Biomedical subjects

O Stendahl

Publications and source records attributed to O Stendahl.

At least 19 recordsLinked to original sources

PubMed2000

Selective inhibition of IgG-mediated phagocytosis in gelsolin-deficient murine neutrophils.

Phagocytosis and the microbicidal functions of neutrophils require dynamic changes of the actin cytoskeleton. We have investigated the role of gelsolin, a calcium-dependent actin severing and capping protein, in peripheral blood neutrophils from gelsolin-null (Gsn-) mice. The phagocytosis of complement opsonized yeast was only minimally affected. In contrast, phagocytosis of IgG-opsonized yeast was reduced close to background level in Gsn- neutrophils. Thus, gelsolin is essential for efficient IgG- but not complement-mediated phagocytosis. Furthermore, attachment of IgG-opsonized yeast to Gsn- neutrophils was reduced ( approximately 50%) but not to the same extent as ingestion ( approximately 73%). This was not due to reduced surface expression of the Fcgamma-receptor or its lateral mobility. This suggests that attachment and ingestion of IgG-opsonized yeast by murine neutrophils are actin-dependent and gelsolin is important for both steps in phagocytosis. We also investigated granule exocytosis and several steps in phagosome processing, namely the formation of actin around the phagosome, translocation of granules, and activation of the NADPH-oxidase. All these functions were normal in Gsn- neutrophils. Thus, the role of gelsolin is specific for IgG-mediated phagocytosis. Our data suggest that gelsolin is part of the molecular machinery that distinguishes complement and IgG-mediated phagocytosis. The latter requires a more dynamic reorganization of the cytoskeleton.

Actins↗

PubMed2000

Activation of human neutrophils by Mycobacterium tuberculosis H37Ra involves phospholipase C gamma 2, Shc adapter protein, and p38 mitogen-activated protein kinase.

Recent studies have shown that human neutrophils play a significant protective role in mycobacteria infection. When encountered with mycobacteria, neutrophils exhibit the typical early bactericidal responses including phagocytosis and generation of reactive oxygen intermediates (ROI), but the underlying mechanisms are largely unknown. The present study shows that stimulation of neutrophils with an attenuated strain of Mycobacterium tuberculosis H37Ra (Mtb) led to a tyrosine kinase-dependent ROI production in these cells. Stimulation with Mtb induces a rapid and transient tyrosine phosphorylation of several proteins, one of which was identified as phospholipase C gamma 2 (PLC gamma 2). Several tyrosine-phosphorylated proteins were associated with the PLC gamma 2 precipitates from Mtb-stimulated neutrophils, of which pp46 was characterized as the Shc adapter protein. A role for PLC gamma 2-Shc association in the generation of ROI is supported by the observations that stimulation with Mtb causes the activation of p38 mitogen-activated protein kinase (MAPK), a downstream target of the Shc/Ras signaling cascade, and that the effect of genistein on ROI production coincided with its ability to inhibit both PLC gamma 2-Shc association and p38 MAPK activation. Moreover, pretreatment of neutrophils with a PLC inhibitor markedly suppresses the Mtb-stimulated ROI production as well as p38 MAPK activation in these cells. Taken together, these results indicate that stimulation of neutrophils with Mtb triggers the tyrosine phosphorylation of PLC gamma 2 and its association with Shc, and that such association is critical for the Mtb-stimulated ROI production through activating p38 MAPK.

Calcium↗

PubMed2000

Activation of cAMP-dependent protein kinase is necessary for actin rearrangements in human neutrophils during phagocytosis.

We have investigated the role of cAMP and cAMP-dependent protein kinase (cAPK) in neutrophil phagocytosis. Inhibition of cAPK with H-89 reduced complement- and IgG-dependent phagocytosis to 83 and 46%, respectively. Fluorescence intensity measurements of phalloidin-stained actin in neutrophils showed a reduced amount of filamentous actin (F-actin) in pseudopods and around the phagosome in cells treated with H-89 or cAMP-elevating agents (forskolin and rolipram). The amount of phosphotyrosine-containing proteins was also reduced in pseudopods and around the phagosome. Taken together, the data show that cAMP/cAPK regulates F-actin reorganization during receptor-mediated phagocytosis, particularly triggered by IgG-FcR interaction. Our results support the hypothesis that active subcortical reorganization of F-actin is a prerequisite for FcR-mediated phagocytosis, but is less important during CR3-mediated ingestion.

