Interleukin-6 induction by Helicobacter pylori in human macrophages is dependent on phagocytosis

BACKGROUND: The colonization of the gastric mucosa with Helicobacter pylori is accompanied by elevated levels of proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, and IL-8. The aim of our study was to determine the mechanisms of IL-6 stimulation in phagocytes upon H. pylori infection. MATERIALS AND METHODS: We investigated the secretion of IL-6 by different professional phagocytes from murine and human origin, including granulocyte- and monocyte-like cells and macrophages derived from human peripheral blood monocytes (PBMCs). The influence of viability, phagocytosis, and the impact of different subcellular fractions of H. pylori bacteria were evaluated. RESULTS: IL-6 levels induced by H. pylori were low in cell lines derived from murine and human monocytes and in human granulocyte-like cells. By contrast, macrophages derived from human PBMCs were highly responsive to both H. pylori and Escherichia coli. IL-6 induction was blocked by inhibition of actin-dependent processes prior to infection with H. pylori, but not with E. coli or E. coli lipopolysaccharide (LPS). Using cell fractionation, the most activity was found in the H. pylori membrane. H. pylori LPS exhibited a 10(3)- to 10(4)-fold lower biologic activity than E. coli LPS, suggesting a minor role for toll-like receptor 4 (TLR4)-mediated signalling from the exterior. CONCLUSIONS: From these data, we conclude that macrophages may be a major source of IL-6 in the gastric mucosa upon H. pylori infection. The IL-6 induction by H. pylori in these cells is a multifactorial process, which requires the uptake and presumably degradation of H. pylori bacteria.     

Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+.     

Coronin function is required for chemotaxis and phagocytosis in human neutrophils

Coronins are a family of conserved actin-associated proteins that have been implicated in a variety of cellular processes dependent on actin rearrangements. In this study, we show that in primary human neutrophils, coronins-1-4 and -7 are expressed. Coronin-1 accumulates at the leading edge of migrating neutrophils and at the nascent phagosome. Inhibition of coronin function by transduction of a dominant-negative form of the protein leads to inhibition of chemotaxis and a reduction in neutrophil spreading and adhesion. This inhibition appears to correlate with changes in the distribution of F-actin structures within the cell. In addition, phagocytosis is inhibited, but neither secretion nor activation of the NADPH oxidase appears to be affected. Together, these results show that coronins are required for actin-dependent changes in cell morphology that lead to migration and phagocytosis.     

Oligomerization of Clostridium perfringens epsilon-toxin is dependent upon membrane fluidity in liposomes

Clostridium perfringens epsilon-toxin binds to receptors on MDCK cells and forms a heptamer in membranes. The mechanism behind the oligomerization of epsilon-toxin was studied using carboxyfluorescein (CF)-loaded liposomes composed of various phosphatidylcholines (PCs). The toxin caused CF to leak from liposomes in a dose-dependent manner. The toxin-induced leakage of CF, binding of the toxin to liposomes, and formation of a functional oligomer increased as the phase-transition temperature (Tm) of the PC used in the liposomes decreased. Surface plasmon resonance analysis using an HPA sensorchip (BIAcore) also revealed that the binding of the toxin to liposomes increased with a decrease in the Tm of the PC used in liposomes. The oligomer that was formed in 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID)-treated liposomes was labeled, indicating that it inserts into a hydrophobic region. Furthermore, the rate of epsilon-toxin-induced CF leakage was enhanced by treatment with phosphatidylethanolamine or diacylglycerol, which is known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. We show that membrane fluidity in the liposome plays an important role in the binding of the toxin to liposomes, insertion into the hydrophobic region in the bilayer of liposomes, and the assembly process in the bilayer.     

Piroxicam enhances phagocytosis of Escherichia coli by human polymorphonuclear neutrophils

Human polymorphonuclear neutrophils were exposed to piroxicam, 1.5 uM, 15 uM and 150 uM during phagocytosis of radiolabelled Escherichia coli in vitro. Preincubation of the cells for 30 minutes before phagocytosis stimulated the uptake of E. coli at all concentrations. The elimination of substances of bacterial origin from the neutrophis in the postingestion phase was, however, not influenced by piroxicam.     

Quantification of phagocytosis in human neutrophils by flow cytometry.

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.     

