Helicobacter pylori antigens as potential modulators of lymphocytes’ cytotoxic activity
Article first published online: 13 FEB 2012
© 2012 The Societies and Blackwell Publishing Asia Pty Ltd
Microbiology and Immunology
Volume 56, Issue 1, pages 62–75, January 2012
How to Cite
Rudnicka, K., Włodarczyk, M., Moran, A. P., Rechciński, T., Miszczyk, E., Matusiak, A., Szczęsna, E., Walencka, M., Rudnicka, W. and Chmiela, M. (2012), Helicobacter pylori antigens as potential modulators of lymphocytes’ cytotoxic activity. Microbiology and Immunology, 56: 62–75. doi: 10.1111/j.1348-0421.2011.00399.x
- Issue published online: 13 FEB 2012
- Article first published online: 13 FEB 2012
- Accepted manuscript online: 1 NOV 2011 04:25AM EST
- Received 11 April 2011; revised 7 October 2011; accepted 13 October 2011.
- cytotoxic lymphocytes;
- H. pylori;
Helicobacter pylori (H.p) colonizes human gastric mucosa and causes gastric and duodenal ulcer disease or gastric cancer. Various H.p compounds may modulate the host immune response in regards to tolerance of the infection or disease development. The aim of this study was to determine whether H.p lipopolysaccharide (LPS) and glycine acid extract antigens (GE) or E. coli LPS influence the cytotoxic activity of peripheral blood lymphocytes from H.p infected – H.p (+) or uninfected – H.p (−) individuals, in the presence or absence of exogenous interleukin (IL)12. Individual H.p status was defined by the urea breath test. Lymphocytes, stimulated or not with H.p, and control antigens, with or without IL-12, were used as effector cells and epithelial HeLa cells as targets. The cytotoxicity of lymphocytes was expressed as the percentage of dead target cells unable to reduce tetrazolium salt. The supernatants from HeLa/lymphocyte cultures were used for detection of the cellular cytotoxicity markers granzyme B and caspase 8. The natural cytotoxic activity of lymphocytes from H.p (+) was less than that of H.p (−) donors. This may have been due to fewer natural killer cells of CD3−CD56+Nkp46+ phenotype in H.p (+) in comparison to H.p (−) subjects. H.p GE and standard E. coli LPS enhanced the cytotoxicity of lymphocytes towards target cells whereas H.p LPS downregulated this activity. The decrease in lymphocyte cytotoxicity in response to H.p LPS correlated with a lack of IL-2 and IL-12 production, inhibition of interferon-γ production, and low IL-10 secretion by mononuclear leukocytes. IL-12 significantly enhanced the natural as well as H.p LPS and H.p GE driven cytotoxic capacity of lymphocytes. In conclusion, H.p LPS may negatively modulate natural cytotoxic activity and cytokine secretion by immunocompetent cells and thus be involved in the maintenance of infection and development of gastric pathologies.
- Cag A
cytotoxin associated gene A antigen
Culture Collection University of Gothenborg
cluster of differentiation molecules
- E. coli
glycine acid extract
Henrietta Lacks cervical cancer epithelial cell line
inducible nitric oxide synthase
- IFN- γ
Lewis blood group related antigens
major histocompatibility complex
natural killer lymphocytes
peripheral blood mononuclear cells
T helper lymphocytes
toll like receptor
tumor necrosis factor alpha
urea breath test
- Vac A
Helicobacter pylori is a major causative agent of chronic gastritis, gastroduodenal ulcers and it is involved in the development of gastric cancers (1, 2). The course of H. pylori infection depends on the effectiveness of the host immune responses against this pathogen, both innate (unspecific) and adaptive (specific). During H. pylori infections, the dense infiltration of the gastric mucosa with immunocompetent cells indicates that the complex interactions between bacterial and host immune cells may be important for both gastric pathologies and the efficiency of pathogen elimination (3). Th1 effector lymphocytes dominate in H. pylori infected gastric mucosa (4–6). However, the in situ cytokine patterns in gastric mucosal biopsies from H. pylori positive and H. pylori negative patients indicate a dual effect of H. pylori on Th1 response. That is, stimulation of gastric inflammation by increased IFN-γ production and inhibition of the response by increased amounts of IL-10 may modulate the inflammatory and cytotoxic effects of the T cell response (5–7). An in vivo neutralization study and unsuccessful immunization of Th1 deficient mice against H. pylori have confirmed the role of IFN-γ in the development of gastric inflammation and H. pylori elimination (5, 8). However, the roles of CD8+ and NK cells, which are IFN-γ producers and express cytotoxic capacity, in the course of H. pylori infections are not clear. In H. pylori positive individuals, there are H. pylori-reactive memory CD8+ T cells that proliferate and produce IFN-γ and granzyme A after activation (9). Both B cells and DCs pulsed with H. pylori antigens can activate these cells. The main function of NK cells is to promote MHC-unrestricted killing of target cells infected