TRYPTASE PROFILE OF MAST CELLS OF THE GASTRIC MUCOSA IN H. PYLORI INFECTION

Full Text

To date, H. pylori infection accounts for at least half of the human population [1], but most infected people do not have clinical manifestations [2, 3]. Colonization and long-term infection of H. pylori in the human stomach almost always leads to chronic gastritis, peptic ulcer or gastric tumor [1]. Various types of immunocompetent cells, including mast cells (MCs) are actively involved in the pathogenesis of gastritis associated with H. pylori [2, 3]. Tryptase is a specific TK protease and is involved in the formation of physiological and pathological processes in the body [4]. MC through tryptase activity initiate inflammation, recruitment of granulocytes, lead to an increase in capillary permeability, changes in intercellular signaling, activity of other cells and noncellular components [4]. Tryptase TK can promote both wound healing and the development of intraorgan fibrosis by regulating the migration and synthetic capacity of fibroblasts [5]. This fact is important to take into account in the aspect of the pathogenesis of Helicobacter-associated gastritis. MCs may be involved in the formation of extracellular traps for the development of antibacterial effects [6]. In this regard, the aim of the work was to evaluate the tryptase profile of MC in H. Pylori-associated inflammation using the method of double immunolabeling with simultaneous detection of MC tryptase and H. pylori.

Material and research methods

Sample preparation of biopsy specimens included fixation with a neutral solution of 10% formalin for 24-48 hours, the use of an MTP histological processor (SLEE, Germany), and paraffin embedding in the Tissue-Tek® TEC™ 5 modular system (Sakura Seiki Co.Ltd. Japan) and making histological sections on a semi-automatic rotary microtome Accu-Cut® SRM™ 200 (Sakura Finetek Europe BV, the Netherlands). Gastrobiopsy specimens from 19 patients with unknown H. pylori infection status were subjected to double immunofluorescent labeling to detect localization of tryptase-positive mast cells and H. pylori strains [7, 8]. Rabbit monoclonal antibodies to H. pylori (ab#172611, dilution 1:500) and mouse monoclonal antibodies to tryptase (ab#2378, dilution 1:2000) were used as primary antibodies, which were incubated overnight. Secondary antibodies were then applied: Goat Anti-Rabbit IgG H&L (Cy3®) and Anti-Mouse IgG H&L (Alexa Fluor® 488), which were developed with the DAPI Staining Solution reagent for 24 s and a fluorescent imaging medium was applied. For review microscopy, micropreparations were stained with hematoxylin and eosin, as well as Giemsa stain. The presence of H. pylori infection of the gastric mucosa was determined using an immunohistochemical method [7]. To identify H. pylori, sections were deparaffinized and incubated with rabbit monoclonal antibodies (ab172611, 1:500 dilution) overnight. AmpliStainTM anti-Rabbit 1-Step HRP #AS-R1-HRP secondary goat anti-rabbit antibodies were then applied and detected with the ImmPACTTM DAB Peroxidase Substrate Kit (#SK-4105) according to the manufacturer's instructions. As a result, upon completion of staining by this method, various forms of H. pylori with a variable degree of contamination were visualized on micropreparations of the stomach. Separately, immunohistochemical detection of TK tryptase was performed with mouse monoclonal antibodies (ab2378, dilution 1:2000) overnight. HRP-conjugated secondary antibodies (Goat anti-Mouse IgG H&L, #AS-M1-HRP) were then applied and detected with the ImmPACTTM DAB Peroxidase Substrat Kit (#SK-4105). Staining with this technique on micropreparations of the stomach visualized tryptase-positive MC, separately located granules, or fragments of the cytoplasm. Gastrobiopsy samples were studied using a ZEISS Axio Imager microscope. A2 with a system of photodocumentation of images. The obtained images were processed using the ZEN 2.3 program (Carl Zeiss, Germany). Determination of the number of tryptase-positive MCs in the biopsy material was carried out using planimetric analysis in the field of view using an x20 objective. For calculations, 60 fields of view were used whenever possible, and the images were documented with a Camera Axiocam 506 color bright-field color microscopy camera. Subsequently, the obtained numerical values ​​were recalculated to obtain quantitative data reflecting the distribution density of TC per mm2 of biopsy material. Statistical analysis was performed using the SPSS software package (V. 13.0). Results are presented as mean (M) ± m (standard error of the mean).