Nonopsonic phagocytosis of Mycobacterium kansasii by human neutrophils depends on cholesterol and is mediated by CR3 associated with glycosylphosphatidylinositol-anchored proteins

Receptors involved in the phagocytosis of microorganisms under nonopsonic conditions have been little studied in neutrophils. Complement receptor type 3 (CR3) is a pattern recognition receptor able to internalize zymosan and C3bi-coated particles. We report that Abs directed against CR3 strongly inhibited nonopsonic phagocytosis of Mycobacterium kansasii in human neutrophils. In these cells CR3 has been found associated with several GPI-anchored proteins localized in cholesterol-rich microdomains (rafts) of the plasma membrane. Cholesterol sequestration by nystatin, filipin, or beta-cyclodextrin as well as treatment of neutrophils with phosphatidylinositol phospholipase C to remove GPI-anchored proteins from the cell surface markedly inhibited phagocytosis of M. kansasii, without affecting phagocytosis of zymosan or serum-opsonized M. kansasii. Abs directed against several GPI-anchored proteins inhibited phagocytosis of M. kansasii, but not of zymosan. N:-acetyl-D-glucosamine, which is known to disrupt interactions between CR3 and GPI proteins, also strongly diminished phagocytosis of these mycobacteria. In conclusion, phagocytosis of M. kansasii involved CR3, GPI-anchored receptors, and cholesterol. In contrast, phagocytosis of zymosan or opsonized particles involved CR3, but not cholesterol or GPI proteins. We propose that CR3, when associated with a GPI protein, relocates in cholesterol-rich domains where M. kansasii are internalized. When CR3 is not associated with a GPI protein, it remains outside of these domains and mediates phagocytosis of zymosan and opsonized particles, but not of M. kansasii.     

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils

Neutrophils are initially the predominant cells involved in the host defence of bacterial infections, including periodontal disease. Aggressive periodontitis is associated with Actinobacillus actinomycetemcomitans, a Gram-negative capnophilic microorganism. Infections caused by A. actinomycetemcomitans are not resolved by the host immune response despite the accumulation of neutrophils at the site of inflammation. To better understand the role of natural host defence mechanisms in A. actinomycetemcomitans infections, the interaction of phenotypically diverse strains of this pathogen with human neutrophils was assessed directly using techniques such as genetic labelling with the gene for green fluorescent protein, fluorescence-activated cell sorting and fluorescence imaging. The study included clinical isolates of A. actinomycetemcomitans represented by self-aggregating, biofilm-associated and isogenic planktonic variants. Data obtained showed that complement-mediated phagocytosis of A. actinomycetemcomitans was generally inefficient regardless of strain-specific serotype or leukotoxin production. Furthermore, the majority of ingested bacteria remained viable after exposure to neutrophils for 1 h. Interestingly, uptake of antibody-opsonized bacteria resulted in the rapid cell death of neutrophils. This was in contrast to ingestion of complement-opsonized bacteria, which did not affect neutrophil viability. The methods used in this study provided reliable and reproducible results with respect to adherence, phagocytosis and killing of A. actinomycetemcomitans when encountering human neutrophils.     

Effect of lipid physical state on the rate of peroxidation of liposomes.

The effect of cholesterol on the rate of peroxidation of arachidonic acid and 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) in dimyristoylphosphatidylcholine (DMPC) liposomes was examined above and below the phase transition temperature (Tm) of the lipid. The rate of peroxidation of arachidonic acid was more rapid below the phase transition temperature of the host lipid. At a temperature below the Tm (4 degrees C), increasing concentrations of cholesterol reduced the rate of peroxidation of arachidonic acid as judged by the production of thiobarbituric acid reactive substances. Above Tm (37 degrees C), cholesterol increased the rate of peroxidation of the fatty acid. Similarly, PAPC was peroxidized more rapidly at 4 degrees C than at 37 degrees C. However, cholesterol had little effect on the rate of peroxidation of PAPC at 4 degrees C. The rate of peroxidation of arachidonic acid was related to the lipid bilayer fluidity as judged by fluorescence anisotropy measurements of diphenylhexatriene. The rate of peroxidation increased slowly with increasing rigidity of the probe environment when the bilayer was relatively fluid and more rapidly as the environment became more rigid. The increase in the rate of peroxidation of arachidonic acid in the less fluid host lipid was unrelated to differences in iron binding or to transfer of arachidonic acid to the aqueous phase. Decreasing the concentration of arachidonic acid in DMPC to less than 2 mol% dramatically decreased the rate of peroxidation at 4 degrees C, suggesting that formation of clusters of fatty acids at 4 degrees C is required for rapid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of osmotic gradient on the physical properties of membrane lipids in liposomes

Osmotic swelling of multilamellar liposomes was shown to produce structural changes in liposomal membranes. Creation of transmembrane osmotic gradients results in an increased lateral mobility of membrane incorporated hydrophobic probe, pyrene. This is accompanied by some decrease in order parameter of membrane phospholipid polar head group regions. The perturbations are more marked in hydrophobic than in polar regions of the membranes. It is suggested that some functional changes in biomembranes arising upon osmotic swelling may be associated with structural alterations similar to those observed in liposomes.     