by viruses or bacteria as well as tumor cells. Moreover, NK cells are potent producers of cytokines, including IFN-γ, which can activate several immune processes such as phagocytosis and antigen presentation. NK cells can be activated not only by virus and bacteria-infected or cancer-transformed cells, but also by cytokines such as phagocyte-derived IL-12, Th1- derived IL-2 and by regulatory IL-10. Multiple studies have indicated that IL-10 augments the cytotoxic activity of NK cells, whereas it has no such effect on the cytotoxicity of CD8+ lymphocytes (10, 11). During H. pylori infections the predominance, heterogeneity, and distribution of NK cells at different sites within the gastric mucosa reflect a potential functional role of these cells during H. pylori infections (12). Lindgren et al. showed that a large majority of NK cells of human gastrointestinal mucosa lack CD8 expression, in contrast to peripheral blood NK cells (13). Although the cytotoxic capacities of CD8+ and CD8− NK cells were equal, only CD8− NK cells were able to produce IFN-γ in response to H. pylori cell lysates. These authors suggest that the CD8−CD16−CD56bright NK cells in the gastric mucosa are adapted to respond to bacterial infections by cytokine production and in this way may modulate the course of innate defense. Recently, it has been shown that NK cells can also be activated directly by microbial compounds (14), for instance the interactions of H. pylori with NK lymphocytes result in robust IFN-γ secretion (15). However, H. pylori bacteria produce several factors that affect host immune cells and can potentially downregulate host responses, thus maximizing the persistence of the microbes. The roles of VacA, CagA and H. pylori arginase in diminishing activation and proliferation of T lymphocytes has been established (16–22).
H. pylori LPS is an important proinflammatory factor with potential immunomodulatory activity. In previous studies, we showed that H. pylori LPS has anti-phagocytic and anti-proliferative activity, which may promote chronicity of infection (23, 24). Direct cytotoxic properties of H. pylori LPS and its ability to modulate the function of macrophages as antigen presenting cells have been suggested. In this study, we asked whether H. pylori LPS and the surface antigens of these bacteria present in GE influence the natural cytotoxic activity of human peripheral blood lymphocytes from H.p (+) or H.p (−) donors towards epithelial HeLa cells in vitro. We have also considered the potential modulatory effect of exogenous IL-12, a crucial activator of NK cells. Moreover, we have investigated production of the effector cytokines IFN-γ, IL-2, IL-12 and IL-10 by PBMCs in response to H. pylori GE antigens or H. pylori LPS. We thought that if some H. pylori compounds downregulate the lymphocyte cytotoxic activity against epithelial target cells, this may result in diminished elimination of infected, mutated or damaged host cells. Since H. pylori may temporarily survive in host cells (both macrophages and gastric epithelial cells), it is possible that H. pylori compounds interfere with the mechanisms of intracellular killing during phagocytosis or with the cytotoxic capacity of NK cells or T CD8+ lymphocytes. If so, H. pylori driven downregulation of lymphocyte cytotoxic functions may promote chronic infections and facilitate the development of gastric pathologies.
MATERIALS AND METHODS
Forty four healthy volunteers (aged 25–50 years) were included in the study, which was approved by the Local Ethics Committee. All participants gave their written informed consent. H. pylori status was estimated using a capsule-based 13C UBT as previously described (25) and by an in-house anti-H. pylori IgG ELISA. GE from the reference H. pylori strain CCUG 17874 (Culture Collection University of Gothenborg, Sweden) and rabbit anti-human IgG antibodies labeled with HRP (Dako, Glostrup, Denmark) were used as previously described (26, 27). The protein content in GE was 98.4% (NanoDrop 2000c Spectrophotometer, ThermoScientific, Waltman, WY, USA). The plates were coated with GE containing surface antigens (18 hr, 4°C), and the serum samples diluted 1:500. The results were expressed as OD measured at 450 nm wave length. The ELISA cut-off was defined as two SDs above the mean OD of control negative sera from H.p (−) subjects.
The HeLa cell line, purchased from The European Cell Culture Collection (Salisbury Wiltshire, UK), was the source of target cells for the cytotoxic assay. The cells were incubated for 3–4 days, 37°C, 5% CO2 in complete RPMI 1640 medium containing 10% heat inactivated FCS and standard antibiotics, and then treated with 0.25% trypsin, centrifuged and washed twice with a culture medium. The target cells were adjusted to a density of 2 × 105 cells/mL and distributed (100 μL/well) into 96-well plates. Before being used in the cytotoxic assay, the cells were incubated for 24 hr, 37°C, 5% CO2 in order to obtain a monolayer of adherent cells.
