Helicobacter pylori antigens as potential modulators of lymphocytes’ cytotoxic activity

Microbiol Immunol 2012; 56: 62–75doi:10.1111/j.1348-0421.2011.00399.x

ORIGINAL ARTICLE

Helicobacter pylori antigens as potential modulatorsof lymphocytes’ cytotoxic activityKarolina Rudnicka1, Marcin Włodarczyk1, Anthony P. Moran2†, Tomasz Rechcinski3, Eliza Miszczyk1,Agnieszka Matusiak1, Ewelina Szczesna1, Maria Walencka1, Wiesława Rudnicka1

and Magdalena Chmiela1

1Department of Immunology and Infectious Biology, University of Łodz, Banacha 12/16, 90-237, 2National University of Ireland, Galway, Ireland and3Medical University, Łodz, Poland

ABSTRACTHelicobacter pylori (H.p) colonizes human gastric mucosa and causes gastric and duodenal ulcer dis-ease or gastric cancer. Various H.p compounds may modulate the host immune response in regards totolerance of the infection or disease development. The aim of this study was to determine whether H.plipopolysaccharide (LPS) and glycine acid extract antigens (GE) or E. coli LPS influence the cytotoxicactivity of peripheral blood lymphocytes from H.p infected – H.p (+) or uninfected – H.p (−) individ-uals, in the presence or absence of exogenous interleukin (IL)12. Individual H.p status was defined bythe urea breath test. Lymphocytes, stimulated or not with H.p, and control antigens, with or withoutIL-12, were used as effector cells and epithelial HeLa cells as targets. The cytotoxicity of lymphocyteswas expressed as the percentage of dead target cells unable to reduce tetrazolium salt. The supernatantsfrom HeLa/lymphocyte cultures were used for detection of the cellular cytotoxicity markers granzyme Band 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 oflymphocytes towards target cells whereas H.p LPS downregulated this activity. The decrease in lympho-cyte cytotoxicity in response to H.p LPS correlated with a lack of IL-2 and IL-12 production, inhibitionof interferon-γ production, and low IL-10 secretion by mononuclear leukocytes. IL-12 significantly en-hanced 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 immunocompetentcells and thus be involved in the maintenance of infection and development of gastric pathologies.

Key words cytotoxic lymphocytes, H. pylori, lipopolysaccharide.

Helicobacter pylori is a major causative agent of chronicgastritis, gastroduodenal ulcers and it is involved in thedevelopment of gastric cancers (1, 2). The course of

† deceasedCorrespondenceMagdalena Chmiela, Department of Immunology and Infectious Biology, University of Łodz, Banacha 12/16, 90-237 Łodz, Poland.Tel: +48 42 6354186; fax: +48 42 6655818; email address: [email protected]

Received 11 April 2011; revised 7 October 2011; accepted 13 October 2011.

List of Abbreviations: Cag A, cytotoxin associated gene A antigen; CCUG, Culture Collection University of Gothenborg; CD, cluster of differentiationmolecules; DC, dendritic cells; E. coli, Escherichia coli; FasL, Fas ligand; FITC, fluorescein isothiocyanate; GE, glycine acid extract; H.p, Helicobacter pylori;HeLa, Henrietta Lacks cervical cancer epithelial cell line; HRP, horseradish peroxidase; iNOS, inducible nitric oxide synthase; IFN- γ, interferon gamma;Ig, immunoglobulin; IL, interleukin; Lewis, Lewis blood group related antigens; LPS, lipopolysaccharide; MHC, major histocompatibility complex;MTT, tetrazolium salt; NK, natural killer lymphocytes; OD, optical density; PAI, pathogenicity island; PBMC, peripheral blood mononuclear cells; PE,phycoerythrin; PE-Cy5, phycoerythrin-cyanin 5; SD, standard deviation; Th, T helper lymphocytes; TLR, toll like receptor; TNF-α, tumor necrosis factoralpha; UBT, urea breath test; Vac A, vacuolating cytotoxin.

H. pylori infection depends on the effectiveness of thehost immune responses against this pathogen, both in-nate (unspecific) and adaptive (specific). During H. pylori

