Animals

The experiments were approved by the Ethics Committee of Experiments on Animals, University of Gothenburg. Thirty specific pathogen free (SPF) male Mongolian gerbils (Charles River, Uppsala, Sweden), seven weeks of age, were used. They were randomly separated into three groups of equal size, consisting of one control group and two groups that were to be infected with H. pylori. Five animals were kept in each cage and the room was regulated with respect to temperature (18-22°C), humidity (about 55%) and light/dark cycle (12/12h). The gerbils had free access to food (2016F, Harlan Tek. Lab, Blackthorn, England) and drinking water. They were allowed one week of acclimatization before the inoculation.

Bacterial strains and inoculation

Two different H. pylori strains were used for inoculation, the TN2GF4 strain [16] and the Sydney strain 1 (SS1) [17]. Both strains are known to cause a chronic active inflammation in the gerbil gastric antral and corporal mucosa. The bacteria were grown on Columbia plates. These cultures were then used to inoculate 500 ml Brucella broth containing 5% newborn calf serum, 10 μg/ml vancomycin and 5 μg/ml trimetoprim. The flasks were incubated for 24 hours under micro aerobic conditions at 37°C. The bacteria were examined by phase contrast microscopy before being administered to the animals. The gerbils were infected with 0.5 ml of the bacterial suspension or Brucella broth only (controls) using a feeding needle. Viable counts were made in the suspension to determine the actual infectious doses, which were 7 × 10⁷ units and 4 × 10⁷ units for SS1 and TN2GF4, respectively.

Time course and sacrifice

Following a 24-hr fasting period with free access to drinking water, five animals from each group were sacrificed six or 12 months after inoculation. The animals were anesthetized by intraperitoneal injection of medetomodin (0.5 mg/kg) and ketamin (75 mg/kg). A midline laparotomy was performed and the stomachs were then removed and opened along the major curvature. One half was used for culture, and specimens were collected from the other half for histological analyses and western blot analysis. The animals were sacrificed by cardiac incision. Histopathological findings and bacteriological status have been reported previously [14].

Immunohistochemistry

The full wall specimens (antrum and corpus) were collected immediately after the opening of the stomachs, fixed in Histofix (Histolab products AB, Gothenburg, Sweden) at room temperature (RT) overnight and then rinsed in PBS for 24 hours. Specimens were subsequently dehydrated, embedded in paraffin, cut into 5-μ thick sections and mounted on glass slides. Before immunohistocemical staining, sections were deparaffinized and boiled in citric acid buffer (0.01 M, pH 6.0, 10 min). The Immunocruz TM Staining System (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for the immunohistochemistry protocol. After inhibition of endogenous peroxidase activity, non specific binding of secondary antibodies was blocked by incubation with normal goat serum for 30 min at RT. The following two polyclonal primary antibodies, dilutions and incubation times were then used: rabbit anti-AT1R (N-10, Santa Cruz Biotech, 1:100 in PBS, 2 hr at RT) and rabbit anti-AT2R (H-143, Santa Cruz Biotech, 1:100 in PBS, 2 hr at RT). After incubation with primary antibodies, sections were washed three times in PBS and probed with a biotinylated secondary antibody; the complex was detected using horseradish peroxidase (HRP)-streptavidin and the color was developed using 3,3'-diaminobenzidine (DAB).

An unexpected, strong staining of cells with a typical appearance of endocrine cells was noted after immunohistochemical staining with this peroxidase based method. Immunoflourescens labelling was done to further rule out the possibility that this staining was a result of endogenous biotin, of endogenous peroxidase activity or of unspecific binding of the secondary antibody. The following protocol was used: after boiling in citric acid buffer, non specific binding of secondary antibodies was blocked by incubation with normal goat serum (sc-2043, Santa Cruz Biotech) for 30 min at RT. Sections were incubated with primary antibodies as above. The slides were then washed three times in PBS and probed with a secondary antibody of Alexa Flour 488 conjugated goat anti-rabbit IgG (A-11034, Molecular Probes Inc, Eugene, OR, USA), diluted 1:400 in PBS for 1 hr at RT. The slides were next washed three times in PBS and mounted with fluorescence mounting medium (DakoCytomation, Glostrup, Denmark), after which images were captured with a fluorescence microscope. To confirm the location of AT1R in endocrine cells, double immunostaining was performed using the above described peroxidase based protocol for AT1R and the above described immunoflorescens labelling protocol for a primary antibody directed against Chromogranin A (C-20, Santa Cruz Biotech, 1:100 in PBS, 2 hr at RT). Non specific binding of the secondary antibody (Alexa Flour 568-conjugated donkey anti-goat IgG, A-11057, Molecular Probes Inc, 1:800 in PBS, 1 hr at RT) was blocked by incubation with normal donkey serum (sc-2044, Santa Cruz Biotech). Preabsorpion with an excess (5×) blocking peptide (sc-1173P, Santa Cruz Biotech) was performed as a control for the specificity of the AT1R antibody, while the primary antibody was omitted for the AT2R.

