild, moderate and marked atrophy were 22.13%, 24%, 22.18% and 16.8% respectively [Figure 3].

Helicobacter pylori Density

Sixteen patients did not show the presence of
H. pylori in the biopsy and twenty six showed the presence of a mild infiltration with H. pylori. The mean NFκB positivity for absent and mild H. pylori infiltration was 22.38% and 20.96% respectively. There was no correlation between the mean NFκB positivity and the presence or grade of the H. pylori infiltration.


   Discussion Top


NFκB was first identified as a regulator of expression of the kappa light chain gene in murine B lymphocytes but has subsequently been found in many different cells. [2] NFκB is made up of protein dimers that bind to a common sequence motif known as the κB site. The activated form of NFκB is a heterodimer, which usually consists of two proteins: a p65 subunit and a p50 subunit. In unstimulated cells, NFκB is found in cytoplasm and is bound to IκB, which prevents it from entering nuclei. When cells are stimulated, specific kinase phosphorylate IκB results in IκB's rapid degradation by proteasomes. The release of NFκB from IκB would result in passage of NFκB into nuclei, where it binds to specific sequences in promoter regions of target genes and further activates target genes. [12],[13],[14],[15],[16]

In the present study, we find that high expression of NF-κB/p65 is detected in the tissues with prominent neutrophilic infiltrate. We also observe that expression of NF-κB/p65 is mainly localized in cytoplasm. Furthermore, the intensity of NFκB staining correlates with the density of the neutrophilic infiltrate. This finding suggests that the activation of NFκB is the primary factor which is responsible for the initial inflammatory response. This finding is in consonance with other studies. [17],[18] Similar to other reports, the localization of NFκB is predominantly in epithelial cells and not neutrophils. [17] This study, therefore, supports an important role for epithelial NF-kB activation in the production of neutrophil chemoattractants like IL-8.

We did not find any correlation of the NFκB staining with the H. pylori density. This is contrary to what has been reported in literature. [17],[18],[19] This is also in contrast to what we observed in sections which we stained for both NFκB and H. pylori. Localization of NFκB is predominantly seen when the cells are in close proximity to the bacterium. The explanation of this discrepancy is possibly because of the small sample size and the fact that none of the slides evaluated had more than a mild H. pylori infiltration.

There was no correlation with the intensity of the chronic inflammatory infiltrate and the intensity of NFκB staining. This is in consonance with what as been reported by Brink et al. [17] There was also no correlation with the eosinophillic infiltrates. To the best of our knowledge, such a finding has not been reported in literature. This suggests that the activation of NFκB is not the primary factor in the recruitment of eosinophils.

We found a negative correlation with the grade of atrophy. This is contrary to some of the reports which found a positive correlation with atrophy. [18] We had previously reported that atrophy occurred only in the later stages of the disease. [20] We had also reported that although the grade of atrophy correlated well with the density of the chronic inflammatory infiltrate, there was no correlation with the neutrophilic infiltrate. Our present findings suggest that with the advance in the disease, the amount of NFκB staining decreases. The role of the NFκB molecule appears to be predominantly in the early stages of the disease when it is responsible for the induction of the neutrophilic infiltrate. There is no role of NFκB in the later stages of the disease which is indicated by the inverse NFκB staining in the presence of atrophy.

In conclusion, it can be stated that epithelial NNF-κB activation is strongly correlated with the neutrophil influx in gastritis, a clear illustration of the importance of NF-κB activation in chemokine production. The lack of correlation with the chronic inflammatory infiltrate and the negative correlation with of NF-κB staining with atrophy indicates that the molecule has no role to play in the chronicity of the disease process.