Peripheral blood mononuclear cells from H.p (+) and H.p (−) donors were separated by Histopaque 1077 gradient centrifugation (Sigma, St Louis, MI, USA), washed twice, adjusted to a density of 2.5 × 106/mL and distributed into 24-well culture plates (1 mL/well). H. pylori LPS from the reference strain of H. pylori CCUG 17874 (courtesy of A.P. Moran), or standard E. coli LPS derived from O55:B5 strain (Sigma), were added to the selected wells to a final concentration of 25 ng/mL, whereas GE antigens were used in a protein concentration of 5 μg/mL. The H. pylori LPS was prepared by hot phenol-water extraction after pretreatment of the bacterial biomass with protease. Then, the LPS crude preparation was purified by RNase, DNase and proteinase K treatment and by ultracentrifugation, as previously described (28). As shown by chromogenic limulus amebocyte lysate test (Lonza, Braine-Alleud, Belgium), the GE preparation contained a concentration of LPS of 0.001 EU/mL, whereas the purified H. pylori LPS was used in a concentration of 0.7 EU/mL. To some wells, IL-12 (R&D Systems, Minneapolis, MN, USA) was added (2 ng/mL). After incubation (24 hr, 37°C, 5% CO2) the supernatants were collected and stored frozen at −70°C for further cytokine measurement. The non-adherent lymphocytes were collected, washed twice, adjusted to a density of 2 × 107 cell/mL in a complete culture medium, and used as effector cells in the cytotoxic assay. The viability of lymphocytes from each donor was established before and after stimulation of the cells with bacterial antigens. The viability of the effector cells unstimulated or stimulated was equal to 100%.
Phenotype analysis of freshly isolated or culture propagated lymphocyte subsets
Peripheral blood mononuclear cells from four H.p (+) and four H.p (−) individuals were isolated and stimulated as described in the Materials and Methods section (effector cells). After stimulation, PBMCs were detached from the surfaces of the cell culture plates by treatment with cold PBS on ice for 30 min., collected by centrifugation, washed twice with PBS, and adjusted to 1 × 106 cells/mL. Fluorescence labeling was performed by incubating the cells at 4°C (in the dark) in PBS for 30 min. The cells were subsequently washed twice and resuspended in 500 μL PBS and acquired on a flow cytometer (LSR2, Becton Dickinson, Sparks, MD, USA). Ten thousand events were collected and analyzed by FlowJo softwere. The surface expression of cell differentiation markers was evaluated with the following fluorescently-conjugated antibodies: FITC, PE and PE-Cy5-conjugated isotype controls (eBioscience, San Diego, CA, USA), CD16-FITC (eBioCB16 clone, eBioscience), Nkp46-PE (9 E2 clone, BioLegend, Franklin Lakes, NJ, USA), CD56-FITC (MEM 188 clone, Diaclone, San Diego, CA, USA), CD3-PE-Cy5 (B 199.2 Ab, Serotec, Raleigh, NC, USA), and CD25-PE (BC96 clone, eBioscience). The analysis was performed on cells gated as lymphocytes, using triple-color flow cytometry by determination of the percentage of positive cells.
Mixed target/effector cell cultures
The lymphocytes obtained from the PBMC fraction as described in the previous section, untreated (incubated in the RPMI culture medium alone) or preincubated with H. pylori LPS, E. coli LPS or H. pylori GE, in the presence or absence of exogenous IL-12, were added to wells containing settled target cells (100:1 ratio adjusted experimentally) and then incubated for 4 hr, 37°C, 5% CO2. In the next stage, the lymphocytes were removed and the remaining cells washed out from the target cells using a culture medium. The effectiveness of washing was controlled under an inverted microscope. In all experiments wells containing HeLa cells alone (i.e without lymphocytes) were included as controls for target cell viability. The MTT reducing capacity of the cells, which was estimated from the standard curve, was in the range of 1000–1200 OD units, which corresponded to 100% cell viability.
The cytotoxic activity of the lymphocytes was estimated on the basis of the live target cells’ ability to reduce MTT by using the TACS MTT Cell Proliferation Assay (R&D Systems), as recommended by the manufacturer. The intensity of MTT reduction was estimated spectrophotometrically at 570 nm wavelength. The correlation between the number of viable target cells and the absorbance intensity was used for construction of the standard curve. The magnitude of lymphocyte cytotoxicity was expressed as a percentage of dead target cells.
The cytotoxic activity of the lymphocytes against the target cells was additionally established on the basis of granzyme B and caspase 8 concentrations in the supernatants from mixed target/effector cell cultures using the commercially available immunoenzymatic assays Granzyme B ELISA kit (Diaclone, Gen-Probe) and human caspase 8 platinum ELISA assay (eBioscience), according to recommended procedures. The sensitivity of granzyme B ELISA was 20 pg/mL, whereas the detection limit of caspase 8 ELISA was 100 pg/mL.