The gerbil AT1R and AT2R have >90% amino acid homology sequence identity with human, rat and mouse Ang II receptors [18, 19]. In gerbils, as in rats and mice, there are two AT1R subtypes (AT1aR and AT1bR) that are encoded by different genes but with 95% amino acid sequence homology. These receptor subtypes are known to be differentially localized and regulated but have a similar affinity for Ang II. Because of high amino acid sequence homology between the gerbil AT1aR and AT1bR, we assumed equal binding affinity to the AT1R subtypes for the AT1R antibody used in the present study. Moreover, the antibodies used for staining Ang II receptors are established by the manufacturer in mice, rats and humans. Therefore, to confirm the specificity of these antibodies in gerbils, sections from paraffin embedded blocks of normal gerbil and human adrenal glands were stained as above (except that both primary antibodies were diluted 1:50 in PBS) and the distribution patterns for the Ang II receptor antibodies were studied. The adrenal distribution studies showed that the anti-AT1R antibody predominantly stained adrenal cortical cells in the zona glomerulosa (staining of the capsule surrounding the gland, vascular smooth muscle cells (VSMCs) and endothelial cells was also noted) in both gerbil and human sections. The anti-AT2R antibody stained adrenal medulla cells and cells in the zona glomerulosa in gerbil and human adrenal gland (staining of VSMCs and endothelial cells was also noted using this antibody). These results agree with previously reported studies [20], and the strong similarity of staining patterns in the gerbil and the human adrenal gland support a general usefulness of the antibodies in gerbil tissues.

Western blot

After opening the stomachs, full thickness antral wall specimens were immediately collected, frozen in liquid nitrogen and stored at -70°C. The specimens were homogenized on ice (Polytron, PT-MR 2100, Kinematica, Switzerland) in buffer A (10% glycerol, 20 mM Tris-HCL pH 7.3, 100 mM NaCl, 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 nM EGTA, 10 mM sodium orthovanadate, 10 μg/ml leupeptin and 10 μg/ml aprotinin) [21] and centrifuged at 30,000 g for 30 min at 4°C. The pellets were resuspended in buffer B (1% NP-40 [Sigma-Aldrich, Stockholm, Sweden] in buffer A) and stirred at 4°C for 1 hr before centrifugation at 30,000 g for 30 min at 4°C. The supernatants were analyzed for protein content by the method of Bradford [21] and stored at -70°C until further analysis. Samples were diluted in SDS buffer and heated at 70°C for 10 min before being loaded on a NuPage 10% BisTris gel (Invitrogen, Carlsbad, CA, USA). One lane was loaded with prestained molecular weight standards (SeeBlue™, Invitrogen AB, Lidingo, Sweden), and whole cell lysates of PC-12 (for AT1R; sc-2250, Santa Cruz Biotech), HEP G2 (for AT2R; sc-2227, Santa Cruz Biotech) and HL60 (for myeloperoxidase (MPO); 12-354, Upstate Lake Placid, NY, USA) were used as positive controls. Polyvinyldifluoride membranes (Amersham, Buckinghamshire, UK) were incubated with polyclonal rabbit antibodies directed against AT1R (N-10, Santa Cruz Biotech), AT2R (H-143, Santa Cruz Biotech) or MPO (07-496, Upstate). An alkaline phosphatase conjugated goat anti-rabbit antibody (sc-2007, Santa Cruz Biotech for the AT1R and AT2R antibodies, and IgG 12-448, Upstate, for the MPO antibody) and CDP-Star substrate (Tropix, Bedford, MA, USA) were used to identify immunoreactive proteins by chemiluminescense. Images were captured by a cooled CCD camera (LAS-1000; Fujifilm, Tokyo, Japan) and semi quantification was done by comparing samples to positive controls using the Gauge 3.3 software (Fujifilm, Tokyo, Japan).