 
   References Top

1.Cossart P, Boquet P, Normark S, Rappuoli R. Cellular microbiology emerging. Science 1996;271:315-6.  Back to cited text no. 1      
2.Barnes PJ, Karin M. Nuclear factor-κB: A pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997;336:1066-71.  Back to cited text no. 2      
3.Baeuerle PA. IkB- Nuclear Factor Kappa B (NF-κB) structures: At the interface of inflammation control. Cell 1998;95:729-31.  Back to cited text no. 3      
4.Ghosh S, May MJ, Kopp EB. Nuclear Factor Kappa B (NF-κB) and Rel proteins: Evolutionary conserved mediators of immune responses. Annu Rev Immunol 1998;16:225-60.  Back to cited text no. 4      
5.Sha WC. Regulation of immune responses by NF-κB/Rel transcription factors. J Exp Med 1998;187:143-6.  Back to cited text no. 5      
6.Drumm B, Sherman P, Cutz E, Karmali M. Association of Campylobacter pylori on the gastric mucosa with antral gastritis in children. N Engl J Med 1987;316:1557.  Back to cited text no. 6      
7.Blaser MJ, Parsonnet J. Parasitism by the "slow" bacterium Helicobacter pylori leads to altered gastric homeostasis and neoplasia. J Clin Invest 1994;94:4-8.  Back to cited text no. 7      
8.Sharma SA, Tummuru MK, Blaser MJ, Kerr LD. Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-kB in gastric epithelial cells. J Immunol 1998;160:2401-7.  Back to cited text no. 8      
9.Noach LA, Bosma NB, Jansen J, Hoek FJ, van Deventer SJ, Tytgat GN. Mucosal tumor necrosis factor-a, interleukin 1b and interleukin- 8 production in patients with Helicobacter pylori infection. Scand J Gastroenterol 1994;29:425-9.   Back to cited text no. 9      
10.Moorchung N, Srivastava AN, Gupta NK, Achyut BR, Mittal B. The Histopathology of Chronic gastritis. Indian J Pathol Microbiol 2007;50:18-24.  Back to cited text no. 10  [PUBMED]  Medknow Journal  
11.Wotherspoon AC, Doglioni C, Diss TC, Pan L, Moschini A, de Boni M, et al. Regression of primary low grade gastric lymphoma of mucosal associated lymphoid tissue type after eradication of Helicobacter pylori. Lancet 1993;342:575-7.  Back to cited text no. 11      
12.Siebenlist U. Signal transduction. Barriers come down. Nature 2001;412:601-2.  Back to cited text no. 12      
13.Isomoto H, Mizuta Y, Miyazaki M, Takeshima F, Omagari K, Murase K, et al. Implication of NF-kappaB in Helicobacter pylori-associated gastritis. Am J Gastroenterol 2000;95:2768-76.  Back to cited text no. 13      
14.Foryst-Ludwig A, Naumann M. p21-activated kinase 1 activates the nuclear factor kappa B (NF-kappa B)-inducing kinase-lkappa B kinases NF-kappa B pathway and proinflammatory cytokines in Helicobacter pylori infection. J Biol Chem 2000;275:39779-85.  Back to cited text no. 14      
15.Naumann M. Nuclear factor-kappa B activation and innate immune response in microbial pathogen infection. Biochem Pharmacol 2000;60:1109-14.  Back to cited text no. 15      
16.Maeda S, Akanuma M, Mitsuno Y, Hirata Y, Ogura K, Yoshida H, Shiratori Y, Omata M. Distinct mechanism of Helicobacter pylori- mediated NF-kappa B activation between gastric cancer cells and monocytic cells. J Biol Chem 2001;30:44856-64.  Back to cited text no. 16      
17.van Den Brink GR, ten Kate FJ, Ponsioen CY, Rive MM, Tytgat GN, van Deventer SJ, et al. Expression and activation of NF-kappa B in the antrum of the human stomach. J Immunol 2000;164:3353-9.  Back to cited text no. 17      
18.Doger FK, Meteoglu I, Ozkara E, Erkul ZK, Okyay P, Yόkselen V. Expression of NF-kappaB in Helicobacter pylori infection. Dig Dis Sci 2006;51:2306-9.  Back to cited text no. 18      
19.Isomoto H, Miyazaki M, Mizuta Y, Takeshima F, Murase K, Inoue K, et al. Expression of nuclear factor-kappaB in Helicobacter pylori-infected gastric mucosa detected with southwestern histochemistry. Scand J Gastroenterol 2000;35:247-54.  Back to cited text no. 19      
20.Moorchung N, Srivastava AN, Gupta NK, Ghoshal UC, Achyut BR, Mittal B. Cytokine gene polymorphisms in the pathogenesis of chronic gastritis. Singapore Med J 2007;48:447-54.  Back to cited text no. 20      

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Correspondence Address:
Nikhil Moorchung
Department of Pathology, Armed Forces Medical College, Sholapur Road, Pune-411 040
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.68255

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