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Keywords:

  • HP-NAP;
  • Helicobacter pylori;
  • Transmigration;
  • Endothelial cells;
  • Inflammation

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark of Helicobacter pylori infection in humans. In this study, we examined the ability of H. pylori to induce transendothelial migration of neutrophils using a transwell system consisting of a cultured monolayer of human endothelial cells as barrier between two chambers. We showed for the first time that live H. pylori, but not formalin-killed bacteria, induced a significantly increased transendothelial migration of neutrophils. H. pylori conditioned culture medium also induced significantly increased transendothelial migration, whereas heat-inactivated culture filtrates had no effect, suggesting that the chemotactic factor was proteinaceous. Depletion of H. pylori-neutrophil activating protein (HP-NAP) from the culture filtrates resulted in significant reduction of the transmigration. Culture filtrates from isogenic HP-NAP deficient mutant bacteria also induced significantly less neutrophil migration than culture filtrates obtained from wild-type bacteria. HP-NAP did not induce endothelial cell activation, suggesting that HP-NAP acts directly on the neutrophils. In conclusion, our results demonstrate that secreted HP-NAP is one of the factors resulting in H. pylori induced neutrophil transendothelial migration. We propose that HP-NAP contributes to the continuous recruitment of neutrophils to the gastric mucosa of H. pylori infected individuals.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Infection with Helicobacter pylori gives rise to active chronic gastritis in virtually all infected subject. In some individuals, the infection eventually leads to the development of ulcer disease or gastric adenocarcinoma. Several bacterial and host factors seem to play important roles in determining the severity of the inflammation and the outcome of the infection [1,2]. Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark in human H. pylori infections. A strong correlation exists between gastric neutrophil infiltration, mucosal damage, and development of duodenal ulcer (DU) disease in H. pylori infections [3,4]. There is also strong evidence that H. pylori is able to activate neutrophils; in some studies, the strains isolated from DU patients were shown to be more potent activators of neutrophils than other isolates [3,5–9]. One of the bacterial factors shown to mediate neutrophil activation is the H. pylori neutrophil activating protein (HP-NAP) [5]. Evans et al. [5,9] first characterized the HP-NAP protein and showed that it stimulated the release of reactive oxygen species by human neutrophils. HP-NAP is dodecameric and is able to bind iron. It has sequence similarities with iron-binding proteins in other bacterial species [10,11]. HP-NAP is a cytosolic protein expressed by virtually all H. pylori isolates [12]. It is released into the supernatant by autolysis, upon which a fraction of HP-NAP is then bound to carbohydrates on the external surface of the outer membrane [5,13,14].

While the mechanisms behind the continuous recruitment of neutrophils to H. pylori infected tissues in vivo remain to be fully understood, endothelial cell function and activation appear to be key components in this process. Endothelial cells are involved in a wide range of physiological reactions including blood coagulation, blood pressure regulation, and inflammatory responses. One important function is to direct circulating leukocytes to sites of inflammation via a series of steps that are independent of the stimulatory agent [15]. During the inflammatory process, or upon in vitro stimulation with TNF-α, IL-1 or bacterial products, endothelial cells respond rapidly (within a few hours) by upregulation of adhesion molecules such as VCAM-1, ICAM-1, and E-selectin as well as production of proinflammatory cytokines and chemokines [16], all of which contribute to leukocyte recruitment.

