Glycobiology, 2000, Vol. 10, No. 11 1171-1181
© 2000 Oxford University Press
Inhibition of nonopsonic Helicobacter pylori–induced activation of human neutrophils by sialylated oligosaccharides
Institute of Medical Biochemistry, Göteborg University, P.O. Box 440, SE 405 30 Göteborg, Sweden, and 2Department of Clinical Microbiology and Immunology, Örebro Medical Centre Hospital, SE 701 85 Örebro, Sweden
Received on February 11, 2000; revised on June 15, 2000; accepted on June 21, 2000.
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Certain strains of Helicobacter pylori have nonopsonic neutrophil-activating capacity. Some H.pylori strains and the neutrophil-activating protein of H.pylori (HPNAP) bind selectively to gangliosides of human neutrophils. To determine if there is a relationship between the neutrophil-activating capacity and the ganglioside-binding ability, a number of H.pylori strains, and HPNAP, were incubated with oligosaccharides, and the effects on the oxidative burst of subsequently challenged neutrophils was measured by chemiluminescence and flow cytometry. Both by chemiluminescence and flow cytometry a reduced response was obtained by incubation of H.pylori with sialic acid–terminated oligosaccharides, whereas lactose had no effect. The reductions obtained with different sialylated oligosaccharides varied to some extent between the H.pylori strains, but in general 3'-sialyllactosamine was the most efficient inhibitor. Challenge of neutrophils with HPNAP gave no response in the chemiluminescence assay, and a delayed moderate response with flow cytometry. Preincubation of the protein with 3'-sialyllactosamine gave a slight reduction of the response, while 3'-sialyllactose had no effect. The current results suggest that the nonopsonic H.pylori–induced activation of neutrophils occurs by lectinophagocytosis, the recognition of sialylated glycoconjugates on the neutrophil cell surface by a bacterial adhesin leads to phagocytosis and an oxidative burst with the production of reactive oxygen metabolites.
Key words: lectinophagocytosis/Helicobacter pylori/neutrophil activation/sialylated oligosaccharides/neutrophil gangliosides
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Introduction |
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There is convincing evidence that infection with Helicobacter pylori plays a major role in the development of chronic superficial gastritis, peptic ulcer disease (PUD), atrophic gastritis and gastric cancer (Kuipers, 1997
). It is one of the world’s most common bacterial infections, however only a minority of infected individuals will develop clinically overt gastroduodenal disease. The reasons for this are unclear. In addition to the heavy urease production and flagella for swimming through the mucus layer, factors present in all clinical isolates and commonly supposed to be necessary for the colonization of the stomach and for survival in this hostile environment (Labigne et al., 1991; Leyning et al., 1992), there is increasing evidence that some strains are more virulent than others. The expression of two major phenotypic markers have attracted most attention; vacuolating cytotoxin (VacA), a protein with a molecular weight of 87 kDa and determined by particular alleles of the vacA gene (Atherton et al., 1997; van Doorn et al., 1998), and CagA, a protein with a molecular weight of 116–120 kDa and determined by particular alleles of the so-called cag pathogenicity island (Crabtree et al., 1991; Censini et al., 1996). These two markers are generally considered virulence factors as expression of one or the other or both have been associated with PUD and gastric cancer (Covacci et al., 1993; Blaser et al., 1995), but there are also studies with conflicting results (Ito et al., 1997; Pan et al., 1997).
Particular strains of H.pylori have nonopsonic neutrophil activating capacity (NAC). Since such strains are more often isolated from individuals with PUD than from persons with chronic gastritis only, the NAC might be considered a virulence factor (Rautelin et al., 1993). H.pylori strains with the NAC marker are also associated with more severe inflammation in patients with PUD or gastritis (Rautelin et al., 1996; Hansen et al., 1999). The NAC can occur independently of cagA and/or VacA (Rautelin et al., 1994; Crabtree et al., 1995a). However, coexpression of NAC and cagA, or of NAC, cagA and VacA, enhances the association to PUD (Crabtree et al., 1995a; Danielsson et al., 2000). Infection with proinflammatory cagA positive strains will enhance the production of interleukin-8 (IL-8) in gastric epithelium (Crabtree et al., 1995b). As interleukin-8 is the strongest chemoattractant for neutrophils known (Crabtree, 1998) infection with cagA and NAC positive H.pylori will lead to activation of neutrophils to an oxidative burst with the production of reactive oxygen metabolites (ROM) and release of biologically active enzymes. These factors may be of importance for tissue damage and might explain that coexpression of cagA and NAC will enhance the association to PUD (Crabtree et al., 1995a; Danielsson et al., 2000).