Results

Morphological analysis of the mucosal lining of gastric biopsies from patients with unknown H. pylori infection status began with immunohistochemical staining to determine bacterial infection. In 12 out of 19 samples, bacteria with varying degrees of contamination were found; in 7 patients, the bacterial agent was not detected. In the group of infected patients (n=12), there was a statistically significant increase in the number of tryptase-positive mast cells by 2.03 times (177.99±30.55 88.58±11.49, p<0.05). The quantitative indicators of mast cells in the group of patients with an unidentified pathogen (n=7), but with signs of a chronic inflammatory process of the gastric mucosa, were statistically significantly lower compared to the group of infected patients. Invasion of mast cells containing tryptase into the glandular epithelium was noted, in the lumen of which bacterial strains with a high degree of contamination were found. ,21±2.58, p<0.05). In addition, there was a higher content of isolated localized tryptase-positive MC granules in the gastric stroma and separately located MC fragments, which suggests activation of the formation of autonomous tryptase-containing sites under infection conditions. , providing an expansion of the area of ​​formation of selective effects.

Discussion

Using the technology of double immunofluorescent labeling of tryptase TK and H. pylori, it was possible to identify features in relation to the histotopography of tryptase, with the formation of areas of accumulation of large free-lying granules around the glands with a pronounced degree of H. pylori contamination. The high density of MC in patients with Helicobacter pylori infection can be regarded as additional morphological evidence of gastritis activity compared with the group of patients with a negative status. Obviously, such a format for controlling local homeostasis in the presence of H. pylori has its own advantages, since, in general, simultaneous accumulation of tryptase in a larger number of mucosal loci is achieved, and, accordingly, the efficiency of regulation of a specific tissue microenvironment increases. Atrophic phenomena were noted in two gastric biopsies of patients infected with H. pylori with the highest amount of TK.

Conclusion

Gastrobiopsy specimens revealed colocalization of tryptase-positive mast cells and H. pylori strains, which indicates a close involvement in the development of inflammatory reactions of the gastric mucosa. In patients infected with H. pylori (n=12), there was a statistically significant increase in the number of tryptase-positive mast cells by 2.03 times (177.99±30.55 88.58±11.49, p<0.05) . The quantitative indicators of mast cells in the group of patients with an unidentified pathogen (n=7), but with signs of a chronic inflammatory process of the gastric mucosa, were statistically significantly lower compared to the group of infected patients. The role of TC in this process is ambiguous, due to the peculiarities of the cellular microenvironment, depends on the degree of damage to the mucous membrane, bacterial contamination and the duration of the inflammatory process.

About the authors

Victoria S. Aralova

Voronezh State Medical University named after N.N. Burdenko;Research Institute of Experimental Biology and Medicine

Email: vikaaralova2014@mail.ru
ORCID iD: 0000-0002-7290-9390
SPIN-code: 9051-2635
Russian Federation, 394036, Russia, Voronezh, Student, 10;394066 Voronezh, Moskovsky prospect, 185

Lyubov N. Antakova

Research Institute of Experimental Biology and Medicine

Email: tsvn@bk.ru
ORCID iD: 0000-0001-5212-1005
SPIN-code: 3936-3381
Russian Federation, 394066 Voronezh, Moskovsky prospect, 185

Victoria V. Shishkina

Research Institute of Experimental Biology and Medicine

Author for correspondence.
Email: 4128069@gmail.com
ORCID iD: 0000-0001-9185-4578
SPIN-code: 9339-7794
Russian Federation, 394066 Voronezh, Moskovsky prospect, 185

References