Quantification of cytokines by immunoassays
After stimulation of PBMC with H. pylori LPS, E. coli LPS or H. pylori GE the concentrations of cytokines IFN- γ, IL-2, IL-10 and IL-12 were estimated in the culture supernatants by using commercially available specific ELISA assays and standard procedures. In the assays the detection limits were 7 pg/mL for IL-2 and IFN-γ (Quantikine ELISA, R&D Systems), 5 pg/mL for IL-10 (Diaclone), and 3.2 pg/mL for IL-12 (human IL-12 p70 platinum ELISA, eBioscience).
For statistical analysis of the data, mean arithmetic values (x) and SDs were calculated. Statistica 5.5 PL software with non-parametric tests was used: Mann-Whitney's U test (for impaired data) to verify the hypothesis that the two compared samples came from two statistically different populations; χ2 test for prevalence comparison of the analyzed variables in the studied groups. Differences were considered significant when P < 0.05.
Natural and antigen driven cytotoxic activity of peripheral blood lymphocytes from H. pylori uninfected and H. pylori infected donors
The majority of the investigated lymphocytes revealed natural cytotoxic activity towards target cells (Fig. 1). The natural (spontaneous) cytotoxic activity of the lymphocytes was expressed as a percentage of dead target cells cocultured with unstimulated lymphocytes. Figure 1 shows the degree of lymphocyte cytotoxicity in general and the cytotoxic capacity above 10% of dead targets. The population of lymphocytes with stronger natural and H. pylori GE driven cytotoxic activity was significantly larger in H.p (−) than in H.p (+) donors, P= 0.03. We demonstrated a similar tendency for lymphocytes stimulated with H. pylori LPS, although the difference was not statistically significant. However, after stimulation of the cells with H. pylori LPS, the lymphocytes from H.p (−) donors showed significantly greater average cytotoxic capacity than did lymphocytes from H.p (+) donors (Fig. 2). As is shown in Figure 2, the average percentage of dead target cells after incubation with LPS-stimulated lymphocytes was 8.5% for H.p (−) donors and 4.3% for H.p (+) donors, P = 0.03. When stimulated with H. pylori LPS, the vast majority (18/22) of the lymphocytes from H.p (+) donors had a cytotoxic capacity of less than 10% whereas only 5/22 lymphocyte cultures from H.p (−) donors expressed cytotoxicity of less than 10% (Fig. 1).
After stimulation of the effector cells with H. pylori GE, the lymphocytes from H.p (−) donors also showed a greater cytotoxic capacity than did the lymphocytes from H.p (+) subjects (P = 0.01) (Fig. 2). Moreover, LPS from E. coli, but not H. pylori LPS, significantly enhanced the intensity of lymphocyte cytotoxicity in both groups of donors, whereas H. pylori GE only did so in H.p (−) individuals (Fig. 2). By comparison, in response to H. pylori LPS the cytotoxic activity of lymphocytes was even slightly less than the natural cytotoxic capacity of the lymphocytes from both H.p (+) and H.p (−) donors (Fig. 2).
Granzyme B and caspase 8 concentrations in the supernatants from mixed cultures of target cells with effector lymphocytes
There was a strong correlation between the cytotoxic capacities of the lymphocytes as estimated by MTT reduction assay and by quantification of granzyme B concentration (Fig. 3). The concentrations of granzyme B in the cultures containing unstimulated lymphocytes from H.p (−) were higher than in the cultures with unstimulated lymphocytes from H.p (+) donors. This may confirm the greater natural cytotoxic activity of lymphocytes from H.p (−) versus H.p (+) individuals. Moreover, significantly diminished granzyme B concentrations were detected in the mixed cultures of target cells with lymphocytes from H.p (+) or H.p (−) individuals stimulated with H. pylori LPS than in cultures of unstimulated lymphocytes. In contrast, granzyme B concentrations were significantly higher in cultures of target cells containing lymphocytes prestimulated with H. pylori GE or E. coli LPS than in control cultures containing unstimulated lymphocytes. Similarly, there was a correlation between the average cytotoxicity as estimated by the MTT reduction assay and the concentrations of caspase 8 (Fig. 4). It is worth mentioning that caspase 8 concentrations were lower in the cultures of target cells with H. pylori LPS stimulated lymphocytes than in the mixed cultures of targets and unstimulated lymphocytes from both groups of donors; however, we found a statistically significant difference only for H.p (+) individuals. Moreover, we found that caspase 8 was produced more intensively in cultures of target cells containing lymphocytes stimulated with E. coli LPS, but not H. pylori LPS, from H.p (+) and H.p (−) donors, P= 0.04 and P= 0.01, respectively.