62 c© 2012 The Societies and Blackwell Publishing Asia Pty Ltd

H. pylori and lymphocyte cytotoxicity

infections, the dense infiltration of the gastric mucosa withimmunocompetent cells indicates that the complex inter-actions between bacterial and host immune cells may beimportant for both gastric pathologies and the efficiency ofpathogen elimination (3). Th1 effector lymphocytes dom-inate in H. pylori infected gastric mucosa (4–6). However,the in situ cytokine patterns in gastric mucosal biopsiesfrom H. pylori positive and H. pylori negative patients in-dicate a dual effect of H. pylori on Th1 response. That is,stimulation of gastric inflammation by increased IFN-γproduction and inhibition of the response by increasedamounts of IL-10 may modulate the inflammatory andcytotoxic effects of the T cell response (5–7). An in vivoneutralization study and unsuccessful immunization ofTh1 deficient mice against H. pylori have confirmed therole of IFN-γ in the development of gastric inflamma-tion and H. pylori elimination (5, 8). However, the rolesof CD8+ and NK cells, which are IFN-γ producers andexpress cytotoxic capacity, in the course of H. pylori in-fections are not clear. In H. pylori positive individuals,there are H. pylori-reactive memory CD8+ T cells thatproliferate and produce IFN-γ and granzyme A after ac-tivation (9). Both B cells and DCs pulsed with H. pyloriantigens can activate these cells. The main function of NKcells is to promote MHC-unrestricted killing of target cellsinfected by viruses or bacteria as well as tumor cells. More-over, NK cells are potent producers of cytokines, includingIFN-γ, which can activate several immune processes suchas phagocytosis and antigen presentation. NK cells canbe activated not only by virus and bacteria-infected orcancer-transformed cells, but also by cytokines such asphagocyte-derived IL-12, Th1- derived IL-2 and by reg-ulatory IL-10. Multiple studies have indicated that IL-10augments the cytotoxic activity of NK cells, whereas it hasno such effect on the cytotoxicity of CD8+ lymphocytes(10, 11). During H. pylori infections the predominance,heterogeneity, and distribution of NK cells at differentsites within the gastric mucosa reflect a potential func-tional role of these cells during H. pylori infections (12).Lindgren et al. showed that a large majority of NK cellsof human gastrointestinal mucosa lack CD8 expression,in contrast to peripheral blood NK cells (13). Althoughthe cytotoxic capacities of CD8+ and CD8− NK cells wereequal, only CD8− NK cells were able to produce IFN-γin response to H. pylori cell lysates. These authors suggestthat the CD8−CD16−CD56bright NK cells in the gastricmucosa are adapted to respond to bacterial infections bycytokine production and in this way may modulate thecourse of innate defense. Recently, it has been shown thatNK cells can also be activated directly by microbial com-pounds (14), for instance the interactions of H. pylori withNK lymphocytes result in robust IFN-γ secretion (15).

However, H. pylori bacteria produce several factors thataffect host immune cells and can potentially downregu-late host responses, thus maximizing the persistence of themicrobes. The roles of VacA, CagA and H. pylori arginasein diminishing activation and proliferation of T lympho-cytes has been established (16–22).

H. pylori LPS is an important proinflammatory factorwith potential immunomodulatory activity. In previousstudies, we showed that H. pylori LPS has anti-phagocytic and anti-proliferative activity, which may pro-mote chronicity of infection (23, 24). Direct cytotoxicproperties of H. pylori LPS and its ability to modulate thefunction of macrophages as antigen presenting cells havebeen suggested. In this study, we asked whether H. py-lori LPS and the surface antigens of these bacteria presentin GE influence the natural cytotoxic activity of humanperipheral blood lymphocytes from H.p (+) or H.p (−)donors towards epithelial HeLa cells in vitro. We have alsoconsidered the potential modulatory effect of exogenousIL-12, a crucial activator of NK cells. Moreover, we haveinvestigated production of the effector cytokines IFN-γ,IL-2, IL-12 and IL-10 by PBMCs in response to H. pyloriGE antigens or H. pylori LPS. We thought that if someH. pylori compounds downregulate the lymphocyte cyto-toxic activity against epithelial target cells, this may resultin diminished elimination of infected, mutated or dam-aged host cells. Since H. pylori may temporarily survive inhost cells (both macrophages and gastric epithelial cells),it is possible that H. pylori compounds interfere with themechanisms of intracellular killing during phagocytosis orwith the cytotoxic capacity of NK cells or T CD8+ lympho-cytes. If so, H. pylori driven downregulation of lymphocytecytotoxic functions may promote chronic infections andfacilitate the development of gastric pathologies.

MATERIALS AND METHODS

Subjects

Forty four healthy volunteers (aged 25–50 years) were in-cluded in the study, which was approved by the LocalEthics Committee. All participants gave their written in-formed consent. H. pylori status was estimated using acapsule-based 13C UBT as previously described (25) andby an in-house anti-H. pylori IgG ELISA. GE from thereference H. pylori strain CCUG 17874 (Culture Collec-tion University of Gothenborg, Sweden) and rabbit anti-human IgG antibodies labeled with HRP (Dako, Glostrup,Denmark) were used as previously described (26, 27). Theprotein content in GE was 98.4% (NanoDrop 2000c Spec-trophotometer, ThermoScientific, Waltman, WY, USA).The plates were coated with GE containing surface

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antigens (18 hr, 4◦C), and the serum samples diluted 1:500.The results were expressed as OD measured at 450 nmwave length. The ELISA cut-off was defined as two SDsabove the mean OD of control negative sera from H.p (−)subjects.

Target cells

The HeLa cell line, purchased from The European CellCulture Collection (Salisbury Wiltshire, UK), was thesource of target cells for the cytotoxic assay. The cells wereincubated for 3–4 days, 37◦C, 5% CO2 in complete RPMI1640 medium containing 10% heat inactivated FCS andstandard antibiotics, and then treated with 0.25% trypsin,centrifuged and washed twice with a culture medium. Thetarget cells were adjusted to a density of 2 × 105 cells/mLand distributed (100 μL/well) into 96-well plates. Beforebeing used in the cytotoxic assay, the cells were incubatedfor 24 hr, 37◦C, 5% CO2 in order to obtain a monolayerof adherent cells.