Cyclic AMP-Dependent Protein Kinases↗

PubMed2000

Involvement of the beta2-integrin CD18 in apoptosis signal transduction in human neutrophils.

OBJECTIVE AND DESIGN: To examine the hypothesis that an accelerated rate of neutrophil apoptosis occurs following beta2-integrin activation, and further investigate the signal transduction pathways involved. MATERIAL: Human polymorphonuclear neutrophils. TREATMENT: Neutrophils were challenged with pansorbins coated with antibodies towards the beta2-integrin subunit CD18 in a proportion of 1:100 with or without the inhibitors diphenylene iodonium (10 M), cytochalasin B (5 microg/ml), genistein (10 nM), herbimycin A (10 M) and Z-VAD-FMK (10 microM). METHODS: Measurement of phosphatidylserine exposure and DNA fragmentation in flow cytometry and assessment of H2O2-production through spectrofluorometry. The results were analysed using Mann Whitney U test and Kruskal Wallis. RESULTS: Pansorbins coated with antibodies to CD18 induce apoptosis in neutrophils (p<0.01), and activate the production of reactive oxygen species (p<0.01). Pre-treatment with the inhibitors have no effect on anti-CD 18 induced apoptosis. CONCLUSION: Anti-CD18 pansorbins induce apoptosis in neutrophils through an alternative pathway not involving reactive oxygen species and independent of tyrosine phosphorylation, cytoskeletal reorganisation and caspases.

Adult↗

PubMed2000

The influence of retinoic acid and retinoic acid derivatives on beta2 integrins and L-selectin expression in HL-60 cells in vitro.

A decreased expression of the beta2-integrin CD11b molecules on peripheral neutrophils from patients with pustular psoriasis occurred during treatment with retinoid compounds. Since this effect could not be mimicked in vitro with isolated peripheral neutrophils, the effect of retinoid compounds on cell differentiation was investigated. The promyelocytic cell line, HL60, was used to study what effect different retinoid compounds had on the cell surface expression of CD11b and L-selectin (CD62L) molecules, complement-mediated phagocytosis, adhesion and the oxidative burst. Retinoid-differentiated cells showed a significantly lower expression of CD11b and CD62L, and a decreased phagocytosis and oxidative burst compared to DMSO-differentiated HL60 cells or peripheral blood neutrophils. The diminished expression of beta2-integrins or L-selectin did not affect their adhesion to non-activated or lipopolysaccharide-activated endothelial cells in vitro but may however affect adhesion to vascular endothelium under shear forces during blood flow. These results suggest that retinoid treatment could affect several early steps in the inflammatory process.

CD18 Antigens↗

PubMed1999

Particles binding beta(2)-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis.

Complement-opsonised particles are readily ingested by human neutrophils through a complement receptor-mediated process leading to phagolysosome fusion and production of oxidative metabolites. To investigate the complement receptor 3 (CR3)-associated signal system involved, cells were challenged with protein A-positive, heat-killed Staphylococcus aureus to which antibodies with specificity for the subunits of the beta(2)-integrins, i.e. anti-CD11b (the alpha subunit of CR3) and anti-CD18 (the beta subunit of CR3), were bound through their Fc moiety. Despite not being ingested by the neutrophils, the surface associated anti-CD18- and anti-CD11b-coated particles were able to activate the neutrophil NADPH-oxidase. Also anti-CD11a- (the alpha subunit of LFA-1) and to a lesser extent anti-CD11c- (the alpha subunit of CR4) coated particles were able to trigger the NADPH-oxidase. The NADPH-oxidase was activated without extracellular release of reactive oxygen species. The activity was inhibited by cytochalasin B, suggesting a necessary role for the cytoskeleton in the signalling pathway that activates the oxidase. We show that particle-mediated cross-linking of beta(2)-integrins on the neutrophil surface initiates a signalling cascade, involving cytoskeletal rearrangements, leading to an activation of the NADPH-oxidase without phagosome formation or extracellular release of reactive oxygen species.