Microscopic morphometry

Tissue specimens from the antral wall were fixed in Histofix (Histolab Products AB) and embedded in paraffin. Four-μ thick sections were mounted on coded glass slides and routinely stained with haematoxylin/eosin (H&E). The degree of mucosal infiltration of mononuclear and polymorphonuclear leucocytes was studied in the light microscope. The lamina propria will tend to increase in volume as a result of the influx of inflammatory cells. Therefore, the relative volume of the lamina propria was assessed by morphometry to reflect the infiltration of both mononuclear and polymorphonuclear leucocytes. This was carried out by H.F.H. in a light microscope, with a 10 × 10-square grid inserted into the eyepiece and a ×25 objective lens. Using the point counting method [22], the volume density of the lamina propria was determined and expressed in per cent of the mucosa (in this case defined as the region between the mucosal surface and the bottom of the glands). Mucosal volume is here defined as epithelial layer + lamina propria. The mucosal infiltration of polymorphonuclear leucocytes (PMNs) was assessed by P.H. in a light microscope, with a 10 × 10-square grid inserted into the eyepiece, a ×50 oil immersion objective lens (N.A. 1.4) and an oil immersion top lens of the condenser (N.A. 1.4). PMNs were identified as roughly round cells in lamina propria with a darkly stained multilobulated or bilobulated nucleus. The number of PMNs in a square region of the mucosa was determined, as was the total number of squares over the lamina propria; the number of PMNs is expressed per 1 mm² lamina propria. The counting started from the bottom of the glands and ended after assessment of one to seven full swaths of the mucosa, resulting in a minimum of 0.077 mm²lamina propria analyzed per section. Systematic sampling with a random start was used for selection of the areas to be studied in both analyses. Areas with lymphoid follicles were not included.

Statistical analysis

The Kruskal-Wallis test and the Mann-Whitney U-test assessed significance in the differences of protein expression between the control, the TN2GF4-infected and the SS1-infected groups. The Mann-Whitney U-test assessed significance in differences in mucosal infiltration of inflammatory cells between the TN2GF4-infected and SS1-infected gerbils. A linear relationship between AT1R-protein and PMNs, and a linear relationship between AT1R and ln (MPO) in the antral mucosa, were tested with Pearson correlation. All tests were two-tailed, and statistical significance was set at p < 0.05. All the statistical analyses were carried out using SPSS, version 15.0 (SPSS Inc. Chicago, Illinois).

Localization of AT1R and AT2R protein

Immunoreactivity to the AT1R and the AT2R proteins was observed in all gerbil specimens studied, and no staining was seen in the control sections. The AT1R antibody generally induced a more distinct staining than the AT2R antibody.

Several tissue compartments in both the corpus and antrum, independent of the presence or absence of H. pylori infection, exhibited immunoreactivity to both of the Ang II receptor subtypes (see Table 1 for an overview of the results and Figure 1 for representative sections. See also Additional file 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 for high resolution images). Thus, immunoreactivity to both the AT1R and the AT2R were found in endothelial cells of vessels located in the lamina propria, the submucosa (Figure 1B) and the muscularis propria, as well as in smooth muscle cells of the muscularis mucosae and muscularis propria (Figure 1C and 1G) and in some mesenchymal cells located in the lamina propria and submucosa. Weak staining of both receptors was also noted, mainly in the basal part of most epithelial cells. Distinct staining of intramural neural structures was not observed. Immunoreactivity only to AT1R was observed in the vascular smooth muscle cells of vessels in the submucosa, the muscularis propria and the serosa of both the corpus and antral parts of the stomach (Figure 1A and 1G). Interestingly, a strong immunostaining for AT1R appeared in a subset of cells mainly in the base of the antral glands. These cells showed a typical endocrine cell appearance, i.e. they were quite small, with the cell bodies close to the basement membrane; a narrow string of cytoplasm was observed in some cases (Figure 1G, 1H and 1I). Such AT1R staining could not be found in endocrine-like cells in the oxyntic mucosa. Double immunostaining for AT1R and for the pan-endocrine marker Chromogranin A [23] confirmed the location of AT1R in a subpopulation of endocrine cells in the gerbil antral mucosa (Figure 1M and 1N).