Effector cells

Peripheral blood mononuclear cells from H.p (+) andH.p (−) donors were separated by Histopaque 1077 gra-dient centrifugation (Sigma, St Louis, MI, USA), washedtwice, adjusted to a density of 2.5 × 106/mL and dis-tributed into 24-well culture plates (1 mL/well). H. py-lori LPS from the reference strain of H. pylori CCUG17874 (courtesy of A.P. Moran), or standard E. coli LPSderived from O55:B5 strain (Sigma), were added to theselected wells to a final concentration of 25 ng/mL,whereas GE antigens were used in a protein concentra-tion of 5 μg/mL. The H. pylori LPS was prepared by hotphenol-water extraction after pretreatment of the bacterialbiomass with protease. Then, the LPS crude preparationwas purified by RNase, DNase and proteinase K treat-ment and by ultracentrifugation, as previously described(28). As shown by chromogenic limulus amebocyte lysatetest (Lonza, Braine-Alleud, Belgium), the GE prepara-tion contained a concentration of LPS of 0.001 EU/mL,whereas the purified H. pylori LPS was used in a concen-tration of 0.7 EU/mL. To some wells, IL-12 (R&D Sys-tems, Minneapolis, MN, USA) was added (2 ng/mL). Af-ter incubation (24 hr, 37◦C, 5% CO2) the supernatantswere collected and stored frozen at −70◦C for furthercytokine measurement. The non-adherent lymphocyteswere collected, washed twice, adjusted to a density of 2 ×107 cell/mL in a complete culture medium, and used aseffector cells in the cytotoxic assay. The viability of lym-phocytes from each donor was established before and afterstimulation of the cells with bacterial antigens. The via-bility of the effector cells unstimulated or stimulated wasequal to 100%.

Phenotype analysis of freshly isolated orculture propagated lymphocyte subsets

Peripheral blood mononuclear cells from four H.p (+)and four H.p (−) individuals were isolated and stimulatedas described in the Materials and Methods section (effec-tor cells). After stimulation, PBMCs were detached fromthe surfaces of the cell culture plates by treatment withcold 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 thecells at 4◦C (in the dark) in PBS for 30 min. The cells weresubsequently washed twice and resuspended in 500 μLPBS and acquired on a flow cytometer (LSR2, BectonDickinson, Sparks, MD, USA). Ten thousand events werecollected and analyzed by FlowJo softwere. The surfaceexpression of cell differentiation markers was evaluatedwith the following fluorescently-conjugated antibodies:FITC, PE and PE-Cy5-conjugated isotype controls (eBio-science, San Diego, CA, USA), CD16-FITC (eBioCB16clone, 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 aslymphocytes, using triple-color flow cytometry by deter-mination of the percentage of positive cells.

Mixed target/effector cell cultures

The lymphocytes obtained from the PBMC fraction as de-scribed in the previous section, untreated (incubated inthe RPMI culture medium alone) or preincubated withH. pylori LPS, E. coli LPS or H. pylori GE, in the pres-ence or absence of exogenous IL-12, were added to wellscontaining settled target cells (100:1 ratio adjusted exper-imentally) and then incubated for 4 hr, 37◦C, 5% CO2.In the next stage, the lymphocytes were removed and theremaining cells washed out from the target cells using aculture medium. The effectiveness of washing was con-trolled under an inverted microscope. In all experimentswells containing HeLa cells alone (i.e without lympho-cytes) were included as controls for target cell viability.The MTT reducing capacity of the cells, which was esti-mated from the standard curve, was in the range of 1000–1200 OD units, which corresponded to 100% cell viability.

Cytotoxicity assay

The cytotoxic activity of the lymphocytes was estimatedon the basis of the live target cells’ ability to reduce MTTby using the TACS MTT Cell Proliferation Assay (R&DSystems), as recommended by the manufacturer. Theintensity of MTT reduction was estimated spectrophoto-metrically at 570 nm wavelength. The correlation between



the number of viable target cells and the absorbance in-tensity was used for construction of the standard curve.The magnitude of lymphocyte cytotoxicity was expressedas a percentage of dead target cells.

The cytotoxic activity of the lymphocytes against thetarget cells was additionally established on the basis ofgranzyme B and caspase 8 concentrations in the su-pernatants from mixed target/effector cell cultures us-ing the commercially available immunoenzymatic assaysGranzyme B ELISA kit (Diaclone, Gen-Probe) and humancaspase 8 platinum ELISA assay (eBioscience), accordingto recommended procedures. The sensitivity of granzymeB ELISA was 20 pg/mL, whereas the detection limit ofcaspase 8 ELISA was 100 pg/mL.

Quantification of cytokines byimmunoassays

After stimulation of PBMC with H. pylori LPS, E. coli LPSor H. pylori GE the concentrations of cytokines IFN- γ,IL-2, IL-10 and IL-12 were estimated in the culture super-natants by using commercially available specific ELISAassays and standard procedures. In the assays the detec-tion limits were 7 pg/mL for IL-2 and IFN-γ (QuantikineELISA, R&D Systems), 5 pg/mL for IL-10 (Diaclone), and3.2 pg/mL for IL-12 (human IL-12 p70 platinum ELISA,eBioscience).