Modulation of lymphocyte cytotoxic activity by exogenous interleukin-12
Exogenous IL-12 alone and in combination with H. pylori antigens GE or LPS positively modulated the cytotoxic activity of lymphocytes from both H.p (+) and H.p (−) donors (Fig. 5). Prestimulation of the lymphocytes from both studied groups with exogenous IL-12 resulted in a higher percentage of dead targets in the cytotoxic assay, in comparison to unstimulated lymphocytes. The cytotoxic responses of lymphocytes from H.p (+) donors stimulated with H. pylori GE or H. pylori LPS were significantly higher in the presence of IL-12 (P < 0.05), whereas the lymphocytes from H.p (−) donors showed an increase in their cytotoxicity after stimulation with IL-12 alone or with IL-12 and H. pylori LPS (Fig. 5). The increase in the cytotoxic activity of lymphocytes in the presence of exogenous IL-12 was correlated with increased production of granzyme B, an effector cytotoxicity protein (Fig. 6).
Concentrations of effector cytokines interferon-γ, and interleukin-2, 10 and 12 in peripheral blood mononuclear cell cultures from H. pylori infected or uninfected donors in response to H. pylori glycine acid extract antigens and H. pylori lipopolysaccharide
The PBMC from both H.p (+) and H.p (−) donors produced IFN-γ spontaneously (cells in the culture medium alone) and, to a similar degree, after stimulation with H. pylori GE, in the presence or absence of exogenous IL-12 (Fig. 7a). IFN-γ was not detected in cultures of PBMC stimulated with H. pylori LPS. Exogenous IL-12 slightly upregulated IFN-γ production in response to H. pylori LPS in PBMC cultures from H.p (−) donors, but the difference was not statistically significant. The concentration of IFN-γ in PBMC in response to E. coli LPS was about five times higher than that in PBMC in the culture medium alone, P= 0.0003 for H.p (+) donors and P= 0.0004 for H.p (−) donors. Exogenous IL-12 slightly upregulated IFN-γ production in response to H. pylori GE in PBMC cultures from H.p (+) donors and in response to H. pylori LPS in PBMC cultures from H.p (−) donors.
Spontaneous secretion of IL-2 by PBMC was below the detection limit (Fig. 7b). IL-2 was detected in the PBMC cultures from both H.p (+) and H.p (−) donors only after stimulation of the cells with E. coli LPS, but not with H. pylori GE or H. pylori LPS. In the presence of exogenous IL-12 PBMC from H.p (−) donors reacted with IL-2 production in response to H.p GE (Fig. 7b).
As shown in Figure 7c, we detected IL-10 in each type of culture. There was no difference in IL-10 secretion by PBMC from H.p (+) and H.p (−) donors. Stimulation of leukocytes from H.p (−) and H.p (+) individuals with H. pylori GE antigens was accompanied by > 35 fold and > 16 fold induction of IL-10 secretion, respectively. By comparison, stimulation of PBMC with H. pylori LPS also resulted in significant enhancement of IL-10 production in H.p (+) and H.p (−) donors, but only by 2- and 5-fold, respectively. Furthermore, GE driven IL-10 production was significantly stronger than that induced by H. pylori LPS, in PBMC cultures from both groups of donors. IL-12 supplementation did not alter IL-10 secretion (Fig. 7c).
The presence of IL-12 in the leukocyte culture supernatants with or without stimulation (GE, H. pylori LPS) was assessed by specific ELISA but IL-12 was detected neither in the cultures stimulated with H. pylori GE or LPS nor in the nonstimulated leukocyte cultures in both groups of donors.
Phenotypic characteristics of freshly isolated or culture propagated lymphocyte subsets of H. pylori infected and uninfected donors
There was no difference between H.p (+) and H.p (−) donors in the total number of freshly isolated leukocytes positive for lymphocyte CD3 marker (CD3+). However, in H.p (+) subjects the number of CD3− natural killer cells positive for CD56 (CD56+) and NKp46 (NKp46+) markers was only about half that of H.p (−) individuals (Table 1). There was no difference between H.p (−) and H.p (+) subjects in the number of NK cells with expression of a CD25 marker, an IL-2 receptor (CD3−CD56+CD25+). However, the number of CD3+CD25+ lymphocytes was doubled in H.p (+) donors (Table 1). Furthermore, we found that H.p LPS caused stronger expansion of the CD3+CD25+ lymphocyte subpopulation in in vitro cell cultures then did H.p GE (Table 2).