The factor(s) of NAC positive H.pylori responsible for the activation of neutrophils, as determined by luminol-enhanced chemiluminescence (CL), are heat-labile and dependent of whole non-disintegrated organisms, that is, the oxidative burst of neutrophils was not demonstrated after heating the organisms at 50°C or after disintegration by sonication (Rautelin et al., 1993). Its relation to the neutrophil-activating protein of H.pylori, identified by Yoshida et al. (1993) and termed HPNAP by Evans et al. (1995), is unclear. This protein upregulates CD11b/CD18, induces adhesion of neutrophils to endothelial cells and production of reactive oxygen radicals as determined by nitro-benzo-tetrazolium. HPNAP binds to glycosphingolipids with terminal NeuAc
3Galß4GlcNAcß3Galß4GlcNAcß sequence present in human neutrophils (Teneberg et al., 1997). The role of HPNAP to predict clinical outcome has not been reported.
Certain H.pylori strains bind to gangliosides, polyglycosylceramides and glycoproteins of human neutrophils (Miller-Podraza et al., 1999). A terminal 3-linked sialic acid is pivotal for this interaction (Johansson and Karlsson, 1998). Sialylated carbohydrate receptors might be involved in the nonopsonic activation of neutrophils by H.pylori, which may be a corollary to the lectinophagocytosis, i.e, interactions between bacterial lectins and phagocyte cell surface glycoconjugates leading to attachment of nonopsonized bacteria to phagocytic cells, followed by phagocytosis. This has been described for type 1-fimbriated Escherichia coli and piliated Neisseria gonorrhoeae with PII opacity associated outer membrane proteins (Öhman et al., 1982; Rest et al., 1985; Ofek and Sharon, 1988).
The aim of the present study was to determine whether lectin–carbohydrate interactions are involved in the nonopsonic H.pylori–induced activation of human neutrophils. A number of nonopsonized or opsonized H.pylori strains, and HPNAP, were incubated with oligosaccharides, and the effects on the oxidative burst of subsequently challenged neutrophils was measured by chemiluminescence and flow cytometry.
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Glycosphingolipid binding assays
The results from binding of 35S-labeled H.pylori to glycosphingolipids on thin-layer chromatograms (Figure 1) are summarized in Table I. All strains bound to the reference gangliotriaosylceramide (lane 3) as reported previously (Ångström et al., 1998
). The reference strain NCTC 11637 (Figure 1B) also bound selectively to slow-migrating gangliosides of human neutrophils (lane 1), in line with previous reports (Johansson and Karlsson, 1998; Miller-Podraza et al., 1999). An identical binding pattern was obtained with the clinical isolate S-032 (not shown). Binding of the strains S-002, S-008, and C-7050 to slow-migrating gangliosides of human neutrophils was also occasionally obtained (exemplified in Figure 1C). However, the binding of these three strains was considerably weaker, although the same amounts of bacteria and radioactivity were used.
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As reported previously (Teneberg et al., 1997
) the neutrophil-activating protein of H.pylori had a more restricted binding pattern than the bacterial cells, and selectively recognized two minor ganglioside double bands in the ganglioside fraction of human neutrophils (Figure 1D).
Effects of oligosaccharides on the oxidative burst
Challenge of human neutrophils with H.pylori type I strains, NCTC 11637 and S-032, resulted in a rapid and strong chemiluminescence (CL) response (exemplified in Figures 2 and 3). Similar responses were obtained with the intermediate type strains S-002 and S-008, while the type II strain C-7050 gave no detectable response. The findings with CL were confirmed with flow cytometry (FC) of hydroethidine-loaded neutrophils (exemplified in Figures 4 and 5). Microscopy of acridine orange stained slides of the mixtures of H.pylori and neutrophils showed a rapid attachment and phagocytosis (within 2–5 min) of both the type I and the intermediate type NAC positive strains. After 15–30 min, there was also an obvious agglutination of the neutrophils with large numbers of phagocytosed organisms (data not shown).