Statistical analysis

For statistical analysis of the data, mean arithmetic val-ues (x) and SDs were calculated. Statistica 5.5 PL softwarewith non-parametric tests was used: Mann-Whitney’s Utest (for impaired data) to verify the hypothesis that thetwo compared samples came from two statistically differ-ent populations; χ2 test for prevalence comparison of theanalyzed variables in the studied groups. Differences wereconsidered significant when P < 0.05.

RESULTS

Natural and antigen driven cytotoxic activityof peripheral blood lymphocytes fromH. pylori uninfected and H. pylori infecteddonors

The majority of the investigated lymphocytes revealed nat-ural cytotoxic activity towards target cells (Fig. 1). Thenatural (spontaneous) cytotoxic activity of the lympho-cytes was expressed as a percentage of dead target cells co-cultured with unstimulated lymphocytes. Figure 1 showsthe degree of lymphocyte cytotoxicity in general and thecytotoxic capacity above 10% of dead targets. The popula-tion of lymphocytes with stronger natural and H. pylori GEdriven 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. py-lori LPS, although the difference was not statistically sig-nificant. However, after stimulation of the cells with H.pylori LPS, the lymphocytes from H.p (−) donors showedsignificantly greater average cytotoxic capacity than didlymphocytes from H.p (+) donors (Fig. 2). As is shown inFigure 2, the average percentage of dead target cells afterincubation 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 ma-jority (18/22) of the lymphocytes from H.p (+) donorshad a cytotoxic capacity of less than 10% whereas only5/22 lymphocyte cultures from H.p (−) donors expressedcytotoxicity of less than 10% (Fig. 1).

After stimulation of the effector cells with H. pyloriGE, the lymphocytes from H.p (−) donors also showed agreater cytotoxic capacity than did the lymphocytes fromH.p (+) subjects (P = 0.01) (Fig. 2). Moreover, LPS fromE. coli, but not H. pylori LPS, significantly enhanced theintensity of lymphocyte cytotoxicity in both groups ofdonors, whereas H. pylori GE only did so in H.p (−) in-dividuals (Fig. 2). By comparison, in response to H. pyloriLPS the cytotoxic activity of lymphocytes was even slightlyless than the natural cytotoxic capacity of the lymphocytesfrom both H.p (+) and H.p (−) donors (Fig. 2).

Granzyme B and caspase 8 concentrationsin the supernatants from mixed cultures oftarget cells with effector lymphocytes

There was a strong correlation between the cytotoxic ca-pacities of the lymphocytes as estimated by MTT reduc-tion assay and by quantification of granzyme B concen-tration (Fig. 3). The concentrations of granzyme B inthe cultures containing unstimulated lymphocytes fromH.p (−) were higher than in the cultures with unstim-ulated lymphocytes from H.p (+) donors. This mayconfirm the greater natural cytotoxic activity of lympho-cytes from H.p (−) versus H.p (+) individuals. More-over, significantly diminished granzyme B concentrationswere detected in the mixed cultures of target cells withlymphocytes from H.p (+) or H.p (−) individuals stim-ulated with H. pylori LPS than in cultures of unstim-ulated lymphocytes. In contrast, granzyme B concen-trations were significantly higher in cultures of targetcells containing lymphocytes prestimulated with H. py-lori GE or E. coli LPS than in control cultures containingunstimulated lymphocytes. Similarly, there was a correla-tion between the average cytotoxicity as estimated by theMTT reduction assay and the concentrations of caspase8 (Fig. 4). It is worth mentioning that caspase 8 concen-trations were lower in the cultures of target cells with H.pylori LPS stimulated lymphocytes than in the mixed cul-tures of targets and unstimulated lymphocytes from both


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Fig. 1. The cytotoxicity of peripheral blood lymphocytes from H.p (+) and H.p (−) donors, nonstimulated or stimulated with bacterialantigens H. pylori GE, H. pylori LPS and E. coli LPS.

groups of donors; however, we found a statistically signif-icant difference only for H.p (+) individuals. Moreover,we found that caspase 8 was produced more intensively incultures of target cells containing lymphocytes stimulatedwith E. coli LPS, but not H. pylori LPS, from H.p (+) andH.p (−) donors, P = 0.04 and P = 0.01, respectively.

Modulation of lymphocyte cytotoxic activityby exogenous interleukin-12

Exogenous IL-12 alone and in combination with H. py-lori antigens GE or LPS positively modulated the cyto-

toxic activity of lymphocytes from both H.p (+) andH.p (−) donors (Fig. 5). Prestimulation of the lympho-cytes from both studied groups with exogenous IL-12resulted in a higher percentage of dead targets in the cyto-toxic assay, in comparison to unstimulated lymphocytes.The cytotoxic responses of lymphocytes from H.p (+)donors stimulated with H. pylori GE or H. pylori LPS weresignificantly higher in the presence of IL-12 (P < 0.05),whereas the lymphocytes from H.p (−) donors showedan increase in their cytotoxicity after stimulation with IL-12 alone or with IL-12 and H. pylori LPS (Fig. 5). Theincrease in the cytotoxic activity of lymphocytes in the



Fig. 2. The intensity of natural and antigen-driven H. pylori GE, H. pylori LPS or E. coli LPS cytotoxicity of lymphocytes from H.p (+) orH.p (−) donors towards target cells estimated on the basis of their viability in the MTT reduction assay.

