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Indian Journal of Medical Microbiology, Vol. 28, No. 3, July-September, 2010, pp. 233-237 Original Article Comparison of Helicobacter pylori and Escherichia coli in induction of TNF-α mRNA from human peripheral blood mononuclear cells E Ahmadzadeh1, H Zarkesh-Esfahani1, R Roghanian1, F Navab Akbar2 1 Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, I.R, Iran Keywords: E. coli , H. pylori, PBMCs, TNF-α Introduction Helicobacter pylori (H pylori) is recognized as a human-specific gastric pathogen and carcinogen that colonizes the mucus layer in the stomachs of at least half of the world′s population. [1],[2] Most infected individuals are asymptomatic. However, in some subjects, the infection causes acute or chronic gastritis or peptic ulceration and plays an important role in the development of peptic ulcer, gastric adenocarcinoma, mucosa-associated lymphoid tissue lymphoma and primary gastric non-Hodgkin lymphoma. Indeed, H pylori is recognized as a category 1 (definite) human carcinogen. [3] H pylori is a micro-aerophilic, gram-negative, flagellated bacillus. [2] As in other gram-negative bacteria, lipopolysaccharides (LPS) form the main component of the cell surface of H pylori and have been correlated with virulence factors. [3] Outer membrane-derived LPS of most gram-negative bacteria are powerful stimulators of the host immune response. However, the LPS of H pylori has low immunological activity and, thereby, contributes to the chronicity of infection. [2],[4],[5] The LPS molecule is composed of three moieties: lipid A, core, and O-polysaccharide chain. Lipid A is central to the biological and immunological activities associated with LPS. H pylori lipid A is underphosphorylated (lacking phosphate at position 4) and has both 3-hydroxyhexadecanoic and 3-hydroxyoctadecanoic acids. [6],[7],[8] LPS activates monocytes and macrophages to produce cytokines such as tumour necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) that, in turn, serve as endogenous inflammatory mediators.[9] There have been suggestions that H pylori possesses LPS with a lower virulence as compared to the typical bacterial endotoxins such as Escherichia coli (E coli) LPS [4],[5],[10] However, most biological activities of LPS of H pylori, such as its effect on peripheral blood mononuclear cells (PBMCs), which are clinically important in sustaining local tissue inflammation and injury, are still little understood. [2] Gastric mucosal levels of proinflammatory cytokines such as interleukin-1β (IL-1β), IL-6, interleukin-8 (IL-8), and TNF-α have been reported to be increased in H pylori-infected subjects. Cytokines such as IL-1β, IL-4, IL-6, IL-8, interferon-gamma (IFN-γ), and TNF-α may be of importance for the pathological mechanisms of H pylori infection. TNF-α is a cytokine that is produced mainly by activated macrophages. It is a key mediator in host defence against bacterial infection and in septic shock syndrome induced by LPS. [11] In this study, we used a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method to compare the abilities of H pylori and E coli0 in the induction of expression of TNF-α mRNA from human PBMCs. This information can help us better understand the mechanism by which H pylori prevents a severe immune response and thereby facilitates its stay in the stomach. Materials and Methods Bacterial strains and culture Urease-positive samples of gastric antral biopsies were cultured on brucella agar (Merck, Darmstadt, Germany) supplemented with 5% (v/v) sheep blood, vancomycin (6 mg/L) (Sigma-Aldrich, Steinheim, Germany), trimethoprim (5 mg/L) (Sigma-Aldrich, Steinheim, Germany), and amphotericin B (2 mg/L) (Fungizone; Gibco) under microaerophilic conditions (85% N 2 , 10% CO 2 , 5% O 2 ) at 37°C. H pylori were identified according to colony morphology, microscopic observation after Gram′s staining, and biochemical analysis (urease and catalase tests). [12] H pylori were then harvested at between 48 and 72 hours after inoculation of plates, resuspended in sterile phosphate buffered saline (PBS), and enumerated by absorbance at 600 nm [1 optical density (OD) 600 nm equal to 2.4 x 10 8 colony-forming units/mL]. [13],[14] The bacteria were then diluted to the final concentration of 10 8 bacteria/mL in RPMI1640 (Sigma, Munich, Germany) medium containing 10% foetal calf serum (Gibco, Invitrogen Corporation, Paisley, UK). E coli (PTCC 1335) was cultured to produce a suspension of 10 8 organisms/mL. For some experiments, bacteria (H pylori and E