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The Cl responses obtained after incubation of NAC positive H.pylori strains with 3'- or 6'-sialyllactose, or with other sialylated oligosaccharides (see Table II for the structures of the saccharides used in the inhibition assays), were reduced as compared with the reference (neutrophils and H.pylori only) with regard to the measured peak values (mV) and the times to reach the peak (min). Incubation with lactose at corresponding molarities gave no inhibitory effects (data not shown). The results are summarized in Tables III and IV, and typical experiments exemplified in Figures 2 and 3.
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The reductions of the CL responses were dose dependent with the tested oligosaccharide concentrations; 36–50% inhibition with 1 mM sialyllactose versus 11–21% with 0.1 mM concentrations (Figure 2, Table III). When using concentrations of 3'-sialyllactose >1 mM only marginal increases of the inhibitory effect was obtained. Preincubation of the NAC positive strains NCTC 11637 and S-002 with 10 mM lactose had only minimal inhibitory effect (Table IV).
As can be seen from Table IV there were some variations in the inhibitory effects both with regard to H.pylori strains and sialylated oligosaccharides used. Thus, 3'-sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine had approximately the same inhibitory capacity for the NCTC 11637 strain, in terms of lower peak values and delayed time to reach the peak. However, examination of the same parameters for the strains S-032 and S-002 showed that 3'-sialyllactosamine was the most potent inhibitor of the oxidative burst induced by these strains.
Sialyl-Lex also had inhibitory capacity, but the effect was less pronounced than the effects of 3'-sialyllactose and 3'-sialyllactosamine.
The inhibitory effects of the CL responses by sialylated oligosaccharides were confirmed with FC of HE-loaded neutrophils challenged with NAC positive H.pylori strains. With this technique the maximal response is reached after 15–30 min because it only measures the internal response, whereas the CL measures both the external and internal ones. After 40–45 min the responses correspond to the start values. The measurements given in the Figures were therefore restricted to 0 min, 15 min, and 30 min. A rapid response within 15 min was obtained when using live H.pylori organisms in this assay (Figure 4A). Again, 3'-sialyllactosamine was the most effective inhibitor for the S-032 strain, and at 1 mM gave 
50% reduction of the FC response at 30 min (Figure 5A). In this assay 3'-sialyllactose and 3'-sialyllactosamine gave the most pronounced reductions of the FC responses induced by the strain NCTC 11637 (50% and 60% inhibition at 1 mM; Figure 5B), while 1 mM 6'-sialyllactose and 6'-sialyllactosamine gave 30% and 25% reduction, respectively (Figure 5C).
No CL response was demonstrated after challenge of neutrophils with HPNAP (data not shown). However, a moderate response was demonstrated at 30 min with FC of HE-loaded neutrophils (Figure 4B). This was in contrast with whole and live H.pylori organisms which in this assay, as with CL, gave responses within 15 min. Preincubation of HPNAP with different oligosaccharides resulted in only slight inhibitions; 25% with 3'-sialyllactosamine and 10% with 3'-sialyllactose.
Opsonized organisms of the type II NAC negative strain C-7050 induces, in contrast to nonopsonized organisms, a rapid and relatively strong oxidative burst of challenged neutrophils, as measured with CL or FC. Preincubation of such opsonized organisms with different oligosaccharides resulted in only slight inhibitions, that is, <10% at 1 mM concentrations (Figure 6A), and none at all with lower concentrations.
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Challenge of neutrophils with fMLP or PMA, two well documented activators of neutrophils, resulted in rapid CL responses which was confirmed with FC of HE loaded neutrophils. Preincubation of fLMP or PMA with the sialylated oligosaccharides resulted in only slight inhibitions; <10% in assays with fMLP and approximately 15–20% with PMA (exemplified in Figure 6B).