Fig. 3. Comparison of granzyme B concentration in the supernatants from mixed cultures of target cells and lymphocytes from H.p (+)and H.p (−) donors, nonstimulated or stimulated with bacterial antigens H. pylori GE, H. pylori LPS or E. coli LPS.


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Fig. 4. Comparison of caspase 8 concentration in the supernatants from mixed cultures of target cells and lymphocytes from H.p (+)and H.p (−) donors, nonstimulated or stimulated with bacterial antigens H. pylori GE, H. pylori LPS or E. coli LPS.

presence of exogenous IL-12 was correlated with increasedproduction of granzyme B, an effector cytotoxicity protein(Fig. 6).

Concentrations of effector cytokinesinterferon-γ, and interleukin-2, 10 and 12 inperipheral blood mononuclear cell culturesfrom H. pylori infected or uninfected donorsin response to H. pylori glycine acid extractantigens and H. pylori lipopolysaccharide

The PBMC from both H.p (+) and H.p (−) donors pro-duced IFN-γ spontaneously (cells in the culture mediumalone) 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 PBMCstimulated with H. pylori LPS. Exogenous IL-12 slightlyupregulated IFN-γ production in response to H. pyloriLPS in PBMC cultures from H.p (−) donors, but the dif-

ference was not statistically significant. The concentrationof IFN-γ in PBMC in response to E. coli LPS was about fivetimes higher than that in PBMC in the culture mediumalone, P = 0.0003 for H.p (+) donors and P = 0.0004for H.p (−) donors. Exogenous IL-12 slightly upregulatedIFN-γ production in response to H. pylori GE in PBMCcultures from H.p (+) donors and in response to H. pyloriLPS in PBMC cultures from H.p (−) donors.

Spontaneous secretion of IL-2 by PBMC was below thedetection limit (Fig. 7b). IL-2 was detected in the PBMCcultures from both H.p (+) and H.p (−) donors onlyafter stimulation of the cells with E. coli LPS, but notwith H. pylori GE or H. pylori LPS. In the presence ofexogenous IL-12 PBMC from H.p (−) donors reactedwith IL-2 production in response to H.p GE (Fig. 7b).

As shown in Figure 7c, we detected IL-10 in each typeof culture. There was no difference in IL-10 secretion byPBMC from H.p (+) and H.p (−) donors. Stimulation of



Fig. 5. The influence of IL-12 on the cytotoxic activity of lymphocytes from H.p (+) or H.p (−) donors nonstimulated or stimulated withH. pylori GE or H. pylori LPS towards HeLa cells.

leukocytes from H.p (−) and H.p (+) individuals with H.pylori GE antigens was accompanied by >35 fold and >16fold induction of IL-10 secretion, respectively. By com-parison, stimulation of PBMC with H. pylori LPS alsoresulted in significant enhancement of IL-10 productionin H.p (+) and H.p (−) donors, but only by 2- and 5-fold,respectively. Furthermore, GE driven IL-10 productionwas significantly stronger than that induced by H. pyloriLPS, in PBMC cultures from both groups of donors. IL-12supplementation did not alter IL-10 secretion (Fig. 7c).

The presence of IL-12 in the leukocyte culture super-natants with or without stimulation (GE, H. pylori LPS)was assessed by specific ELISA but IL-12 was detectedneither in the cultures stimulated with H. pylori GE orLPS nor in the nonstimulated leukocyte cultures in bothgroups of donors.

Phenotypic characteristics of freshly isolatedor culture propagated lymphocyte subsets ofH. 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 killercells 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 cellswith expression of a CD25 marker, an IL-2 recep-tor (CD3−CD56+CD25+). However, the number ofCD3+CD25+ lymphocytes was doubled in H.p (+)donors (Table 1). Furthermore, we found that H.p LPScaused stronger expansion of the CD3+CD25+ lympho-cyte subpopulation in in vitro cell cultures then did H.pGE (Table 2).

DISCUSSION

Some of the mechanisms that control the immune re-sponse during H. pylori infections are related to pathogenvirulence factors. The role of H. pylori surface adhesins,LPS and vacA, in avoiding the engulfment and intracellu-lar killing of the pathogen by phagocytes has been shown(23, 29, 30). In this study, we have demonstrated that H.


K. Rudnicka et al.

Fig. 6. The influence of IL-12 on granyzme B concentration in supernatants from target cells and lymphocytes from H.p (+) or H.p (−)donors nonstimulated or stimulated with H. pylori GE or H. pylori LPS.

pylori antigens may modulate positively or negatively theMHC-unrestricted cytotoxic activity of peripheral bloodlymphocytes towards tumor epithelial HeLa cells. HeLacells were selected for this study because this is a standardepithelial cell line recommended for the estimation of thelymphocyte cytotoxicity. Cell lines derived from humangastric epithelial cells such as AGS and Kato III were alsoincluded in this study. Although all cell types responded ina similar way, the most homogenous results were obtained

for the HeLa cells. In a preliminary study we demonstratedthat AgS and Kato III cells may differ concerning their pro-liferative activity in general, and in response to H. pyloriantigens. Lymphocytes isolated from H.p (+) donors, un-stimulated or treated with H. pylori GE or H. pylori LPS,expressed less cytotoxic activity against target cells thandid lymphocytes from H.p (−) individuals. This probablymeans that H.p (+) subjects possess a smaller numberand/or lower activity of natural cytotoxic lymphocytes