We have shown in a previous study that endothelial cells are strongly activated by H. pylori, resulting in increased production of neutrophil recruiting chemokines and expression of adhesion molecules [17]. To further characterize this response, we investigated the effect of H. pylori on the migration of neutrophils through cultured endothelial monolayers. We showed that live bacteria, secreted bacterial proteins containing HP-NAP, and purified HP-NAP all resulted in an increased transendothelial migration of neutrophils. In contrast, culture filtrates depleted of HP-NAP, as well as culture filtrates produced by isogenic HP-NAP deficient mutants were less effective in inducing neutrophil transendothelial migration.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1H. pylori strains

H. pylori strains were isolated from the duodenum of adults suffering from DU (strain Hel 333, P12, Table 1), from the stomach of adult asymptomatic subject (strain Hel 312, Table 1) or from a patient suffering from a non-ulcer dyspepsia (strain P1, Table 1), as previously described [3,18]. Isolates were stored in a freeze-drying medium (tryptic soy broth with 15% glycerol) at −80°C. The mouse adapted strain SS1 was originally provided by Dr. Adrian Lee, Sydney, Australia [19]. Characteristics of these strains [12,17] are summarized in Table 1.

Table 1.  Characterization of H. pylori strains used in the study
Straincag PAIaVacAbOrigin
  1. acag PAI, cytotoxin-associated gene pathogenicity island.

  2. bVacA, vacuolating toxin A.

  3. cs, sequence region; m, middle region.

  4. dAS, asymptomatic carrier.

  5. eDU, duodenal ulcer patient.

  6. fNUD, non-ulcer dyspepsia patient.

Hel 312+s1/m1cASd
Hel333s2/m2DUe
P1+s1/m2NUDf
P12+s1/m1DU
SS1+s2/m2DU, mouse adapted

2.2Generation of isogenic HP-NAP deficient mutants

Isogenic P1Δnap and P12Δnap knock-out mutants were constructed by insertion of a chloramphenicol resistance gene cassette (CmR, 1 kb fragment of plasmid pTnMax1) or kanamycin resistance gene cassette (Aph-3, 1.4 kb fragment of plasmid pILL-600) according to a standard protocol [18]. For this purpose, we amplified a fragment of 1.8 kb containing the nap gene and flanking regions with the following primers: 179F 5′-TTTTTGAAGGGCCAATCTTAGAA and 179R 5′-AGCGACGAAGGGTTTTTTG. This product was ligated into pCR2.1 vector (Invitrogen, Karlsruhe, Germany) and the correct integration of the CmR or Aph-3 cassettes in the nap gene was checked by standard PCR. Both mutants were created with terminatorless cassettes, and are non-polar.

2.3H. pylori culture conditions

H. pylori were cultured routinely on Columbia-iso agar plates at 37°C in a microaerophilic milieu (3% O2, 5% CO2 and 92% N2). After four days of incubation, the bacteria were resuspended in 3–5 ml of PBS. The optical density (OD) of the suspension was determined (Shimadzu UV-1201, Lambda Polynom, Stockholm, Sweden), and adjusted to a final OD600nm= 1.0 in PBS, corresponding to approximately 5 × 109H. pylori bacteria ml−1 (5 × 109 CFUs ml−1). In some experiments, PBS resuspended bacteria inactivated by treatment with 0.01 M Formaldehyde at 37°C for 2 h and then at room temperature over night, were used.

2.4Preparation of H. pylori secreted proteins (culture filtrate)

H. pylori strains were grown and resuspended as described above. Bacterial suspension (2.5 ml) was added to 48 ml of M200 medium (Cascade Biologics Inc. OR, USA) containing Brucella broth (Difco Laboratories, Detroit, MA, USA), 5% new born calf serum (Biochrome AG, Berlin Germany), 10μg ml−1 Vancomycin, 5 μg ml−1 Trimothoprim, and 20 U ml−1 Polymyxin B, and the mixture was then incubated for 6.5 h at 37°C in 5% CO2. The time point was chosen based on our previous study showing that human umbilical vein endothelial cells were strongly stimulated by secreted H. pylori proteins produced by a 6.5 h culture [17]. The cultures were sterile filtered, and one aliquot of each culture filtrate was heat-inactivated at 85°C for 35 min. All filtrates were then stored at −80°C until use.