Antibodies↗

PubMed1999

CR3, FcgammaRIIA and FcgammaRIIIB induce activation of the respiratory burst in human neutrophils: the role of intracellular Ca(2+), phospholipase D and tyrosine phosphorylation.

Human neutrophils express two different types of phagocytic receptors, complement receptors (CR) and Fc receptors. In order to characterize the different signaling properties of each receptor we have used non-adherent human neutrophils and investigated CR3, FcgammaRIIA and FcgammaRIIIB for their signaling capacity. Selective activation of each receptor was achieved by coupling specific antibodies to heat-killed Staphylococcus aureus particles, Pansorbins, through their Fc moiety. Despite the fact that these particles are not phagocytosed, we show that addition of Pansorbins with anti-CD18 antibodies recognizing CR3 induced prominent signals leading to a respiratory burst. Stimulation with anti-FcgammaRIIIB Pansorbins induced about half of the response induced by anti-CR3 Pansorbins, whereas anti-FcgammaRIIA Pansorbins induced an even weaker signal. However, FcgammaRIIA induced strong phosphorylation of p72(syk) whereas FcgammaRIIIB induced only a very weak p72(syk) phosphorylation. During CR3 stimulation no tyrosine phosphorylation of p72(syk) was seen. Both phospholipase D and NADPH oxidase activities were dependent on intracellular calcium. This is in contrast to tyrosine phosphorylation of p72(syk) that occurred even in calcium-depleted cells, indicating that oxygen metabolism does not affect p72(syk) phosphorylation. Inhibitors of tyrosine phosphorylation blocked the respiratory burst induced by both FcgammaRIIA and FcgammaRIIIB as well as CR3. This shows that tyrosine phosphorylation of p72(syk) is an early signal in the cascade induced by FcgammaRIIA but not by CR3.

Antibodies, Monoclonal↗

PubMed1999

Expression and function of beta 2 integrin CD11B/CD18 on leukocytes from patients with psoriasis.

The expression of beta 2 integrin CD11b on granulocytes and monocytes from patients with psoriasis vulgaris and pustular psoriasis was examined by flow cytometry. The amount of CD11b expressed on both granulocytes and monocytes was greater in 4 patients with pustular psoriasis than in 16 patients with psoriasis vulgaris. Its expression correlated with the development of pustules on the skin. No difference was seen between healthy blood donors and patients with active psoriasis vulgaris. Three patients with pustular psoriasis were followed during retinoid treatment. Granulocytes and monocytes showed a decrease in CD11b expression after administration of retinoids, in parallel with clearing of the skin. The adherence of granulocytes isolated from psoriasis patients was tested on cultured human umbilical vein endothelium. No significant difference in adherence was observed between control cells and cells from patients with active psoriasis vulgaris. These data indicate that the development of microabscesses in the dermis in psoriasis vulgaris is not related to enhanced beta 2 integrin function. The increased CD11b expression found in patients with pustular psoriasis may, however, serve as a triggering factor for pustule formation in pustular psoriasis.

Aged↗

PubMed1999

Yersinia pseudotuberculosis-induced calcium signaling in neutrophils is blocked by the virulence effector YopH.

Pathogenic species of the genus Yersinia evade the bactericidal functions of phagocytes. This evasion is mediated through their virulence effectors, Yops, which act within target cells. In this study we investigated the effect of Yersinia pseudotuberculosis on Ca2+ signaling in polymorphonuclear neutrophils. The intracellular free calcium concentration in single adherent human neutrophils was monitored during bacterial infection and, in parallel, the encounter between the bacteria and cells was observed. When a plasmid-cured strain was used for infection, adherence of a single bacterium to the cellular surface induced a beta1 integrin-dependent transient increase in the intracellular concentration of free calcium. This was, however, not seen with Yop-expressing wild-type bacteria, which adhered to the cell surface without generating any Ca2+ signal. Importantly, the overall Ca2+ homeostasis was not affected by the wild-type strain; the Ca2+ signal mediated by the G-protein-coupled formyl-methionyl-leucyl-phenylalanine receptor was still functioning. Hence, the blocking effect was restricted to certain receptors and their signaling pathways. The use of different Yop mutant strains revealed that the protein tyrosine phosphatase YopH was responsible for the inhibition. This virulence determinant has previously been implicated in very rapid Yersinia-mediated effects on target cells as the key effector in the blockage of phagocytic uptake. The present finding, that Y. pseudotuberculosis, via YopH, specifically inhibits a self-induced immediate-early Ca2+ signal in neutrophils, offers more-detailed information concerning the effectiveness of this virulence effector and implies an effect on Ca2+-dependent, downstream signals.