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Discussion |
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Adherence of H.pylori to the gastric epithelium is considered to be a virulence factor facilitating the colonization of the stomach, and several different receptor candidates have been described. Thus, the binding of H.pylori to such diverse compounds as sialic acid–containing glycoconjugates (Evans et al., 1988
), phosphatidylethanolamine and gangliotetraosylceramide (Lingwood et al., 1992), the Leb blood group determinant (Borén et al., 1993), heparan sulfate (Ascencio et al., 1993), sulfatide (Saitoh et al., 1991), lactosylceramide (Ångström et al., 1998), and polyglycosylceramides (Miller-Podraza et al., 1996) has been documented. Several of these putative receptors, for example, the Leb blood group determinant, sulfatide, and lactosylceramide, have been identified in the epithelial cells of human stomach. However, the human gastric epithelium has a low content of sialylated glycoconjugates (Filipe, 1979; Madrid et al., 1990). One current hypothesis is that the sialic acid binding capacity is used by the bacterium for interactions with neutrophils (Karlsson, 1998). Interactions with inflammatory cells may appear contradictory in terms of bacterial survival. However, despite the inflammatory response and the local and systemic immune response H.pylori infection is not eliminated, and it has been suggested that the products of the inflammatory response are used by the bacterium as a nutritional source (Blaser, 1996). As a matter of fact H.pylori survives the non-opsonic phagocytosis by neutrophils (Rautelin et al., 1993) All H.pylori strains tested in this study had the capacity to interact with slow-migrating gangliosides of human neutrophils. However, the NAC positive type I strains NCTC 11637 and S-032 gave a strong and highly reproducible binding, while the binding obtained with the NAC positive intermediate type strains S-002 and S008 and the NAC negative type II strain C-7050 was considerably weaker and more infrequent. The reasons for this are unclear. It might be due to variations in the expression of the factor(s) responsible for the interactions, but no definite conclusions can be drawn until more strains have been investigated at molecular and genetic levels.
While the bacterial cells interacted with a number of slow-migrating gangliosides of human neutrophils, the binding of the neutrophil-activating protein of H.pylori was restricted to two minor ganglioside double bands.
The gangliosides of human neutrophils are a complex mixture (Fukuda et al., 1985; Müthing et al., 1996; Stroud et al., 1996a,b). Apart from the GM3 ganglioside, the acid glycosphingolipids have one or several N-acetyllactosamine moieties, where one or more of the N-acetylglucosamines may be substituted with 3-linked fucose(s). The terminal sialic acid may be 6- or 3-linked. Sialylated N-acetyllactosamine sequences are also found in the glycoproteins of human neutrophils (Fukuda, 1985).
Preincubation of NAC positive H.pylori with sialic acid-terminated oligosaccharides resulted in a reduced and delayed CL response, confirmed with FC of HE loaded neutrophils, whereas lactose had no inhibitory effect. The reductions obtained with sialylated oligosaccharides varied to some extent between the different H.pylori strains which might have a bearing to the observed variations of their interactions with slow-migrating gangliosides in the glycosphingolipid binding assays. On the whole, however, 3'-sialyllactosamine was the most efficient inhibitor. There was no obvious relation to the presence or absence of cagA.
Due to the heterogeneity of human neutrophil gangliosides, chemical determination of the precise H.pylori–binding sequence(s) is difficult to achieve. Since the most effective sialylated oligosaccharides used were not able to completely inhibit the H.pylori–induced oxidative burst, it is, however, important to find out the structure of the binding-active sequences of these slow-migrating gangliosides to allow the design of more efficient inhibitors.
A non-optimal composition of the saccharides used may be the reason for the modest inhibition of the HPNAP-induced oxidative burst measured by FC. The H.pylori neutrophil-activating protein binds to NeuAc3Galß4GlcNAcß3Galß4GlcNAcß-terminated gangliosides of human neutrophils, but does not recognize gangliosides with a single N-acetyllactosamine unit as sialylneolactotetraosylceramide NeuAc3Galß4GlcNAcß3Galß4Glcß1Cer (Teneberg et al., 1997). A slight inhibition was, however, observed when the protein was incubated with 3'-sialyllactosamine, and production of NeuAc3Galß4GlcNAcß3Galß4GlcNAcß saccharides for testing of inhibitory power is now underway.