|Subsets of lymphocytes (%± SD)|
|Lymphocytes||Lymphocytes with IL-2R||NK cells||NK cells with IL-2R|
|H. pylori (−)||72.21 ± 11.20||10.11 ± 2.70||10.30 ± 2.30||0.21 ± 0.35|
|H. pylori (+)||70.51 ± 9.30||19.93 ± 0.80||6.80 ± 1.20||0.24 ± 0.26|
|Subsets of lymphocytes in cell cultures in vitro (%± SD)|
|Lymphocytes with IL-2R||NK cells with IL-2R|
|H. pylori (−)|
|nonstimulated||8.61 ± 2.50||0.17 ± 0.21|
|LPS H. pylori||13.45 ± 5.54||0.10 ± 0.11|
|GE H. pylori||6.09 ± 6.63||0.48 ± 0.02|
|LPS E. coli||10.11 ± 2.69||0.19 ± 0.07|
|Lymphocyte donors H. pylori (+)||CD3+CD25+||CD3−CD56+CD25+|
|nonstimulated||10.04 ± 1.60||0.93 ± 0.35|
|LPS H. pylori||16.52 ± 5.00||0.45 ± 0.16|
|GE H. pylori||5.55 ± 0.66||0.82 ± 0.34|
|LPS E. coli||8.78 ± 1.55||0.43 ± 0.21|
Some of the mechanisms that control the immune response during H. pylori infections are related to pathogen virulence factors. The role of H. pylori surface adhesins, LPS and vacA, in avoiding the engulfment and intracellular killing of the pathogen by phagocytes has been shown (23, 29, 30). In this study, we have demonstrated that H. pylori antigens may modulate positively or negatively the MHC-unrestricted cytotoxic activity of peripheral blood lymphocytes towards tumor epithelial HeLa cells. HeLa cells were selected for this study because this is a standard epithelial cell line recommended for the estimation of the lymphocyte cytotoxicity. Cell lines derived from human gastric epithelial cells such as AGS and Kato III were also included in this study. Although all cell types responded in a similar way, the most homogenous results were obtained for the HeLa cells. In a preliminary study we demonstrated that AgS and Kato III cells may differ concerning their proliferative activity in general, and in response to H. pylori antigens. Lymphocytes isolated from H.p (+) donors, unstimulated or treated with H. pylori GE or H. pylori LPS, expressed less cytotoxic activity against target cells than did lymphocytes from H.p (−) individuals. This probably means that H.p (+) subjects possess a smaller number and/or lower activity of natural cytotoxic lymphocytes than do H.p (−) donors. If so, the size of the cytotoxic lymphocyte population and their characteristics might also be important for the control of infection and elimination of damaged or tumor host cells. However, during H. pylori infection bacterial compounds may themselves affect the number or activity of cytotoxic lymphocytes. In this study we demonstrated that, in H.p (+) donors, the number of CD3−CD56+Nkp46+ NK cells among freshly isolated lymphocytes is half that of H.p (−) subjects. O’Keeffee et al. suggested that the role of NK cells in H. pylori infections depends on their predominance, heterogeneity, and distribution in the gastric mucosa (12). According to Lindgren et al., the key players in the innate response to H. pylori are CD8−CD16−CD56bright NK cells, which probably modulate the defense mechanisms indirectly through cytokine production rather than directly by cytotoxic activity (14).
In this study, we have demonstrated downregulation of the natural cytotoxic activity of lymphocytes by H. pylori LPS, but not E. coli LPS or H. pylori GE. A direct toxic effect of H. pylori LPS on lymphocytes from both H.p (+) and H.p (−) donors was excluded under our study conditions, since the viability of effector cells stimulated with H. pylori LPS, as assessed by MTT reduction assay, was the same as the viability of unstimulated cells. In a previous study, Grebowska et al. reported antiproliferative activity of H. pylori LPS towards lymphocytes (31). However, the appearance of this phenomenon was time-dependent. Inhibition of lymphocyte proliferation was observed in 72 hr, but not in 24 hr, cell cultures (31).
We here report that the diminished cytotoxic activity of lymphocytes from H.p (−) and especially H.p (+) donors in response to H. pylori LPS is correlated with attenuation of IFN-γ secretion, increased, but still low, production of IL-10 by PBMC, and lack of IL-2 secretion. In contrast to H. pylori LPS, standard LPS from E. coli stimulated the cytotoxic activity of lymphocytes, which was correlated with enhanced production of IFN-γ and IL-2 by PBMC and lack of enhancement of IL-10 production. Finally, the GE antigenic complex increased the cytotoxicity of lymphocytes. GE driven production of IFN-γ was equal to the spontaneous secretion of this cytokine by PBMC. Although it did not stimulate secretion of IL-2 by PBMC, IL-10 production in response to GE was very strong. It is possible that the mechanisms of lymphocyte cytotoxicity observed in this study may depend on the antigen used for PBMC stimulation, H. pylori LPS or GE, and E. coli LPS. The cytotoxic activity of lymphocytes in response to E. coli LPS was probably related to IFN-γ and IL-2 production by the immune cells, whereas the lymphocyte cytotoxicity in response to H. pylori GE could be a result of IL-10 signaling and IFN-γ production. The inhibition of cellular cytotoxicity in response to H. pylori LPS could be due to a lack of IFN-γ and IL-2 mediated stimulation, as well as low production of IL-10, which is insufficient to stimulate lymphocytes to express their cytotoxic activity.