Fig. 7. Concentrations of (a) IFN-γ, (b) IL-2 and (c) IL-10 in theculture supernatants of PBMCs from H.p (+) or H.p (−) donors,unstimulated or stimulated with H. pylori GE or H. pylori LPS inthe presence or absence of IL-12.

than do H.p (−) donors. If so, the size of the cytotoxic lym-phocyte population and their characteristics might also beimportant for the control of infection and elimination ofdamaged or tumor host cells. However, during H. pyloriinfection bacterial compounds may themselves affect thenumber or activity of cytotoxic lymphocytes. In this studywe demonstrated that, in H.p (+) donors, the numberof CD3−CD56+Nkp46+ NK cells among freshly isolatedlymphocytes is half that of H.p (−) subjects. O’Keeffeeet al. suggested that the role of NK cells in H. pylori in-fections depends on their predominance, heterogeneity,and distribution in the gastric mucosa (12). According toLindgren et al., the key players in the innate response to H.pylori are CD8−CD16−CD56bright NK cells, which proba-bly modulate the defense mechanisms indirectly throughcytokine production rather than directly by cytotoxic ac-tivity (14).

In this study, we have demonstrated downregulation ofthe natural cytotoxic activity of lymphocytes by H. pyloriLPS, but not E. coli LPS or H. pylori GE. A direct toxiceffect of H. pylori LPS on lymphocytes from both H.p (+)and H.p (−) donors was excluded under our study con-ditions, since the viability of effector cells stimulated withH. pylori LPS, as assessed by MTT reduction assay, was thesame as the viability of unstimulated cells. In a previousstudy, Grebowska et al. reported antiproliferative activ-ity of H. pylori LPS towards lymphocytes (31). However,the appearance of this phenomenon was time-dependent.Inhibition of lymphocyte proliferation was observed in72 hr, but not in 24 hr, cell cultures (31).

We here report that the diminished cytotoxic activity oflymphocytes from H.p (−) and especially H.p (+) donorsin response to H. pylori LPS is correlated with attenuationof IFN-γ secretion, increased, but still low, production ofIL-10 by PBMC, and lack of IL-2 secretion. In contrast toH. pylori LPS, standard LPS from E. coli stimulated thecytotoxic activity of lymphocytes, which was correlatedwith enhanced production of IFN-γ and IL-2 by PBMCand lack of enhancement of IL-10 production. Finally, theGE antigenic complex increased the cytotoxicity of lym-phocytes. GE driven production of IFN-γ was equal tothe spontaneous secretion of this cytokine by PBMC. Al-though it did not stimulate secretion of IL-2 by PBMC,IL-10 production in response to GE was very strong. It ispossible that the mechanisms of lymphocyte cytotoxicityobserved in this study may depend on the antigen used forPBMC stimulation, H. pylori LPS or GE, and E. coli LPS.The cytotoxic activity of lymphocytes in response to E. coliLPS was probably related to IFN-γ and IL-2 productionby the immune cells, whereas the lymphocyte cytotoxic-ity in response to H. pylori GE could be a result of IL-10signaling and IFN-γ production. The inhibition of cel-lular cytotoxicity in response to H. pylori LPS could be


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Table 1. The phenotypic characteristics of peripheral blood lymphocytes and natural killer (NK) cells in H. pylori (−) and H. pylori (+) donors

Subsets of lymphocytes (% ± SD)

Lymphocytes Lymphocytes with IL-2R NK cells NK cells with IL-2R

Lymphocyte donors CD3+ CD3+CD25+ CD3−CD56+Nkp46+ CD3−CD56+CD25+

H. pylori (−) 72.21 ± 11.20 10.11 ± 2.70 10.30 ± 2.30 0.21 ± 0.35H. pylori (+) 70.51 ± 9.30 19.93 ± 0.80 6.80 ± 1.20 0.24 ± 0.26

Table 2. The influence of H. pylori LPS, GE or the LPS of E. coli on

expression of CD25-receptor for IL2R on peripheral blood lymphocytes

and NK cell subsets in H. pylori (−) and H. pylori (+) donors

Subsets of lymphocytes in cell cultures in vitro (% ± SD)

Lymphocytes with IL-2R NK cells with IL-2R

Lymphocyte donors CD3+CD25+ CD3−CD56+CD25+

H. pylori (−)nonstimulated 8.61 ± 2.50 0.17 ± 0.21LPS H. pylori 13.45 ± 5.54 0.10 ± 0.11GE H. pylori 6.09 ± 6.63 0.48 ± 0.02LPS E. coli 10.11 ± 2.69 0.19 ± 0.07Lymphocyte donors

H. pylori (+)CD3+CD25+ CD3−CD56+CD25+

nonstimulated 10.04 ± 1.60 0.93 ± 0.35LPS H. pylori 16.52 ± 5.00 0.45 ± 0.16GE H. pylori 5.55 ± 0.66 0.82 ± 0.34LPS E. coli 8.78 ± 1.55 0.43 ± 0.21

due to a lack of IFN-γ and IL-2 mediated stimulation,as well as low production of IL-10, which is insuffi-cient to stimulate lymphocytes to express their cytotoxicactivity.