Tissue Compartment		Cell type	AT1R	AT2R
Mucosa	Epithelium	Most epithelial cells	+ mainly in basal parts	+ mainly in basal parts
		Small epithelial cells in the base of glands	+++ in some endocrine like cells in antrum	n.o.
	Lamina propria	Vasculature	+ in endothelium	+ in endothelium
		Nerve structures	n.o.	n.o.
		Mesenchymal cells	++ in some cells; ++ in leucocytes in H. pylori-infected	+ in some cells; ++ in leucocytes in H. pylori-infected
	Muscularis mucosae	Smooth muscle cells	++	+
Submucosa		Vasculature	+ in endothelium; ++ in VSMCs	+ in endothelium; n.o. in VSMCs
		Nerve structures	n.o.	n.o.
		Mesenchymal cells	++ in some cells; ++ in leucocytes in H. pylori-infected	+ in some cells; ++ in leucocytes in H. pylori-infected
Muscularis propria		Smooth muscle cells	++	+
		Vasculature	+ in endothelium; ++ in VSMCs	+ in endothelium; n.o. in VSMCs
		Nerve structures	n.o.	n.o.
Serosa		Vasculature	n.o. in endothelium; ++ in VSMCs	n.o.

n.o: not observed (or unspecific) immunoreactivity

+: weak immunoreactivity but with distinct location

++: easily recognised immunoreactivity

+++: strong immunoreactivity

VSMCs: vascular smooth muscle cells

The intensity and distribution of the immunoreactivity described above did not seem to be different between H. pylori-negative and H. pylori-infected animals or between the animals sacrificed at six or 12 months after inoculation. However, one obvious difference was noted between H. pylori-negative and H. pylori-infected animals: unlike in the uninfected gerbils, the sections of both the antrum and corpus of infected animals showed an abundance of inflammatory cells in the lamina propria with immunoreactivity to both the AT1R and the AT2R. In one of the TN2GF4-infected animals sacrificed at six months, aggregates of inflammatory cells were also seen in the muscularis propria (Figure 1J, 1K and 1L). No obvious differences in staining patterns or intensity were seen between animals infected with the TN2GF4 strain or the SS1 strain, or between animals sacrificed at six or 12 months after inoculation.

Quantification of AT1R and AT2R protein and relation to inflammatory cells

Due to the similar immunohistochemical appearance of angiotensin II receptors in the corpus and antrum (with the exception of the AT1R expressing subpopulation of endocrine cells mentioned above) at both six and 12 months, quantitative assessments were restricted to antral specimens at 12 months after inoculation.

Both AT1R and AT2R proteins were identified by western blotting in all samples (Figure 2). After 12 months of infection, the AT1R protein expression was significantly higher in SS1-infected animals than in controls at that time (Figure 3). No significant differences in AT2R protein expression were found between the different groups of gerbils (not shown).

Given that H. pylori-infected animals showed an abundance of AT1R expressing inflammatory cells in their gastric mucosa and an increased antral AT1R expression, a quantitative analysis of the inflammatory cells in the mucosa was made to assess whether the up-regulation of AT1R protein in SS1-infected gerbils was related to mucosal infiltration of inflammatory cells.

A linear relationship (r = 0.75, p < 0.01) was seen between the AT1R protein expression in the antrum and the number of polymorphonuclear leucocytes in the antral mucosa after 12 months of H. pylori infection (Figure 4). This relationship was supported by the logarithmic correlation between the antral expression of AT1R and the expression of myeloperoxidase (r = 0.58, p < 0.05) (Figure 5). Myeloperoxidase (MPO) is an enzyme that is abundantly expressed in neutrophils and to a lesser extent in monocytes and certain types of macrophages [24]. Hence, tissue levels of MPO served as protein markers of neutrophil infiltration in this study. If the outlier value marked with a triangle in Figure 5 is excluded from the analysis (not shown in a separate figure) the relationship between AT1R and MPO becomes noticeably stronger (r = 0.86 and p < 0.01).