2.5Depletion of HP-NAP from H. pylori culture filtrate

The culture filtrate was depleted of HP-NAP using magnetic beads (Dynal, Norway) coupled to IgG1 monoclonal antibody (mAb) against HP-NAP (NAP 6:1), the preparation of which has been described previously [12]. Sixty milligrams of beads coated with sheep antibodies to mouse IgG1 were incubated with 120 μg IgG1 mAb NAP 6:1 for 4 h, washed on a magnet, resuspended in 2 ml of the culture filtrate and incubated for 4 h at 4°C on a waddle table. The beads were removed with a magnet and the HP-NAP depleted culture filtrate was stored at −80°C until use. Beads incubated with an IgG1 mAb reacting with cholera toxin were used as controls for unspecific binding. HP-NAP depletion from culture filtrates was confirmed by Western blot analysis using the same NAP-mAb. In brief, samples were separated on a 18% Tris–Glycine SDS–PAGE, and blotted onto a PVDF membrane using standard procedures. After blocking in 2% milk powder diluted in blotting buffer (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), the membrane was probed with monoclonal mouse anti-NAP [12] diluted 1:5 in blotting buffer over night at 4°C. Thereafter the membrane was washed and mouse immunoglobulins were detected with biotinylated rabbit anti-mouse Ig (1:2000, DAKO, E0413), followed by a combination of HRP-conjugated donkey anti-rabbit Ig (1:2000, Amersham, NA9340) and Strep ABComplex/HRP (DAKO). Peroxidase activity was subsequently detected by ECL chemiluminescense (Amersham) according to the manufacturer's instructions and exposure for 15 min on Fuji NP100 film (Fig. 3B).

image

Figure 3. Induction of neutrophil transendothelial migration by HP-NAP. (A) rNAP (10 μg ml−1) was added to the lower wells of Transwell cultures and 106 neutrophils to the upper wells. fMLF (10−8 M) was used as a positive control. Bars indicate geometric mean + SEM of percentage of transmigrated neutrophils. In (B) culture filtrate, HP-NAP depleted (depl.) culture filtrate, or culture filtrate treated with beads coated with a mAb against cholera toxin (sham depleted) were added to the lower wells of Transwell cultures, and 106 neutrophils to the upper wells. Bars indicate geometric mean + SEM of percentage of transmigrated neutrophils relative to the migration induced by the non-treated culture filtrate. Statistical evaluation between negative control and stimulated samples was performed by the Mann–Whitney test. ***p < 0.001, **p < 0.01, *p < 0.05. Data are obtained in triplicates from at least three independent experiments. (C) Depletion of HP-NAP from the culture filtrate is shown as a radiographic photo were lane 1 is rNAP, lane 2 is the HP-NAP depleted culture filtrate from strain Hel 312 and lane 3 is the control of non-depleted culture filtrate from the same strain.

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The distribution of immunogenic H. pylori proteins in the culture filtrates was examined by Western blotting using an H. pylori-reactive polyclonal mouse serum pool (data not shown). With the exception of HP-NAP, the relative distribution of other filtrate proteins remained unchanged during incubation with the beads.

2.6Production of rNAP

rNAP was produced in E. coli as described and provided by Dr. Susanne Nyström [12]. Briefly, E. coli cells were transformed with an expression vector containing the H. pylori nap gene under the control of the T7 promoter. After disruption of the cells, the soluble proteins were applied to a Q-Sepharose column (Pharmacia BioTech, Uppsala, Sweden) in 25 mM Tris–HCl and 50 mM NaCl (pH 8.0). rNAP was found in the breakthrough. The fractions containing rNAP were concentrated and then purified by gel filtration through a Superdex 200 column (Pharmacia). rNAP eluted as a single peak. Purity of the protein was confirmed by SDS–PAGE and Coomassie staining as well as immunoblotting, using mAbs specific for rNAP [20].