Bacterial Adhesion↗

PubMed1999

Localization of intracellular Ca2+ stores in HeLa cells during infection with Chlamydia trachomatis.

Chlamydia trachomatis elementary bodies (EBs) enter epithelial cells within membrane-bound endosomes that aggregate with each other in a calcium-regulated process, but avoid fusion with lysosomes. Annexin III but not I translocates to chlamydial aggregates and inclusions. In this study, we localize the intracellular Ca2+ stores during the course of infection by analyzing the distribution of three intracellular Ca2+ store proteins: calreticulin, type-1 inositol-1,4, 5-trisphosphate receptor (IP3-R), and Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase type 2 (SERCA2) in HeLa cells infected with C. trachomatis serovar L2. In uninfected cells, immunofluorescence staining of the proteins showed a fine granular distributed pattern for all three proteins. After infection with C. trachomatis, calreticulin was found at the periphery of chlamydial aggregates and inclusions from 3 to 48 hours post-infection. In infected cells, SERCA2 was intimately associated with chlamydial inclusions after 3 and 24 hours, but not after 48 hours. Moreover, IP3-R was translocated to and colocalized with EB aggregates and chlamydial inclusions and had a distribution very similar to that of SERCA 2. After 24 hours incubation with chlamydiae, there was a local accumulation of [Ca2+]i (105+/-17 nM) in the proximity of chlamydial inclusions, compared to 50+/-13 nM in other parts of the cell cytoplasm. In the absence of extracellular Ca2+, this local accumulation of Ca2+ increased to 295+/-50 nM after adding 50 microM ATP, and to a similar extent after adding 100 nM thapsigargin (Tg). These data indicate that during infection of HeLa cells with chlamydiae, intracellular Ca2+ stores are redistributed, causing local accumulation of Ca2+ in the vicinity of chlamydial inclusions. These changes may trigger the association of certain proteins such as annexins with chlamydia-containing vesicles, and thereby regulation of membrane-membrane interaction during endosome aggregation and inclusion formation.

Adenosine Triphosphate↗

PubMed1998

Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosis in human neutrophils.

The phagocytic function of neutrophils is a crucial element in the host defence against invading microorganisms. We investigated phagocytosis and intracellular killing of an attenuated strain of Mycobacterium tuberculosis(H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca2+]i and certain cytosolic calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca2+]i. The ability to kill H37Ra decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated from cytoplasm to the proximity of the H37Ra-containing phagosomes, whereas the localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca2+-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca2+-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.

Annexins↗

PubMed1997

Role of annexins in endocytosis of antigens in immature human dendritic cells.