coli) were ultrasonically broken. Bacterial suspension of H pylori was sonicated five times on ice for 5 seconds with 30-seconds intervals and the bacterial suspension of E coli was sonicated three times: for 10 seconds on and 1 minute off. Bacterial concentrations were kept constant in all the experiments. Isolation of peripheral blood mononuclear cells Heparinised venous blood samples drawn from healthy adult volunteers were diluted with the equal amounts of PBS. Mononuclear cells were separated using Ficoll-Hypaque density gradient (Histopaque-1077, Sigma, Deisenhofen, Germany) centrifugation according to the manufacturer′s recommendations. PBMC were cultured at the final concentration of 10 6 cells/mL in RPMI 1640 medium (Sigma, Munich, Germany) supplemented with 10% foetal calf serum (Gibco, Invitrogen Corporation, Paisley, UK), 100 U/mL penicillin, and 100 μg/mL streptomycin in 24-well tissue culture plates (Costar) at 37°C and 5% CO 2 PBMCs treatment Isolated PBMCs were incubated with either different concentrations (10-100 ng/mL) of LPS (positive control) from E coli, serotype 0111:B4 (Sigma, purified by phenol extraction and gel filtration), or bacteria at multiplicity of infection (MOI) of 100 for 4 hours. RNA isolation Total RNA was isolated from treated PBMCs using TRIzol (Gibco, Eggenstein, Germany) in RNase-free condition. Cells were centrifuged at 400 g for 10 minutes and cell pellets were resuspended in TRIzol. Chloroform (Sigma) 0.2 mL per 1 mL of homogenate was added to the solution and centrifuged for 15 minutes after shaking for 15 seconds. The aqueous phase was extracted and stored for 15 minutes at 4°C after adding 0.3 mL of isopropanol (Sigma). The samples were centrifuged again for 15 minutes. The RNA was washed once with 75% ethanol. The extracted RNA was dried briefly and resuspended in 50 μL of DEPC-treated dH 2 O. RT-PCR Complementary DNA (cDNA) was synthesized using standard protocols. One microgram of isolated RNA was mixed with 1.5 μL of random hexamer primers (Fermentas, St. Leon-Rot, Germany) and volume adjusted to 30 μL using DEPC-treated dH 2 O. Then 30 μL of master mix (12 μL buffer, 6 μL dNTP, 6 μL DTT, 6 μL RT enzyme) (Fermentas, St. Leon-Rot, Germany) were added to each tube. The tubes were put on the PCR machine, using the following program for cDNA synthesis: 37°C for 45 minutes, 95°C for 10 minutes, and then cooled down to 4°C. Two microlitres of cDNA were added to 48 μL of PCR master mix (5 μL Mg-free buffer, 3 μL MgCl 2 , 0.5 μL dNTP, 0.25 μL Tag polymerase, 1.5 μL primers, 37.75 μL dH 2O) (Fermentas, St. Leon-Rot, Germany). PCR were performed through 25 cycles for GAPDH and 30 cycles for TNF-α genes each at 94°C for 1 minute, 55° for 1 minute, 72°C for 1 minute. Finally, the PCR products were electrophoresed on 1% agarose (Sigma) gel and visualized using gel documentation system. The mRNA expression levels for cytokine were adjusted to GAPDH (positive control and housekeeping gene), using a densitometry software. Primer sequences for GAPDH and cytokine genes were obtained from a previously published paper. [15] Statistical analysis The statistical significance of the TNF-α production response to treatments was evaluated using Student′s t test. Data are expressed as the mean ± SEM and a P < .05 was considered to indicate statistically significant difference. Results Effects of LPS on TNF-α mRNA expression levels by PBMCs We first performed a dose response to LPS using different doses of LPS, including doses of 10-100 ng/mL of purified LPS from E coli0, serotype 0111:B4. TNF-α mRNA expression was clearly induced by LPS in PBMCs as compared to untreated cells. LPS at the concentration of 10 ng/mL stimulated the TNF-α mRNA expression and higher doses of LPS clearly induced more TNF-α expression [Figure - 1]. This shows the dose-dependent effects of LPS on TNF-α mRNA expression levels by PBMCs. Effects of E coli and H pylori on TNF-α mRNA expression by PBMCs The ability of H pylori to stimulate TNF-α mRNA expression by PBMCs was compared with that of E coli. Human PBMCs (10 6 cells/well) were incubated with either intact bacteria or sonicated bacteria for both E coli (10 8 bacteria/well) and H pylori (10 8 bacteria/well) for 4 hours, and TNF-α gene expression was determined. TNF-α gene expression levels were significantly higher in PBMCs stimulated with E coli than in those stimulated with H pylori at the same number and identical conditions (P < 0.001) [Figure - 2]. The results suggest that sonicated bacteria were substantially (P < 0.001) less stimulatory for