2.7Isolation of neutrophils

Human neutrophils were isolated from buffy coats, or alternatively, from heparinised whole blood obtained from healthy blood donors. After dextran sedimentation at 1g, hypotonic lysis of the remaining erythrocytes, and subsequent centrifugation in a Ficoll-Paque gradient [21], the neutrophils were washed twice and resuspended to a final volume of 1 × 107 ml−1 in Krebs–Ringer phosphate buffer (KRG) containing glucose (10 mM), Ca2+ (1 mM), and Mg2+ (1.5 mM), and kept on ice up to 1.5 h until use.

2.8Sub-culturing and stimulation of endothelial cells

Human umbilical vein endothelial cells (HUVEC), passage 1, were purchased from Cascade Biologics Inc. Cells were grown in 75 cm2 plastic bottles (Nunc, Roskilde, Denmark) in M200 medium supplemented with low serum growth supplement, (LSGS) and penicillin–streptomycin–amphotericin (PSA) (all from Cascade Biologics Inc.). Medium was changed every 48 h during culturing at 37°C, 5% CO2. The medium used in this study contained routinely less than 0.03 EU ml−1 endotoxin as confirmed by Limulus test.

Confluent monolayers were trypsinized using 0.25 mg ml−1 trypsin/EDTA (Cascade Biologics Inc.) and split at a ratio of 1:3. At passage 4, the cells were frozen in fetal calf serum containing 10% DMSO (Sigma) at a density of 106 cells ml−1. HUVEC within passage 4–7 were used throughout the experiments [22] to minimize spontaneous morphological or functional changes as a result of continuous passaging.

HUVEC were grown for four days, after which the medium was replaced with fresh antibiotic-free medium containing 10 μg ml−1 rHP-NAP or 5 × 108 live wild-type P1 or P1Δnap. After 6.5 h incubation, the culture medium was collected and HUVEC were detached from the plate using trypsin. The endothelial expression of ICAM-1, E-selectin and VCAM-1 was determined by flow cytometry, and the IL-8 concentration in the culture medium analyzed by ELISA, as previously described [17].

2.9Transendothelial migration of neutrophils

HUVEC (3 × 105 cells) were added to collagen-coated nets of Transwell chambers (Costar, Badhoevedorp, The Netherlands) with a growth area of 4.7 cm2, and allowed to grow to confluence. When HUVEC reached a confluent monolayer after 24 h as confirmed by microscopy, the medium was changed and incubation continued for another hour at 37°C. This creates a system with two separate chambers, where transport through the endothelial monolayer is the only means of communication between the chambers, thereby allowing a chemotactic gradient of the substances added to the lower chamber to be established. Stimuli were placed in the lower chamber in 1 ml of M200 medium, and purified neutrophils (106 cells in 100 μl KRG medium) were added in the upper chamber to a final volume of 1 ml. The addition of bacteria was carried out 1 h before the addition of neutrophils to allow a gradient of secreted products to be established, whereas all other stimulatory agents were added at the same time as the neutrophils. The chemo-attractant formyl-methionyl-leucyl-phenylalanine (fMLF) was used at a concentration of 10−8 M as a positive control in all experiments and was added in a volume of 100 μl. All stimuli (including bacteria) used were present in the lower chamber throughout the experiments.

After 90 min (optimal duration for reducing spontaneous migration and yet achieving maximum migration of attracted cells) of transendothelial migration, the neutrophil containing medium in the lower chamber was removed and collected in polypropylene plastic tubes on ice. Meanwhile, 2 ml of 0.25% Trypsin/EDTA solution was added in order to detach neutrophils that were adhering to the plastic well or to the bottom of the filter. The cells harvested were sedimented, transferred to a 96-well polypropylene plate and stained for the expression of the complement receptor 3 (CR3) by adding 10 μg ml−1 of the PE-conjugated anti-CR3 antibody (BD, Erembodegem, Belgium) in a total volume of 100 μl PBS containing 1.46 g l−1 EDTA, 2.5 g l−1 bovine serum albumin, 0.2 g l−1 NaN3 and 2% AB+ human serum (FACS-wash), and incubated on ice for 35 min. After washing the pellet twice in FACS-wash buffer the transmigrated cells were fixed in Cell Fix? (BD) and enumerated by flow cytometry (Facs-Calibur, BD) using True Count? beads (BD). Neutrophils were identified by their forward and side scatter characteristics, as illustrated in Fig. 1. Since these parameters vary with the agent used for stimulation, expression of CR3, which is upregulated during transendothelial migration, was used to confirm that gated events were indeed neutrophils.