We have evaluated the uptake of a soluble protein antigen, denitrophenylated human serum albumin (DNP-HSA), and two different intracellular bacteria; Chlamydia trachomatis serovar L2 and Mycobacterium tuberculosis strain H37Ra, by immature human dendritic cells. These were generated by culturing progenitor cells from blood in the presence of cytokines (granulocyte-macrophage colony-stimulating factor and interleukin-4). Dendritic cells play a crucial part in antigen presentation for the induction of T-cell-dependent immune responses in various tissues. Recently, macropinocytic and phagocytic activity has been shown for immature dendritic cells of mouse, rat and human origin. In the present study, macropinocytosis characterized the uptake of the soluble protein-antigen DNP-HSA, whereas the C. trachomatis were ingested via receptor-mediated endocytosis in coated pits, and opsonized M. tuberculosis via phagocytosis. To follow the intracellular routes of the antigens, their positions were compared with the localization of annexins, a family of Ca(2+)-and phospholipid-binding proteins, involved in membrane fusion, aggregation and transport of different vesicles. To elucidate further the intracellular pathway of the antigens, two other proteins, lysosome-associated membrane protein-1 (LAMP-1) and cathepsin D, were labelled. They are known to colocalize with major histocompatibility complex class II compartments in the immature dendritic cells. We observed a distinct translocation of annexin V to DNP-HSA containing endosomes, and annexin III to vesicles with C. trachomatis. Furthermore, annexin III, IV and V redistributed to phagosomes with M. tuberculosis. Both LAMP-1 and cathepsin D colocalized with DNP-HSA endosomes, and with phagosomes with M. tuberculosis. Thus, immature human dendritic cells have the capacity to phagocytose. Moreover, the handling of these antigens by dendritic cells may represent three distinct intracellular pathways, albeit some properties and compartments are shared.

Annexins↗

PubMed1997

Phenotypic alterations in circulating monocytes induced by open heart surgery using heparinized and nonheparinized cardiopulmonary bypass systems.

In this study of 31 patients with coronary bypass surgery, we used flow cytometry to compare heparin-coated and noncoated cardiopulmonary bypass systems on leukocyte activation. We found significant differences between the groups during bypass, with activation of the complement system, measured as elevated levels of C3a desArg, upregulation of granulocyte beta2 integrin (CD11b), and a loss of circulating monocytes when noncoated systems were used. In both groups an early increase in the monocyte cell surface CD62L expression was obvious while the percentage of human leukocyte antigen (HLA)-DR positive monocytes did not alter. The morning after the operation, leukocytosis was present, together with a highly significant reduction in the monocyte expression of CD11b and HLA-DR, indicating the recruitment to the peripheral blood of cells with altered phenotypes. This alteration in phenotype on potent inflammatory cells may be one part of the impaired function of the immunological system reported after major surgery.

Adult↗

PubMed1996

Sulfatide-induced L-selectin activation generates intracellular oxygen radicals in human neutrophils: modulation by extracellular adenosine.

The sulfated form of galactocerebrosides (sulfatides) have recently been established as ligands for L-selectin. In this study we show that exposure of human neutrophils to sulfatides induces a transient generation of oxygen radicals, revealed by the luminol-enhanced chemiluminescence (CL) technique. The CL response was mainly located intracellularly, and was dependent on sulfation of the galactose ring, since non-sulfated galactocerebrosides had no effect. Sulfatides also dramatically amplified the CL response triggered by the chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP). This effect was primarily due to an increased (up to 10-fold) intracellular generation of oxygen metabolites. Removal or blocking of L-selectin with chymotrypsin and monoclonal antibodies, respectively, markedly reduced the effects of sulfatides. Furthermore, sulfatides amplified the CL response triggered by ionomycin, whereas the response induced by phorbol-12-myristate-13-acetate was slightly reduced. The tyrosine kinase inhibitor, genistein, markedly inhibited the oxygen radical production induced by sulfatides, and totally abolished the potentiating effects of sulfatides in fMLP- and ionomycin-stimulated neutrophils. Sulfatides also triggered a transient rise in the intracellular free calcium concentration, [Ca2+]i. Consequently, L-selectin activation through sulfatides appear to affect oxidase activity through a Ca(2+)-dependent pathway involving tyrosine phosphorylation. Adenosine is an anti-inflammatory agent predominately released from the vascular endothelium which might suppress an inappropriate activation of the oxidase during L-selectin-mediated rolling of neutrophils. Indeed, we found that adenosine inhibited the oxidative burst induced by sulfatides, mainly by attenuating the intracellular generation of oxygen radicals. However, 10-100 times higher concentration of exogenous adenosine was required to inhibit the CL response induced by sulfatides to the same extent as the adenosine-mediated inhibition of the fMLP-induced response. This difference in sensitivity to adenosine could be explained by various expression of extracellular adenosine deaminase (ADA), since we found that the ADA-inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) markedly reduced the oxygen radical production caused by sulfatides and almost totally abolished the potentiating effects of sulfatides on the fMLP-induced respiratory burst. In contrary, EHNA only slightly reduced the fMLP-triggered CL response. We suggest that the initial activation of L-selectin prepare the neutrophil for an effective microbicidal activity in the extravascular space. This process might be dependent on a L-selectin-mediated increase in the expression and activity of ADA, which locally reduces the extracellular level of adenosine.