PBMCs than intact bacteria for both E coli and H pylori [Figure - 3]a. Sonicated H pylori were also less potent in stimulating PBMCs as compared to sonicated E coli. [Figure - 3]b. Discussion LPS from most gram-negative bacteria is considered a mitogen that stimulates innate immune responses such as inflammation and production of cytokines, among the most common of which are TNF-α, IL-10, and IFN-γ. LPS is recognized by cells that contain TLR-4 in combination with either soluble or bound CD14 and MD-2. LPS-binding protein brings LPS in contact with CD14, which then aids in the binding of LPS to the soluble co-receptor MD-2. When MD-2 is bound to both LPS and TLR-4, transcription of NF-κB is upregulated, leading to cytokine production.[6] Several studies have investigated the host immune responses during H pylori infection. Most of them have used biopsy samples as the source of cytokines mRNA molecules. [16] In this study, we compared the efficacy of H pylori endotoxin and E coli endotoxin in the stimulation of the cytokines from human mononuclear cells. The result from our study demonstrated that the TNF-α mRNA expression levels by PBMCs were significantly higher in cells stimulated by E coli as compared to those stimulated with H pylori. Previous studies have reported that the ability of H pylori LPS to induce TNF-α production by human peripheral monocytes is at least 1000-fold lower than that of S minnesota. [4] H pylori LPS has been reported to possess 100- to 10000-fold lesser endotoxin activity when compared to other gram-negative species, which is due to the unusual phosphorylation patterns in the lipid A region. [5] Ogawa et al. demonstrated that H pylori natural lipid A possessed low activity in inducing TNF-α production by human PBMC.[10] Pece et al. also demonstrated that H pylori LPS exhibited weak Limulus activity. [17] Perez-Perez et al0. showed that the production of TNF-α, prostaglandin E2 , and nitric oxide in a human promyelomonocytic cell line, THP-1, stimulated with H pylori LPS was lower than that achieved with E coli LPS. [18] In addition, Huang et al. demonstrated that live H pylori cells induced IL-6 andIL-8 but not TNF-α production in epithelial cell lines, while its LPS stimulated a low level of IL-8-producing activity. [19] Salgado et al. demonstrated that H pylori 0LPS from different strains could be divided into two types on the basis of ability to induce the mitogenicity and TNF-α synthesis of cells: one of low biological activity and one of high biological activity. The strains with high activity were demonstrated to belong to the low-virulence genotypes i.e. cagA- and vacA (s1bm2 or s2m2). It was reported that H pylori LPS had a greater ability for inducing PBMC than E coli LPS. [2] Innocenti et al. showed that H pylori LPS induced even higher levels of IL-8 production than E coli LPS, while E coli LPS was a more potent inducer of IL-10 and TNF-α production; also, there was no difference between LPS preparations from different H pylori strains in their ability to induce cytokines and chemokines. LPS from different H pylori strains induce similar responses. [20] Our results suggest that the ability of different H pylori strains isolated from biopsy samples for stimulating TNF-α from PBMCs was significantly lower than that of E coli. Previous studies have demonstrated that compared to intact bacteria, purified LPS was a very poor inducer of IL-1α, IL-6, and TNF-α production and induced no detectable IL-12, suggesting that the conformation of LPS on the outer leaflet of the outer membrane is not totally conserved in purified LPS. A similar conclusion was reached in another study comparing the cytokine-inducing capacity of N meningitidis bacteria with that of purified LPS on mouse macrophages. [21],[22] Our data showed that sonicated bacteria were substantially less stimulatory for PBMCs than intact bacteria for both E coli and H pylori. One important reason for H pylori being able to evade the immune system and sustain a mild inflammation could be its weak LPS activity. Further investigations are needed to clarify the action of H pylori on the production of inflammatory cytokines from human PBMCs. Acknowledgements This study was supported by the office of Graduate Studies of University of Isfahan. The authors would like to thank Dr. Peyman Adibi for his help and providing clinical samples. References
Copyright 2010 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb10070f2.jpg] [mb10070f3.jpg] [mb10070f1.jpg] |
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