image

Figure 1. FACS-analysis of migrating neutrophils. 105 cells were collected after transendothelial migration against culture medium (A), 10−8 M fMLF (B) or 5 × 108 live H. pylori bacteria (C) and enumerated by flow cytometry. Axes show forward (FSC-H) and side scatter (SSC-H) characteristics. In all figures, TrueCount? beads can be seen as indicated by arrow and text in (B). Following encounter with bacteria (C), neutrophils increase their side scatter signal. The figure shows one representative example of all FACS plots analyzed in this study.

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2.10Statistical analysis

Statistical evaluations were performed using the Mann–Whitney test and the Prism software.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Live H. pylori and culture filtrates induce neutrophil transendothelial migration

Neutrophil transendothelial migration towards H. pylori bacteria was analyzed in the Transwell system and quantified using flow cytometry. Five well-characterised H-pylori strains (Hel 312, Hel 333, SS1, P1 and P12; Table 1) were used in our experiments and gave similar results. One representative FACS-dot plot is shown in Fig. 1. Titration experiments demonstrated a dose dependent migration of neutrophils towards H. pylori, with 5 × 108 of bacteria giving a strong and consistent response (data not shown). This amount of bacteria was used in all further experiments. H. pylori strain Hel 312 carrying the cag pathogenicity island (cag PAI+) and vacA s1/m1 allele induced transendothelial migration of significantly (p < 0.01) more neutrophils than the unstimulated control (approximately 25% of the added neutrophils compared to about 5% in the control) (Fig. 2). The H. pylori strain Hel 333 (cag PAI, vacA s2/m2 allele) also induced a substantially increased neutrophil transendothelial migration, where approximately 70% of the total added neutrophils had migrated (p < 0.01, data not shown). In contrast, the same number of formalin-inactivated Hel 312 induced significantly less (p < 0.05) migration compared to live Hel 312 (Fig. 2).

image

Figure 2. Induction of neutrophil transendothelial migration by culture filtrates from H. pylori. 5 × 108 live or formalin killed H. pylori bacteria, culture filtrate, or heat-inactivated culture filtrate, were added to the lower wells of Transwell cultures and 106 neutrophils to the upper wells. fMLF (10−8 M) was used as a positive control. Bars indicate geometric mean + SEM of percentage of transmigrated neutrophils. Statistical evaluation between negative control and stimulated samples was performed by the Mann–Whitney test. ***p < 0.001, **p < 0.01, and *p < 0.05. Data are obtained in triplicates from at least three independent experiments.

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Since live but not dead bacteria could induce neutrophil migration, we sought to test if the effect was caused by secretion of neutrophil chemotactic factors by bacteria into the culture filtrates. We chose to use strain Hel 312, since Hel 333 grows poorly and since the presence of the CagPAI did not seem to influence induction of migration. In addition, H. pylori induced chemokine and adhesion molecule upregulation on endothelial cells is independent of the CagPAI (16). Indeed, when culture filtrate from Hel 312 bacteria was used as stimulatory agent, we could detect a significant level of approximately 20% neutrophil transendothelial migration (p < 0.01), whereas the positive control fMLF induced approximately 40% migration (p < 0.001) (Fig. 2). Heat inactivation of the H. pylori filtrate at 85°C for 35 min significantly abolished the capacity of the culture supernatant to induce transendothelial migration (Fig. 2), indicating that the stimulatory agents are of proteinaceous nature. Similar results were obtained from culture filtrates of strain Hel 333. As a negative control, culture medium treated in the same way but without the addition of bacteria did not affect neutrophil transendothelial migration (data not shown).