Adenosine↗

PubMed1996

Direct or C5a-induced activation of heterotrimeric Gi2 proteins in human neutrophils is associated with interaction between formyl peptide receptors and the cytoskeleton.

The binding of ligands to N-formyl peptide chemoattractant receptors in human neutrophils results in a rapid association of these receptors with a cytoskeletal fraction and a specific activation and release of Gi2 alpha-subunits from this fraction. In the present study we could show that pretreating neutrophils with GDPbetaS prevented the fMet-Leu-Phe-induced association of its receptor with a cytoskeletal fraction and also blocked the release of Gi2 alpha-subunits from the same cytoskeletal fraction. In contrast, direct activation of Gi2 proteins by addition of GTPgammaS or AlF4- not only caused a release of Gi2 alpha-subunits from the cytoskeleton but also an association of formyl peptide receptors with the cytoskeleton. The receptor for complement fragment 5a, which transduces its signaling through the same Gi2 protein, triggers both a release of Gi2 alpha-subunits from the cytoskeleton fraction and, of even greater interest, an association between formyl peptide receptors and the cytoskeleton. The close relationship between the activation and release of Gi2 alpha-subunits from the cytoskeleton and the association of formyl peptide receptors with the cytoskeleton might, however, not be a matter of protein-protein exchange, since the increased binding of formyl peptide receptors to the cytoskeleton occurs more rapidly than the release of Gi2 alpha-subunits from the cytoskeleton. The present findings suggest a possible mechanism for the initiation of formyl peptide receptor desensitization during neutrophil locomotion.

Aluminum Compounds↗

PubMed1996

Release of oxygen metabolites from chemoattractant-stimulated neutrophils is inhibited by resting platelets: role of extracellular adenosine and actin polymerization.

The effect of human platelets on chemoattractant-induced generation of oxygen metabolites in neutrophils was investigated, using luminol-enhanced chemiluminescence (CL). Resting platelets inhibited the extracellular, but not the intracellular, production of oxygen radicals in formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils. Maximal effect was obtained at the physiological neutrophil/platelet ratio of 1/50. Similar results were acquired by adding supernatants of platelets, indicating a role for a soluble factor. Removal of extracellular adenosine by adenosine deaminase (ADA), or blocking of adenosine-receptors by theophylline, antagonized the inhibitory effects of platelets (or the equivalent supernatant) on the neutrophil respiratory burst. In contrast, accumulation of adenosine by apyrase enhanced the inhibition. Exogenous adenosine mimicked the effects of platelets on the fMet-Leu-Phe-induced respiratory burst. To further assess the role of platelet-derived adenosine, the platelets were fixed with paraformaldehyde. We found that fixed platelets, as well as their supernatant, inhibited the fMet-Leu-Phe-induced CL-response to the same extent as viable cells. These effects were also reversed by ADA and theophylline, respectively. A prior removal of adenosine in the platelet suspension by ADA, followed by treatment with erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) to inactivate ADA, did not reverse the inhibitory action of platelets on the fMet-Leu-Phe-induced CL-response in neutrophils. However, if adenosine receptors of neutrophil at the same time were blocked with theophyline, the inhibition was significantly reduced. Platelets markedly increased the generation of adenosine in a neutrophil suspension. The effect was antagonized by S-(4-Nitrobenzyl)-6-thioguanosine (NBTG), but unaffected by alpha, beta-methyl-eneadenosine5'diphosphate (AMP-CP), indicating that the platelet-dependent accumulation of adenosine is due to an increased release of endogenous adenosine from neutrophils and not to a degradation of extracellular AMP. In correlation, NBTG, but not AMP-CP, reversed the platelet-mediated inhibition of the fMet-Leu-Phe-induced CL-response in neutrophils. Consequently, these data suggest that a platelet-derived factor increases the release of endogenously formed adenosine from neutrophils, terminating the production of oxygen radicals. The inhibition of oxidase activity was also associated with a platelet-induced polymerization of actin in the margin of the neutrophils. Treatment of neutrophils with cytochalasin B reversed the effects of platelets, both on F-actin content and CL-response. In summary, resting platelets limit the release of oxygen radicals from chemoattractant-stimulated neutrophils, thus preventing excessive damage to host tissues in the vascular space. This effect is suggested to be associated with an increase generation of neutrophil-derived adenosine enhancing an autoregulatory inhibitory pathway, and a peripheral accumulation of actin filaments forming a barrier for extracellular release of reactive oxygen radicals.