Interleukin-2 is necessary for lymphocyte growth andit plays the roles of a classic NK cell activator by aug-menting their proliferation rate, migration capability andcytotoxic activity (10, 11). Therefore, the lack of IL-2 pro-duction in the PBMC cultures stimulated with H. pyloriLPS might result in impairment of the cell’s condition andtheir cytotoxic capacity. In this study H. pylori LPS did notinfluence the number of CD3−CD56+ NK cells. However,it induced stronger expansion of CD3+CD25+ lympho-cytes than did H. pylori GE and E. coli LPS. Although thenumber of CD3+CD25+ lymphocytes was increased, theywere unable to support the cytotoxic activity of NK cellsin the absence of IL-2. It is also worth mentioning that theconcentrations of IL-10 were very low and the productionof IFN-γ abrogated in the presence of H. pylori LPS. Pre-viously it has been shown that, in the presence of IL-2, H.pylori LPS is an effective stimulator of human lympho-cyte proliferation, although unlike LPS from other Gram-negative bacteria, H.pylori LPS alone shows very weak, ifany, capacity to stimulate human lymphocytes (32).

In contrast to H. pylori LPS, the GE antigen complexincreased the cytotoxicity of lymphocytes. Moreover, GEdid not diminish natural IFN-γ and IL-2 secretion activityof the PBMCs and greatly enhanced production of IL-10.According to Asadullah et al., IL-10 favors the cytotoxicactivity of NK cells since it increases IL-2 induced produc-tion of cytokines such as IFN-γ, granulocyte macrophagecolony stimulating factor and TNF-α (10). It also ampli-fies IL-2 induced proliferation of the CD56−bright NK cellsubpopulation. However, IL-10 may have no effect on theexpression of other NK cell cytotoxicity markers such asperforin, granzyme B, iNOS, and FasL and may not affecttheir upregulation by IL-2. In this study, we report corre-lations between lymphocyte cytotoxicity as measured byMTT reduction assay and quantification of granzyme Band caspase 8 concentrations in the supernatants obtainedafter the cytotoxic assay.

It has also been suggested that IL-10 influences NK cellcytotoxicity through a specific pathway distinct from thatfor activation of NK cells by IL-2 (11). In this study weobserved expression of lymphocyte cytotoxic activity to H.pylori GE and high IL-10 concentrations in PBMC culturesupernatants in response to this antigen complex. On theother hand, the cytotoxicity of lymphocytes stimulatedwith E. coli LPS was correlated with high concentrationsof IL-2.

Other inflammatory cytokines may also regulate thereactivity of immune cells in response to H. pylori LPS.In this study, we focused on IL-12, which is known toupregulate the cytotoxic activity of NK cells. ExogenousIL-12 stimulated both natural and H. pylori antigens- (GEand LPS) driven lymphocyte cytotoxicity. This suggeststhat IL-12 present in the inflammatory milieu during H.pylori infections might promote activation of the effec-tor functions of naturally cytotoxic lymphocytes. SeveralH. pylori factors might be involved in activation of IL-12expression, for instance, cag PAI compounds and the prod-ucts of genes in the H. pylori plasticity region (33–35). Re-cently, intact H. pylori has been shown to promote rapidmaturation and activation of monocyte derived DCs (36)and H. pylori pulsed DCs to cause activation of autolo-gous T cells and expression of IL-2, TNF-α, and IFN-γ.However, most LPS-induced effects on T cells seem to re-sult from indirect interactions between T cells and LPS



stimulated macrophages and from mediators releasedfrom the LPS, activated accessory cells and cytokine acti-vated T cells (37).

In our study, exogenous IL-12 significantly increasedthe 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 didnot provoke IL-2 production or enhancement of IL-10secretion. This suggests that IL-12 alone is an importantand sufficient stimulator of lymphocyte cytotoxic activity.Choel et al. have demonstrated that H. pylori lysates andIL-12 act synergistically to induce IFN-γ production (38).IL-12 also synergizes with IL-2 to induce lymphokine ac-tivated cytotoxicity as well as perforin and granzyme geneexpression in human NK cells. De Blaker et al. (39) andKao et al. (40) have demonstrated that H. pylori secretedfactors inhibit IL-12 production in dendritic cells. Thephenomenon of IL-12 inhibition has been considered amechanism for impairing host defense against H. pylori.In our study IL-12 was detected neither in cultures stim-ulated with H. pylori GE or LPS nor in nonstimulatedleukocyte cultures in both groups of donors. These find-ings are in agreement with those of other authors showingthat IL-10 may inhibit IL-12 production by both phago-cytes and dendritic cells. IL-10 also acts as a protein at themRNA level (41). It has also been shown that LPS is effec-tive in downregulating IL-12 production by mononuclearcells (42). According to Hafsi et al., Th1 effector responsesare more pronounced when membrane preparations ofH. pylori are used (36). This suggests that digestion ofthe bacteria into subcellular fractions might facilitate theappearance of antigenic proteins. Our results show thatH. pylori LPS may downregulate not only the cytotoxiccapacity of lymphocytes, but also production of IFN-γand IL-2, and thus cannot promote IFN-γ dependent ac-tivation of macrophages and IL-2 dependent expansionof lymphocytes. However, exogenous IL-12 seems to beable to induce cellular cytotoxicity even in the absence ofIFN-γ, IL-2 or IL-10. This may confirm the importance ofIL-12 in the regulation of immune responses towards H.pylori. The increase in cytotoxic activity of lymphocytesin the presence of exogenous IL-12 is correlated with in-creased production of granzyme B. It has been shown thatIL-12 binds to IL-12R and, through a signaling pathway,activates various genes encoding mediators involved in thecytotoxicity process such as effector cytotoxicity proteinsincluding granzyme B, cytokines, cytokine receptors, andsignaling molecules (43).