3.2HP-NAP is a potent chemotactic factor for neutrophils in culture filtrates of H. pylori

Since H. pylori culture filtrates induced neutrophil transendothelial migration to the same extent as bacteria, we examined if HP-NAP, a well known neutrophil activating factor [5,7,8], is responsible for the induction of neutrophil migration. rNAP was added to the lower chambers at a concentration of 10 μg ml−1 and neutrophil transendothelial migration was measured. This concentration was selected based on in vitro titration experiments in our model system to give the highest induction of transendothelial migration (data not shown). When rNAP was added to the assay, almost 25% (p < 0.01) of the added neutrophils transmigrated (Fig. 3A). Again, the positive control fMLF induced the highest degree of transendothelial migration, attracting about 35% (p < 0.001) of the added neutrophils in this set of experiments (Fig. 3A). To avoid the possibility of LPS contamination, 200 μg ml−1 rNAP was incubated with 20 μg ml−1 of polymyxin B for 1 h at room temperature. After incubation, 100 μl of the HP-NAP–polymyxin B mixture was added to each well, giving a final concentration of 10 μg ml−1 HP-NAP and 1 μg ml−1 polymyxin B. Limulus endotoxin test performed before and after polymyxin treatment showed that the HP-NAP preparation contained less than 1.4 EU ml−1 of LPS corresponding to 70 pg ml−1E. coli LPS, far less than the LPS concentration needed for induction of transendothelial migration (10,000–25,000 pg ml−1) [23]. Polymyxin itself had no effect on transendothelial migration (data not shown).

Thus, our findings show that rNAP is a potent inducer of transendothelial migration. We next sought to deplete Hel 312 culture filtrate of HP-NAP using magnetic beads coated with a mAb against HP-NAP. This treatment dramatically reduced the capacity of the filtrate to induce neutrophil transendothelial migration (Fig. 3B). In fact, the HP-NAP depleted filtrate induced a 50% reduction in migration (p < 0.001) compared to the untreated culture filtrate. Beads coated with a mAb directed against the Vibrio cholerae toxin B subunit were used as a negative control and had no effect on the capacity to induce transendothelial migration (Fig. 3B). The removal of HP-NAP from culture filtrates is shown as a radiographic image of a western blot gel in Fig. 3C.

3.3Culture filtrates from HP-NAP deletion mutants but not live mutants have decreased ability to recruit neutrophils

To further assess the importance of HP-NAP, we first tried to create an HP-NAP mutant in strain Hel312, but this strain proved impossible to transform, possibly because it is a clinical isolate. We therefore constructed isogenic HP-NAP deletion mutants from strains P1 and P12. There were no significant differences between the wild-type and the mutant strains when analyzing the induction of neutrophil transmigration by intact bacteria (data not shown). However, when the effect of culture filtrates was examined, filtrates produced by the two HP-NAP mutants were reproducibly found to induce approximately 30% less (p= 0.0025, and 0.0286, respectively) neutrophil migration compared to culture filtrates obtained from wild-type bacteria (Fig. 4).

image

Figure 4. Induction of neutrophil transendothelial migration by culture filtrates from HP-NAP deficient mutants. Culture filtrates from H. pylori wild-type (P1 or P12) or HP-NAP deficient mutants (P1Δnap or P12Δnap) were added to the lower wells of Transwell cultures and 106 neutrophils to the upper wells. Bars indicate geometric mean + SEM of percentage of transmigrated neutrophils expressed relative to the migration induced by the respective wild type strains. Statistical evaluation was performed by the Mann–Whitney test. *p < 0.05 and **p < 0.01. Data are obtained in triplicates from at least three independent experiments.