Interleukin-2 is necessary for lymphocyte growth and it plays the roles of a classic NK cell activator by augmenting their proliferation rate, migration capability and cytotoxic activity (10, 11). Therefore, the lack of IL-2 production in the PBMC cultures stimulated with H. pylori LPS might result in impairment of the cell's condition and their cytotoxic capacity. In this study H. pylori LPS did not influence the number of CD3−CD56+ NK cells. However, it induced stronger expansion of CD3+CD25+ lymphocytes than did H. pylori GE and E. coli LPS. Although the number of CD3+CD25+ lymphocytes was increased, they were unable to support the cytotoxic activity of NK cells in the absence of IL-2. It is also worth mentioning that the concentrations of IL-10 were very low and the production of IFN-γ abrogated in the presence of H. pylori LPS. Previously it has been shown that, in the presence of IL-2, H. pylori LPS is an effective stimulator of human lymphocyte proliferation, although unlike LPS from other Gram-negative bacteria, H.pylori LPS alone shows very weak, if any, capacity to stimulate human lymphocytes (32).
In contrast to H. pylori LPS, the GE antigen complex increased the cytotoxicity of lymphocytes. Moreover, GE did not diminish natural IFN-γ and IL-2 secretion activity of the PBMCs and greatly enhanced production of IL-10. According to Asadullah et al., IL-10 favors the cytotoxic activity of NK cells since it increases IL-2 induced production of cytokines such as IFN-γ, granulocyte macrophage colony stimulating factor and TNF-α (10). It also amplifies IL-2 induced proliferation of the CD56−bright NK cell subpopulation. However, IL-10 may have no effect on the expression of other NK cell cytotoxicity markers such as perforin, granzyme B, iNOS, and FasL and may not affect their upregulation by IL-2. In this study, we report correlations between lymphocyte cytotoxicity as measured by MTT reduction assay and quantification of granzyme B and caspase 8 concentrations in the supernatants obtained after the cytotoxic assay.
It has also been suggested that IL-10 influences NK cell cytotoxicity through a specific pathway distinct from that for activation of NK cells by IL-2 (11). In this study we observed expression of lymphocyte cytotoxic activity to H. pylori GE and high IL-10 concentrations in PBMC culture supernatants in response to this antigen complex. On the other hand, the cytotoxicity of lymphocytes stimulated with E. coli LPS was correlated with high concentrations of IL-2.
Other inflammatory cytokines may also regulate the reactivity of immune cells in response to H. pylori LPS. In this study, we focused on IL-12, which is known to upregulate the cytotoxic activity of NK cells. Exogenous IL-12 stimulated both natural and H. pylori antigens- (GE and LPS) driven lymphocyte cytotoxicity. This suggests that IL-12 present in the inflammatory milieu during H. pylori infections might promote activation of the effector functions of naturally cytotoxic lymphocytes. Several H. pylori factors might be involved in activation of IL-12 expression, for instance, cag PAI compounds and the products of genes in the H. pylori plasticity region (33–35). Recently, intact H. pylori has been shown to promote rapid maturation and activation of monocyte derived DCs (36) and H. pylori pulsed DCs to cause activation of autologous T cells and expression of IL-2, TNF-α, and IFN-γ. However, most LPS-induced effects on T cells seem to result from indirect interactions between T cells and LPS stimulated macrophages and from mediators released from the LPS, activated accessory cells and cytokine activated T cells (37).
In our study, exogenous IL-12 significantly increased the cytotoxic activity of lymphocytes in response to H. pylori LPS, and it slightly enhanced secretion of IFN-γ, but only in PBMC from H.p (−) donors. However, it did not provoke IL-2 production or enhancement of IL-10 secretion. This suggests that IL-12 alone is an important and sufficient stimulator of lymphocyte cytotoxic activity. Choel et al. have demonstrated that H. pylori lysates and IL-12 act synergistically to induce IFN-γ production (38). IL-12 also synergizes with IL-2 to induce lymphokine activated cytotoxicity as well as perforin and granzyme gene expression in human NK cells. De Blaker et al. (39) and Kao et al. (40) have demonstrated that H. pylori secreted factors inhibit IL-12 production in dendritic cells. The phenomenon of IL-12 inhibition has been considered a mechanism for impairing host defense against H. pylori. In our study IL-12 was detected neither in cultures stimulated with H. pylori GE or LPS nor in nonstimulated leukocyte cultures in both groups of donors. These findings are in agreement with those of other authors showing that IL-10 may inhibit IL-12 production by both phagocytes and dendritic cells. IL-10 also acts as a protein at the mRNA level (41). It has also been shown that LPS is effective in downregulating IL-12 production by mononuclear cells (42). According to Hafsi et al., Th1 effector responses are more pronounced when membrane preparations of H. pylori are used (36). This suggests that digestion of the bacteria into subcellular fractions might facilitate the appearance of antigenic proteins. Our results show that H. pylori LPS may downregulate not only the cytotoxic capacity of lymphocytes, but also production of IFN-γ and IL-2, and thus cannot promote IFN-γ dependent activation of macrophages and IL-2 dependent expansion of lymphocytes. However, exogenous IL-12 seems to be able to induce cellular cytotoxicity even in the absence of IFN-γ, IL-2 or IL-10. This may confirm the importance of IL-12 in the regulation of immune responses towards H. pylori. The increase in cytotoxic activity of lymphocytes in the presence of exogenous IL-12 is correlated with increased production of granzyme B. It has been shown that IL-12 binds to IL-12R and, through a signaling pathway, activates various genes encoding mediators involved in the cytotoxicity process such as effector cytotoxicity proteins including granzyme B, cytokines, cytokine receptors, and signaling molecules (43).