In contrast to H. pylori LPS, H. pylori surface GE anti-gens are able to induce a cytotoxic response by lympho-cytes and cytokine production by PBMC. Therefore, cy-totoxic 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. pyloriinfections.

Mohammadi et al. have shown that H. pylori culturesupernatants contain a factor that can activate large restinggranular lymphocytes to exhibit cytolytic activity (44).Yun at al. demonstrated that NK cells can be directlyactivated by whole cell H. pylori, whereas H. pylori lysatecan induce IFN-γ secretion by NK cells (45). Additionof a small amount of IL-12 greatly enhances productionof IFN-γ although a bacterial lysate alone is sufficient toinduce activation of cytotoxicity related molecules (45).In our study, the GE antigenic complex was not effectiveat stimulating IFN-γ production by PBMC, however, thismight be due to different antigenic profiles of GE and thewhole cell lysates.

Hafsi et al. proposed that NK-derived IFN-γ mayplay a major role in the homeostasis of the immune re-sponse during H. pylori infection through activation ofmacrophages and differentiation of T cells (36). However,according to Tarkannen et al., the cytotoxic activity of NKcells does not directly affect the bacteria on the surface ofthe epithelium and may therefore be of less importance(15). Despite antibacterial and antiviral activity, naturallycytotoxic lymphocytes also remove damaged or mutatedcells. Thus, inhibition of such clearance by H. pylori com-pounds, for instance LPS, may initiate dangerous gastricpathologies. Recently, it has been shown that H. pylori-derived LPS augments the growth of gastric cancer cellsvia the LPS-TLR4 pathway and attenuates the cytotoxicityof mononuclear cells against gastric cancer cells. Stim-ulation with H. pylori LPS also downregulates perforinproduction in cancer cells cocultured with CD56+ naturalkiller cells. H. pylori LPS induces neither IL-12 nor IFN-γproduction by mononuclear cells (45). Nevertheless, IL-12restores the IFN-γ -producing capacity of H. pylori LPS-stimulated leukocytes. Pellicano et al. have demonstratedthat the presence of IL-12 is crucial for induction of IFN-γproduction in NK cells (46). It is possible that H. pylori in-fection attenuates antitumor activity and IFN-γ mediatedcellular immunity of mononuclear cells and thereby pro-motes proliferation and progression of gastric cancers (47,48). The immunobiological activity of H. pylori LPS maydepend on the presence of Lewis XY determinants, whichmay promote the production of macrophage derivedcytokines such as TNF, IL-8 and IL-12 (49). Bergman etal. reported that Lewis antigen-bearing H. pylori cells pre-dominantly induce IL-10 and promote Th2 cell response,whereas Lewis antigen-negative H. pylori cells promote astrong Th1 cell response (50).

In conclusion, we have demonstrated that the cytotoxiccapacity of lymphocytes from H.p (+) individuals is lessthat of lymphocytes from H.p (−) donors. This could bedue to there being fewer NK cells of CD3−CD56+NKp64+


K. Rudnicka et al.

phenotype in H.p (+) donors. We have also shown thatH. pylori antigens may vary in their ability to activatecytotoxic lymphocytes. H. pylori surface GE antigens areeffective at stimulating lymphocyte cytotoxic activity butH. pylori LPS downregulates the natural cytotoxic capac-ity of the lymphocytes, which correlates with a diminishedability of PBMCs to produce IFN-γ, IL-2 and with littlesecretion of IL-10 in response to LPS. H. pylori LPS didnot influence the number of CD3−CD25+ NK cells, al-though the population of CD3+CD25+ lymphocytes wasincreased in the milieu of H. pylori LPS, but not H. pyloriGE or LPS from E. coli. It is possible that, in the absence ofIL-2, these lymphocytes were not able to support NK cyto-toxic activity. In contrast with H. pylori LPS, GE antigensstrongly stimulate IL-10 production and do not decreasethe ability of immune cells to produce IFN-γ and IL-2.Based on these findings, we propose that H. pylori LPSnegatively modulates the first line of immune defense, in-cluding naturally cytotoxic lymphocytes, and accordinglymay be involved in the maintenance of infection and in thedevelopment of gastric pathologies. However, the presenceof IL-12 in the inflammatory milieu during H. pylori in-fections might be critical for effective positive modulationof cytotoxic activity in response to H. pylori antigens.

ACKNOWLEDGMENTS

This research was supported by the Polish Ministry ofScience and Higher Education (grant N N401 015 136)and the European Union Project “Stipends supportinginnovative research projects for PhD students”.

DISCLOSURE

The authors have no conflicting financial interests.

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Helicobacter pylori antigens as potential modulators of lymphocytes’ cytotoxic activity

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