The findings of the present study suggest that the nonopsonic H.pylori–induced activation of neutrophils is a corollary to lectinophagocytosis, that is, a bacterial protein ligand-neutrophil carbohydrate interaction leading to phagocytosis and an oxidative burst with the production of ROM. They also suggest that sialylated glycoconjugates in human neutrophils might act as receptors for H.pylori. This is further supported by the observation that the sialylated oligosaccharides had no obvious inhibitory effect when neutrophils were activated with fMLP or PMA. It has been shown that fMLP is an agonist that binds to G-protein coupled membrane receptors that mediates the formation of inositol-1,3,4-triphosphate and diacylglycerol which activate protein kinase C (Nakamura and Nischizuka, 1991). PMA directly stimulates protein kinase C (Merritt et al., 1993), and in this way, both activate neutrophils to a rapid oxidative burst.
The specificity of the inhibitory effects of the sialylated oligosaccharides on the NAC of H.pylori was supported by the experiments with opsonized organisms of the NAC negative type II strain C-7050. No inhibition was demonstrated which indicates that the NAC receptor is separate from the C3-receptor of neutrophils for opsonized bacteria.
In parallel with the chemiluminescence and flow cytometry experiments, the effects of oligosaccharides on the H.pylori–induced interleukin-8 production by the AGS gastric cell line were determined. The tested H.pylori strains (NCTC 11637, S-032 and S-008) induced significant production of IL-8 (800–1700 pg/ml) in AGS cells. However, no inhibitory effect was obtained by preincubation of the bacteria with the different sialylated oligosaccharides (data not shown).
In binding studies using solid-phase immobilized glycosphingolipids H.pylori binds to 3'-sialylneolactotetraosylceramide, but does not recognize 6'-sialylneolactotetraosylceramide (Johansson and Karlsson, 1998). However, in the present study an inhibitory effect was obtained both with 3'-sialyllactosamine and 6'-sialyllactosamine. The reason for this is unclear. However, similar discrepancies have been described in lectin hemagglutination inhibition studies, where the best hapten inhibitor might differ from the membrane-bound receptor recognized by the lectin (Sharon and Lis, 1989), since in solution the flexible saccharides might mimic the structure involved in the binding of the lectin to the cell surface.
The neutrophil activating protein (Yoshida et al., 1993), labeled HPNAP (Evans et al., 1995), has been characterized in molecular and genetic terms and shown to be an iron-binding protein belonging to the ferritin family (Tonello et al., 1999). It has been shown to induce expression of integrins, promote adhesion of neutrophils to endothelial cells and production of reactive oxygen radicals (Evans et al., 1995). In our hands the kinetics of HPNAP and NAC of whole and live H.pylori organisms differ when activation of neutrophils was studied with luminol-enhanced CL and FC of HE loaded neutrophils. No oxidative burst was demonstrated with CL for HPNAP, and only a late and relatively modest response with FC. This might be due to too low concentrations of available HPNAP or to hydrolytic degradation during storage, but it might also suggest that HPNAP and NAC may represent two different components of H.pylori. To prove or disprove this the component(s) responsible for the NAC of H.pylori have to be identified and characterized in molecular and genetic terms. Work is now in progress to pursue this.
Sialylated oligosaccharides cause inhibition of the attachment of H.pylori to human gastrointestinal cell lines, and also detachment of adherent bacteria (Simon et al., 1997). In that study, 3'-sialyllactose was the most effective inhibitor while 6'-sialyllactose was less active, and multivalent presentation, 3'-sialyllactose coupled to human serum albumin, resulted in an increased inhibitory effect. In addition, suppression of infection with 3'-sialyllactose in H.pylori–infected rhesus monkeys was recently demonstrated (Mysore et al., 1999). In our study sialylated oligosaccharides reduced the oxidative burst of H.pylori activated neutrophils. The use of sialylated oligosaccharides thus suggests a possible way to interfere with several steps in the pathogenesis of the H.pylori infection. The mechanisms are truly complex, but the activation of neutrophils to oxidative bursts with the production of ROM and release of biologically active enzymes may be crucial for the induction of tissue damages during the infectious process. Inhibition of the activation of neutrophils and reduction of the oxidative burst might therefore be beneficial for preventing such effects.
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Oligosaccharides and other chemicals
Lactose was from J. T. Baker Chem. Co., Phillipsburg, NJ. 3'-sialyllactose and 6'-siallyllactose were purchased from IsoSep, Tullinge, Sweden. Sialyl-Lex hexasaccharide, 3'-sialyllactosamine and 6'-sialyllactosamine were from Dextra Lab. Ltd., Reading, UK. The structures of the oligosaccharides used are summarized in Table I. Formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA) were purchased from Sigma, St. Louis.