Halogenation and proteolysis of complement component C3 on Salmonella typhimurium during phagocytosis by human neutrophils

We examined the fate of C component C3 on the surface of Salmonella typhimurium during ingestion by human neutrophils. Initial experiments showed that C3 fragments and C3-acceptor complexes were the major serum ligands which were surface iodinated by canine myeloperoxidase on serum-incubated rough and smooth isolates of S. typhimurium. In contrast, labeled C3 was not identified when the same organisms were ingested by neutrophils in the presence of 125I-Na, a situation previously shown to iodinate particulate targets via the neutrophil myeloperoxidase-halide-H2O2 system. Pretreatment of neutrophils before phagocytosis with the lipid-soluble protease inhibitor diisopropylfluorophosphate (DFP), but not with other protease inhibitors (p-nitrophenylguanidinobenzoate, leupeptin, pepstatin), substantially blocked proteolysis of 125I-C3 on S. typhimurium strain RG108 during ingestion by neutrophils. Purification of neutrophil phagosomes containing S. typhimurium-bearing 125I-C3 showed that DFP but no other protease inhibitors blocked proteolysis of 125I-C3 within phagosomes. Iodinated C3-acceptor complexes were identified by immunoprecipitation from the detergent-insoluble fraction of phagosomes prepared from DFP-treated cells ingesting S. typhimurium in the presence of 125I-Na. These results show that C3 fragments on the surface of S. typhimurium are the major serum ligands which are halogenated and degraded by proteolysis during phagocytosis by human neutrophils, and suggest that the majority of proteolysis on the ingested target occurs within the neutrophil phagosome.     

Mitochondrial membrane potential is dependent on the oligomeric state of F1F0-ATP synthase supracomplexes

The F1F0-ATP synthase in mitochondria, in addition to its function in energy transduction, has a structural role in determining cristae morphology. This depends on its ability to form dimeric and higher oligomeric supracomplexes. Here we show that mutants of the dimer-specific subunits e and g, which destabilize dimeric and oligomeric F1F0-ATP synthase supracomplexes, have a decreased mitochondrial membrane potential delta psi. The degree of destabilization correlated with the reduction of the membrane potential. The enzymatic activities of F1F0-ATP synthase and cytochrome c oxidase, maximal respiration rate, coupling of oxidative phosphorylation, and tubular mitochondrial morphology were not affected or only to a minor extent. In mutants lacking one or two coiled-coil domains of subunit e, the reduction of the mitochondrial membrane potential was not due to loss of mitochondrial DNA, a reduced capacity of oxidative phosphorylation, or to altered cristae morphology. We propose a role for the supracomplexes of the F1F0-ATP synthase in organizing microdomains within the inner membrane, ensuring optimal bioenergetic competence of mitochondria.     

Degradation of glomerular basement membrane by human neutrophils in vitro

The glomerular basement membrane is susceptible to immunologic injury when immune complexes or anti-basement-membrane antibodies become lodged in its network. We have studied the digestion of glomerular basement membrane prepared from normal human kidney by isolated neutrophils. In the absence of immunoglobulin aggregates or immune complexes, there was little evidence of neutrophil adherence to the membrane, of release of lysosomal enzymes, or of digestion. However, when the basement membrane contained immunoglobin G (IgG) aggregates generated in situ by heating the membrane impregnated with IgG to 63 degrees C, electron micrographs showed neutrophils adherent to the basement-membrane surface and phagocytosis of smaller fragments. Lysosomal enzymes were detectable in the extracellular medium, and measurements of either total protein or hydroxyproline solubilized showed digestion of 80 micrograms basement membrane/h per 10(7) cells. Hydroxyproline solubilization was almost totally inhibited by phenylmethylsulphonyl fluoride, indicating that the neutrophil serine proteinases, elastase and cathepsin G are responsible for degradation. These findings provide direct evidence for the digestion of extracellular matrix by neutrophils stimulated in situ by deposited immune complexes as a contributor to inflammatory tissue damage.     

Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation

Background

Neutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Δψ_m), which is usually generated by the respiratory chain complexes. We investigated the source of Δψ_m in neutrophils, as compared to peripheral blood mononuclear leukocytes and HL-60 cells, and whether neutrophils can still utilise this Δψ_m for the generation of ATP.

Methods and Principal Findings

Individual activity of the oxidative phosphorylation complexes was significantly reduced in neutrophils, except for complex II and V, but Δψ_m was still decreased by inhibition of complex III, confirming the role of the respiratory chain in maintaining Δψ_m. Complex V did not maintain Δψ_m by consumption of ATP, as has previously been suggested for eosinophils. We show that complex III in neutrophil mitochondria can receive electrons from glycolysis via the glycerol-3-phosphate shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were lacking in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercomplex organisation while gaining increased aerobic glycolysis, just like neutrophils.