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3.4HP-NAP does not activate endothelial cells

We then asked whether the effect of HP-NAP was mediated by a direct interaction with the neutrophils or if it was a secondary effect mediated by the endothelial cells. For this purpose, we measured the production of the neutrophil-recruiting chemokines IL-8, and GRO-α, and surface expression of ICAM-1, VCAM-1 and E-selectin by endothelial cells after incubation with 10 μg ml−1 recombinant HP-NAP for 6.5 and 24 h, respectively. This treatment had no effect on chemokine secretion or adhesion molecule expression (data not shown). In addition, the P1 wild-type and P1Δnap mutant were used for endothelial stimulation. Both the wild-type and mutant strains induced a slightly increased production of IL-8 (Fig. 5) and increased expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin compared to unstimulated controls (Fig. 5). Since the effect of the HP-NAP deficient mutant was indistinguishable from that of the wild-type bacteria, the results indicate that HP-NAP exerts transmigratory effects on the neutrophils themselves rather than on the endothelial cells.

image

Figure 5. Effect of HP-NAP on endothelial cells. 5 × 108 live H. pylori P1 wild-type or HP-NAP deficient mutant P1Δnap were added to cultured endothelial cells and incubated for 6 h whereafter IL-8 production (A) was analyzed using ELISA and the expression of ICAM-1 (B), E-selectin (C) and VCAM-1 (D) was evaluated by flow cytometry. Data are obtained in triplicates from at least three independent experiments. Bars indicate geometric mean + SEM of percentage of transmigrated neutrophils. Statistical evaluation was performed by the student's t test. ***p < 0.001, **p < 0.01, and *p < 0.05. There was no significant difference between wild-type and mutant bacteria in any of the assays.

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Endothelial cells have a key function in the recruitment of neutrophils during inflammation due to their ability to produce cytokines and chemokines and expressing adhesion molecules relevant for transmigration. Circulating neutrophils in turn are sensitive to chemotactic factors such as bacterial peptides and chemokines produced at the site of inflammation. When encountering such stimuli, the neutrophils attach to the endothelium, leave the blood stream, and migrate towards increasing amounts of stimulus. During H. pylori infection, a pronounced and continuous recruitment of neutrophils to the inflamed gastric mucosa has been observed [1,2]. Indeed, neutrophil infiltration has been suggested to be one of the factors leading to mucosal damage and DU formation [3,4]. In a recent study, we showed that live H. pylori was able to activate endothelial cells to express several adhesion molecules and neutrophil recruiting CXC-chemokines [17]. In this study, using the transwell system where cultured endothelial cells creates two separate compartments allowing passage from one side to another only through the endothelial monolayer, we extend these findings by showing that live H. pylori as well as bacterial culture filtrates and purified HP-NAP can induce substantial transendothelial migration of human neutrophils in a manner independent of a functional cag PAI or VacA. Furthermore, the chemotactic effect occurred without prior triggering or other stimulatory signals.

Broom et al. [24] previously showed that H. pylori can synthesize and secrete formylated peptides into culture medium. In addition, a study by Craig et al. [25] showed that H. pylori secrete heat-stable and acid resistant neutrophil chemotactic factors into the culture medium, which have a molecular weight of less than 3 kDa, indicating that they might be the same peptides as those detected by Broom et al. [24]. In addition, Nielsen and Andersen [6] showed that sonicates from H. pylori contain chemotactic proteins with a molecular weight between 25 and 35 kDa. The latter results are in agreement with our finding that H. pylori culture filtrates induce extensive neutrophil transendothelial migration, and that heat treatment inhibits this activity, indicating that the stimulatory factor(s) are of proteinaceous nature. Previous studies [6] have shown chemotactic activity of H. pylori sonicates or water extracts, but this is the first study to demonstrate the actual release of chemotactic proteins from H. pylori to the environment.