Helicobacter pylori strains, culture conditions and labeling
The type I strain NCTC 11637 (CagA/cagA+ and VacA+) with NAC, and the type II strain C-7050 lacking these markers, including NAC, were used as reference strains in parallel with the Swedish clinical isolates S-002 (cagA+/VacA-/NAC+), S-008 (cagA–/VacA+/NAC+), and S-032 (cagA+/VacA+/NAC+). According to definitions (Xiang et al., 1995) S-032 is a type I strain, S-002 and S-008 intermediate types. Two of the patients had a diagnosis of PUD (S-002 and S-008) and one patient chronic active gastritis (S-032). The strains were grown in a microaerophilic atmosphere at 37°C for 48 h on brain heart infusion agar with 10% horse blood for 2 or 3 days, alternatively on GC agar plates (GC II agar base; BBL, Cockeysville, MD) supplemented with 1% bovine hemoglobin (BBL), 10% horse serum, and 1% IsoVitalex (BBL), without antibiotics.
For experiments with chemiluminescence and flow cytometry, H.pylori organisms were collected in 0.01 M phosphate buffered 0.15 M saline, pH 7.4 (PBS) and used as described below. Anti–H.pylori positive serum (determined with PylorisetR IgG) was obtained from a member of our staff and used for experiments with opsonized organisms as described below.
The conditions for culture and 35S-labeling of the bacteria were as described previously (Ångström et al., 1998). For binding assays, the bacteria were suspended to 1 x 108 CFU/ml in PBS. The specific activities of the suspensions were approximately 1 c.p.m. per 100 H.pylori organisms.
Neutrophil-activating protein of Helicobacter pylori
Recombinant HPNAP was obtained by the courtesy of Dr. Evans, Houston, Texas. It was kept frozen at –70°C until used.
Chromatogram binding assay
Glycosphingolipids were isolated and characterized by mass spectrometry, 1H NMR, and degradation studies, as described previously (Karlsson, 1987).
Thin-layer chromatography was performed on glass- or aluminum-backed silica gel 60 HPTLC plates (Merck, Darmstadt, Germany). Mixtures of glycosphingolipids (40 µg) or pure compounds (1–4 µg) were separated using chloroform/methanol/0.25% aqueous KCl (50:40:10, by volume) as solvent system. Chemical detection was accomplished by anisaldehyde (Waldi, 1962).
Binding of 35S-labeled H.pylori to glycosphingolipids on thin-layer chromatograms was done as reported previously (Ångström et al., 1998). Dried chromatograms were dipped for 1 min in diethylether/n-hexane (1:5, by volume) containing 0.5% (w/v) polyisobutylmethacrylate (Aldrich Chemical Co., Milwaukee, WI). After drying, the chromatograms were soaked in PBS containing 2% bovine serum albumin (w/v), 0.1% NaN3 (w/v) and 0.1% Tween 20 (v/v) for 2 h at room temperature. The chromatograms were subsequently covered with radiolabeled bacteria diluted in PBS (2–5 x 106 c.p.m./ml). Incubation was done for 2 h at room temperature, followed by repeated washings with PBS. The chromatograms were thereafter exposed to XAR-5 x-ray films (Eastman Kodak, Rochester, NY) for 12 h.
Binding assays with 125I-labeled HPNAP was done as described previously (Teneberg et al., 1997).
Human neutrophil granulocytes
Heparinized blood from healthy blood donors was used to prepare neutrophils by Ficoll-Pacque (Pharmacia/LKB, Uppsala, Sweden) centrifugation in accordance with the method of Böyum (1974), slightly modified as described (Rautelin et al., 1993). For each series of experiments on a particular day neutrophils were prepared and pooled from three blood donors of the same blood group (A Rh+ or O Rh+). Neutrophils were thus obtained from different blood donors at each experiment. They were suspended in PBS supplemented with MgCl2, CaCl2, glucose, and gelatin (PBS-GG) as described previously (Rautelin et al., 1993). The purity and viability of the neutrophils exceeded 95%.