Conclusions

We show that neutrophils can maintain Δψ_m via the glycerol-3-phosphate shuttle, whereby their mitochondria play an important role in the regulation of aerobic glycolysis, rather than producing energy themselves. This peculiar mitochondrial phenotype is acquired during differentiation from myeloid precursors.     

Function of neutral endopeptidase on the cell membrane of human neutrophils

Intact human neutrophils hydrolyzed N-formyl-Met-Leu-[3H]Phe (fMLP) and released Leu-[3H]Phe, cleaving 45-50% of the peptide within 20 min at 37 degrees C. The dipeptide after its release was then hydrolyzed to free amino acids by a dipeptidase (EC 3.4.13.11). This activity, present in plasma membrane-enriched fractions of neutrophil lysates, was also inhibited over 90% by phosphoramidon, an inhibitor of neutral endopeptidase (NEP, EC 3.4.24.11). Dithiothreitol and EDTA inhibited the activity to a comparable degree, suggesting the requirement for a heavy metal cofactor. Bestatin and amastatin, inhibitors of aminopeptidases (but not human kidney NEP), did not inhibit the rate of fMLP degradation but prevented the production of free phenylalanine and enhanced the accumulation of Leu-Phe. Of other inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin slightly enhanced the rate of fMLP hydrolysis by neutrophils, and others tested were ineffective. Rabbit antiserum to homogeneous human kidney NEP reacted specifically with a 100-kDa protein present in sodium dodecyl sulfate-solubilized neutrophils. The Mr of this protein was slightly larger than that of the kidney enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum incubated with intact cells specifically inhibited the degradation of fMLP over 70%. First, we confirm that NEP present on the plasma membrane cleaves fMLP at the Met-Leu bond; then the dipeptide Leu-Phe is cleaved by a dipeptidase. Finally, inhibition of NEP completely blocks fMLP-mediated chemotaxis. Thus, the enzyme may play an important role in modulating chemotactic responses.     

Rap1 activation in collagen phagocytosis is dependent on nonmuscle myosin II-A

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or β1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.     

Differential effect of triiodothyronine and thyroxine on the liposomal membrane in liquid-crystalline and gel state

The effect of thyroid hormones on the degree of order or fluidity of dimyristoyl, dipalmitoyl or egg yolk phosphatidyl choline liposomes was evaluated by fluorescence spectroscopy methods. The freedom of molecular motion above the phase transition temperature was decreased, while below the transition, the mobility was actually increased by the incorporation of triiodothyronine to liposomes. While thyroxine decreases the fluidity in the liquid crystalline state, it cannot increase the fluidity in the gel state.A differential effect of triiodothyronine and thyroxine on the release of the liposomal content was found, depending on the liquid crystalline or gel state of the liposomes. These facts were correlated with the differential incorporation of the hormones to liposomes above and below the phase transition temperature of dimyristoyl and dipalmitoyl phospholipid choline. In gel state, a low incorporation of thyroxine compared with triiodothyronine was found.This work was supported by Grants PID 3-013800/89 from Consejo National de Investigaciones Científicas y Técnicas (CONICET), Fundación Antorchas A-12576/1-000065 and Consejo de Investigaciones de la Universidad National de Tucumán (CIUNT). We thank Dr. G. Rotillo for the space filling models.     

Effects of negatively charged lipids on phagocytosis of liposomes opsonized by complement

Ingestion of liposomes opsonized by specific antibody plus complement was investigated in vitro. Although the antibodies alone (IgM) did not have an opsonizing effect, in the presence of such antibodies uptake and ingestion of liposomes by mouse peritoneal macrophages was enhanced 5- to 10-fold by addition of complement. Phagocytosis of complement-opsonized liposomes was strongly dependent on the charge of the liposomal lipids. The presence of a negatively charged (i.e., acidic) lipid profoundly suppressed the uptake of the liposomes. Each of three acidic liposomal lipids, phosphatidylserine, phosphatidylinositol and dicetyl phosphate, suppressed liposome uptake. We conclude that opsonization of liposomes with complement greatly stimulates ingestion of liposomes by murine macrophages. However, most of the opsonic enhancement conferred by complement can be prevented by the presence of negatively charged membrane lipids.     

Yersinia pseudotuberculosis is resistant to killing by human neutrophils

The interaction between human neutrophils and the Gram negative gastrointestinal pathogen Yersinia pseudotuberculosis was investigated in vitro. Despite the wealth of data describing how Yersinia can affect the function of neutrophils, there are no published studies describing if neutrophil cells can affect the viability of Y. pseudotuberculosis. The wild-type IP32953 strain of Y. pseudotuberculosis was found to be resistant to killing by human neutrophils. Confocal examination and flow-cytometric analysis of this interaction revealed that bacteria were taken up.