In contrast to H. pylori LPS, H. pylori surface GE antigens are able to induce a cytotoxic response by lymphocytes and cytokine production by PBMC. Therefore, cytotoxic lymphocytes in the milieu of H. pylori antigens, such as protein GE antigens, may constitute an important element of the natural immune response during H. pylori infections.
Mohammadi et al. have shown that H. pylori culture supernatants contain a factor that can activate large resting granular lymphocytes to exhibit cytolytic activity (44). Yun at al. demonstrated that NK cells can be directly activated by whole cell H. pylori, whereas H. pylori lysate can induce IFN-γ secretion by NK cells (45). Addition of a small amount of IL-12 greatly enhances production of IFN-γ although a bacterial lysate alone is sufficient to induce activation of cytotoxicity related molecules (45). In our study, the GE antigenic complex was not effective at stimulating IFN-γ production by PBMC, however, this might be due to different antigenic profiles of GE and the whole cell lysates.
Hafsi et al. proposed that NK-derived IFN-γ may play a major role in the homeostasis of the immune response during H. pylori infection through activation of macrophages and differentiation of T cells (36). However, according to Tarkannen et al., the cytotoxic activity of NK cells does not directly affect the bacteria on the surface of the epithelium and may therefore be of less importance (15). Despite antibacterial and antiviral activity, naturally cytotoxic lymphocytes also remove damaged or mutated cells. Thus, inhibition of such clearance by H. pylori compounds, for instance LPS, may initiate dangerous gastric pathologies. Recently, it has been shown that H. pylori-derived LPS augments the growth of gastric cancer cells via the LPS-TLR4 pathway and attenuates the cytotoxicity of mononuclear cells against gastric cancer cells. Stimulation with H. pylori LPS also downregulates perforin production in cancer cells cocultured with CD56+ natural killer cells. H. pylori LPS induces neither IL-12 nor IFN-γ production by mononuclear cells (45). Nevertheless, IL-12 restores the IFN-γ -producing capacity of H. pylori LPS-stimulated leukocytes. Pellicano et al. have demonstrated that the presence of IL-12 is crucial for induction of IFN-γ production in NK cells (46). It is possible that H. pylori infection attenuates antitumor activity and IFN-γ mediated cellular immunity of mononuclear cells and thereby promotes proliferation and progression of gastric cancers (47, 48). The immunobiological activity of H. pylori LPS may depend on the presence of Lewis XY determinants, which may promote the production of macrophage derived cytokines such as TNF, IL-8 and IL-12 (49). Bergman et al. reported that Lewis antigen-bearing H. pylori cells predominantly induce IL-10 and promote Th2 cell response, whereas Lewis antigen-negative H. pylori cells promote a strong Th1 cell response (50).
In conclusion, we have demonstrated that the cytotoxic capacity of lymphocytes from H.p (+) individuals is less that of lymphocytes from H.p (−) donors. This could be due to there being fewer NK cells of CD3−CD56+NKp64+ phenotype in H.p (+) donors. We have also shown that H. pylori antigens may vary in their ability to activate cytotoxic lymphocytes. H. pylori surface GE antigens are effective at stimulating lymphocyte cytotoxic activity but H. pylori LPS downregulates the natural cytotoxic capacity of the lymphocytes, which correlates with a diminished ability of PBMCs to produce IFN-γ, IL-2 and with little secretion of IL-10 in response to LPS. H. pylori LPS did not influence the number of CD3−CD25+ NK cells, although the population of CD3+CD25+ lymphocytes was increased in the milieu of H. pylori LPS, but not H. pylori GE or LPS from E. coli. It is possible that, in the absence of IL-2, these lymphocytes were not able to support NK cytotoxic activity. In contrast with H. pylori LPS, GE antigens strongly stimulate IL-10 production and do not decrease the ability of immune cells to produce IFN-γ and IL-2. Based on these findings, we propose that H. pylori LPS negatively modulates the first line of immune defense, including naturally cytotoxic lymphocytes, and accordingly may be involved in the maintenance of infection and in the development of gastric pathologies. However, the presence of IL-12 in the inflammatory milieu during H. pylori infections might be critical for effective positive modulation of cytotoxic activity in response to H. pylori antigens.
This research was supported by the Polish Ministry of Science and Higher Education (grant N N401 015 136) and the European Union Project “Stipends supporting innovative research projects for PhD students”.
The authors have no conflicting financial interests.
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