Chemiluminescence, flow cytometry, and design of experiments
The oxidative burst of neutrophils challenged with whole and live H.pylori organisms, nonopsonized or opsonized, or with HPNAP, fMLP or PMA was measured with luminol enhanced CL as described previously (Rautelin et al., 1993, 1994; Crabtree et al., 1995; Danielsson and Jurstrand, 1998; Hansen et al., 1999). To each test tube (LKB, Bromma, Sweden) were added 300 µl of PBS-GG, 100 µl of neutrophils (5 x 106/ml) and either 50 µl of nonopsonized or opsonized H.pylori (5 x 108/ml), or 50 µl HPNAP (1.2 µg/ml), or 50 µl fMLP (3 x 10–6 M), or 50 µl PMA (10–6 M), and finally 50 µl of 10–5 M luminol (Sigma). For the inhibition experiments 50 µl of H.pylori (5 x 108/ml), or HPNAP (1.2 µg/ml), or fMLP (3 x 10–6 M), or PMA (10–6 M) were mixed with 50 µl of oligosaccharides at final concentrations of 0.1–1.0 mM for 15 min at 37°C, and 100 µl of the mixture was thereafter transferred to the test tube for CL measuring. The measurements with a luminometer (LKB Wallac 1251, Turku, Finland) were always started within 1 min after the bacterial suspension had been added. The assays were performed at 37°C, and CL from each sample was measured at 60–90 s intervals during a period of 30–45 min. This technique thus measures both the external and internal oxidative bursts of the nonopsonic phagocytosis by neutrophils, which was checked by quenching the external burst in the presence of catalase (2000 U/ml), and the internal one in the presence of azide (1 mM) and horseradish peroxidase (4 U/ml) as described by Lock and Dahlgren (1988). Positive and negative controls were included in each series of experiments.
Flow cytometry (FC) was also used to study the oxidative burst by the method described by Perticarari et al. (1994) The method is based on preincubating the neutrophils with hydroethidine (HE), which converts to ethidium bromide by the oxidative burst of neutrophils following phagocytosis, and emits red fluorescence. This technique thus measures only the internal oxidative burst of phagocytosing neutrophils. Briefly, 100 µl of neutrophils (3 x 106/ml) were preincubated with 20 µl HE (10 µg/ml) (Sigma) for 15 min at 37°C, after which 20 µl H.pylori organisms (1.5 x 108/ml), or 20 µl HPNAP (1.2 µg/ml), or 20 µl fMLP (3 x 10–6M), or 20 µl PMA (10–6 M), were added to the test tubes. For the inhibition experiments, oligosaccharides at final concentrations of 0.1–10 mM were added to H.pylori, HPNAP, fMLP or PMA, and after a period of 15 min at 37°C the mixtures were added to the neutrophils. The oxidative burst was measured with FACScan FC (Becton Dickinson). Data were collected and analyzed using the Lysis II software program (Becton Dickinson), and the fluorescence distribution was displayed as a single or 3-D histogram; alternatively, resting, that is, nontreated neutrophils were gated and the percentage of neutrophils with a burst was determined at time 0 and after 15 and 30 min (see Figure 4).
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This study was supported by the Swedish Medical Research Council (Grants Nos. 12628, 3967, and 10435), Lars Hiertas Memorial Foundation, the Swedish Cancer Foundation, the Wallenberg Foundation, and the Research Foundations of Örebro Medical Centre Hospital.
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CL, chemiluminescence; FC, flow cytometry; FCS, fetal calf serum; fMLP, formyl-methionyl-leucyl-phenylalanine; IL-8, interleukin-8; HE, hydroethidine; HPNAP, neutrophil-activating protein of Helicobacter pylori; NAC, neutrophil activating capacity; ROM, reactive oxygen metabolites; PMA, phorbol myristate acetate; PMNL, polymorphonuclear leukocytes; PUD, peptic ulcer disease. The glycosphingolipid nomenclature follows the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN for Lipids: Eur. J. Biochem. (1977) 79, 11–21; J. Biol. Chem. (1982) 257, 3347–3351; and J. Biol. Chem. (1987) 262, 13–18). It is assumed that Gal, Glc, GlcNAc, and NeuAc are of the D-configuration, Fuc of the L-configuration, and all sugars present in the pyranose form.
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1 To whom correspondence should be addressed
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References |
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