HP-NAP has been shown to be chemotactic to human neutrophils [9], and we therefore depleted HP-NAP from the Hel 312 culture filtrate. This treatment substantially decreased their chemotactic effect. Furthermore, purified HP-NAP induced a large increase in neutrophil transendothelial migration. However, when we assessed the importance of HP-NAP using intact bacteria, there was no significant difference in the induction of migration between wild-type and HP-NAP mutant strains, whereas culture filtrates from the knock-out strains showed significantly reduced ability to attract neutrophils as compared to culture filtrates from wild-type H. pylori. These seemingly contradictory findings might stem from the fact that intact bacteria are more complex than culture filtrate in terms of protein composition – the former might contain other chemotactic factors in addition to HP-NAP. Furthermore, strain differences may explain why HP-NAP exhibits high levels of chemotactic activity in Hel 312 derived culture filtrates but only a lower level of activity in the P1 and P12 culture filtrates. We therefore postulate that P1 and P12 may release other chemotactic factors, which are not produced by Hel 312. It is interesting to note that a corresponding difference has recently been observed when analyzing the effect of VacA blocking of NF-AT in T cells [26]. In this system, while supernatants from wild-type bacteria and not vacA mutants inhibited T cell proliferation, no difference was observed with either live wild-type H. pylori or vacA mutants.

In the original article, Evans et al. [5] describe increased adherence of neutrophils to HUVEC after incubation with H. pylori water extracts, an effect that could partly be blocked by antibodies directed against HP-NAP. However, we could not demonstrate that purified HP-NAP activates the HUVEC to express adhesion molecules or chemokines. Furthermore, there was no difference in the induction of endothelial activation molecules between wild-type and HP-NAP mutant H. pylori in our system. These findings suggest that HP-NAP is transported across the endothelial monolayer and acts directly on the neutrophils, which could in turn influence the endothelial cells to promote transmigration. We have thus extended previous studies on HP-NAP-induced chemotaxis [9] showing that this protein is highly effective in inducing transendothelial migration of neutrophils. Indeed, our findings indicate that HP-NAP is one of the major inducers of transendothelial migration of neutrophils present in H. pylori culture filtrates. However, we do not discount the possibility that additional chemotactic H. pylori products could act in parallel, or in synergy with HP-NAP in mediating neutrophil chemotaxis in vivo. Besides, chemokines such as IL-8 and ENA-78, which have been detected in H. pylori positive individuals, are also likely to contribute to neutrophil recruitment [27,28].

Our results also showed that sufficient amounts of HP-NAP to induce transendothelial migration were released into the culture medium by live H. pylori within only 6 h of incubation. It is not known if HP-NAP can gain access to the lamina propria during infection in vivo. Given that H. pylori urease is released in a similar fashion, and can be detected in the infected gastric tissues, we propose that HP-NAP might be able to penetrate into the lamina propria. In addition, active transport of HP-NAP across epithelial monolayers has recently been demonstrated in vitro [29]. Our laboratory has recently shown that expression of HP-NAP is much higher in vivo than in vitro, suggesting that HP-NAP is an important factor in H. pylori induced gastritis [30]. Furthermore, no clinical isolate of H. pylori lacking the gene encoding HP-NAP has been reported, further supporting the postulation that this protein confers a selective advantage for the bacteria in vivo.

In conclusion, H. pylori have the ability to induce significant transendothelial migration of neutrophils through a monolayer of human endothelial cells. Live bacteria and bacterial filtrates may induce neutrophil migration in different manners. HP-NAP thus may play a significant role in neutrophil migration when present in bacterial filtrates. Therefore, we suggest that in vivo where live bacteria in the lamina propria are scarce, HP-NAP may be a major factor contributing to the continuous recruitment of neutrophils to the gastric mucosa in H. pylori-associated gastritis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We thank Dr. Susanne Nyström for kindly providing the rNAP used in this study, Dr. Mattias Magnusson for help with western blot analyses, and Dr. Terry Kwok for critical reading of the manuscript. This study was supported by grants from the Swedish Science Council (Grant No. 06X–13428) and the Faculty of Medicine at Göteborg University.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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