Actin Cytoskeleton↗

PubMed1996

Killing of phagocytosed Staphylococcus aureus by human neutrophils requires intracellular free calcium.

The mobilization of intracellular calcium plays an important role in regulating neutrophil activation. With this in mind we investigated the effect of intra- and extracellular calcium on the ability of human neutrophils to kill complement-opsonized Staphylococcus aureus. We found that a rise in intracellular calcium is necessary for efficient killing of phagocytosed S. aureus. In the presence of extracellular calcium, killing of ingested bacteria in calcium-buffered neutrophils compared with normal cells was slightly reduced. Calcium buffering had no effect on phagocytic uptake by the neutrophils, but did decrease the generation of toxic oxygen metabolites, measured as chemiluminescence (CL). In nondepleted and calcium-depleted cells, removal of extracellular calcium did not affect ingestion but did cause a marked decrease in the ability to kill the bacteria. In parallel, the CL response was substantially reduced or completely blocked. These data show that calcium is not a prerequisite for phagocytosis of S. aureus by human neutrophils, but does play a vital role in the post-ingestion killing of the bacteria by regulating the generation of toxic oxygen metabolites.

Blood Bactericidal Activity↗

PubMed1996

Platelets enhance Fc(gamma) receptor-mediated phagocytosis and respiratory burst in neutrophils: the role of purinergic modulation and actin polymerization.

The interaction of platelets with neutrophil granulocytes is considered to play an important role in the inflammatory process, and the present study was focused on platelet-induced modulation of Fcgamma receptor-mediated functions in neutrophils. We found that phagocytosis and the respiratory burst (measured as luminol-enhanced chemiluminescence), triggered in neutrophils by immunoglobulin G (IgG)-opsonized yeast particles, were potentiated by platelets and that maximal enhancement was achieved at a physiological neutrophil/platelet ratio of about 1:50 to 1:100. Platelets both increased the intra- and extracellular generation of oxygen radicals as well as the release of myeloperoxidase from stimulated neutrophils. The presence of platelets also induced a cortical actin polymerization in neutrophils, which might explain the increased phagocytic capacity. Platelets appear to affect neutrophil function in a contact-independent manner that most likely involves ATP, indicated by the following: (1) platelet supernatants, but not fixed platelets, affected neutrophil function in the same way as viable platelets; (2) platelets raised the extracellular ATP level four- to fivefold; (3) exogenous ATP mimicked the effects of platelets on actin polymerization, phagocytosis, and the respiratory burst in neutrophils; (4) hydrolysis of extracellular ATP with apyrase or blocking of ATP receptors with suramin reversed the platelet-induced enhancement of neutrophil function. An increased accumulation of extracellular adenosine, induced by inhibiting endogenous adenosine deaminase or adding exogenous adenosine, reversed the effects of platelets. The platelet-induced potentiation of the respiratory burst was inhibited by the tyrosine kinase inhibitor genistein, suggesting that tyrosine phosphorylation is involved. However, platelets did not significantly affect the Fcgamma receptor-triggered calcium response in neutrophils. In conclusion, we show that platelets, through an ATP-dependent mechanism, potentiate IgG-mediated ingestion and production of oxygen metabolites in neutrophils.

Actins↗