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PubMed2006

In vivo regulation of neutrophil apoptosis by C5a during sepsis.

Delayed neutrophil apoptosis is characteristic of sepsis and may accentuate organ injury. It has been shown that PI-3K and MAPK pathways provide survival signaling in neutrophils. In this study, we demonstrate that neutrophils isolated from septic rats are resistant to apoptosis in comparison with the cells from normal animals. In contrast to normal serum, septic sera induced strong phosphorylation of AKT and p44/42 in neutrophils obtained from normal rats, resulting in marked resistance of these cells to apoptosis. Protection from apoptosis by septic sera was abrogated completely by inhibition of PI-3K and partially diminished by MEK inhibition. Increased neutrophil survival in septic rats was associated with increased levels of Bcl-xL in neutrophils and decreased levels of Bim expression. In vivo blockade of C5a in cecal ligation and puncture rats by anti-C5a antibody markedly restored the susceptibility of neutrophils to undergo apoptosis. C5a activated AKT and p44/42 and also enhanced X-linked inhibitor of apoptosis expression in neutrophils. LPS and C5a were able to induce Bcl-xL expression. Thus, neutrophil survival signals derived from effects of septic sera could be linked to activation of ERK1/2 and PI-3K, increased antiapoptotic protein expression, and ultimately, delayed neutrophil apoptosis.

Animals↗
PubMed1991

Thick filter neutrophil chemotaxis performed in the absence of albumin.

Migration through a filter is a technique widely used for the study of cell chemotaxis. Since the original description of the technique by Boyden in 1962 (J. Exp. Med. 115, 453) using neutrophils and thick filters prepared from cellulose nitrate, albumin has been required in the incubation medium to support chemotaxis. However, the binding to albumin of compounds affecting chemotaxis can reduce their free concentration. We developed two procedures for studying neutrophil chemotaxis under reduced or albumin-free conditions. In one, cellulose nitrate filters were pretreated with albumin by a novel procedure and chemotaxis was carried out in albumin-free medium. As tested with the chemoattractant LTB4 and human neutrophils, the procedure resulted in full chemotaxis, measured by the number of cells crossing the filter, with an EC50 of 0.43 nM for LTB4. The LTB4-receptor antagonist LY 223982 inhibited the chemotactic action of LTB4 with a Ki of 62 nM in the albumin-pretreated filter system, thus showing 58 times greater potency than in medium containing 0.5% albumin. The second procedure makes use, for the first time, of a relatively new filter (Hydrophilic Durapore). This filter has the same dimensions and pore rating of the cellulose nitrate filter but did not require pretreatment with albumin to support chemotaxis in the albumin-free medium. LTB4 stimulated neutrophil chemotaxis across this filter with an EC50 of 0.29 nM. LY 223982 had a Ki of 11 nM, thus exhibiting a potency even greater than in the albumin-pretreated cellulose nitrate filter system. fMLP and C5a also stimulated chemotaxis in the absence of albumin. These results suggest that albumin is not obligatory for neutrophil chemotaxis through thick filters. The role of albumin in the chemotaxis assay using cellulose nitrate filters may be to counteract the adherence of cells or chemotactic agents to the filters.

Albumins↗
PubMed1990

Production of superoxide anion and hydrogen peroxide by human neutrophilic granulocytes during chemotactic migration towards f-Met-Leu-Phe, C5a, leukotriene B4, monocyte-derived chemotaxin/IL-8 and platelet-activating factor.

During the chemotactic migration of human neutrophilic granulocytes towards the chemotactic factors f-Met-Leu-Phe, C5a, leukotriene B4 (LTB4), monocyte-derived chemotaxin (MOC/IL-8) and platelet-activating factor (PAF) in Boyden chambers, the production of superoxide anion and hydrogen peroxide was measured by superoxide dismutase-inhibitable cytochrome C reduction and oxidation of p-OH-phenylacetic acid, respectively. With the exception of 10(-6) M PAF, none of the factors at optimal chemotactic concentrations induced the production of O2- or H2O2 in amounts significantly different from neutrophilic granulocytes migrating at random. At 20-50 times the optimal chemotactic concentration some O2- and H2O2 production was observed with f-Met-Leu-Phe, C5a and LTB4, but not with MOC/IL-8. Superoxide dismutase, catalase or a combination of the two added to both compartments of the Boyden chambers did not affect the random or chemotactic migration towards any of the chemotactic factors. The results suggest that chemotactic migration and the production of reactive oxygen metabolites by human neutrophilic granulocytes are unrelated events.

Chemokine CCL2↗
PubMed2006

Divergent signaling pathways in phagocytic cells during sepsis.

Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is mediated by a group of chemoattractants, especially CXC chemokines. Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP), as reflected by rising levels of MIP-2 and cytokine-induced neutrophil chemoattractant-1 in bronchoalveolar lavage fluids. Alveolar macrophages are primed and blood neutrophils are down-regulated for production of MIP-2 and cytokine-induced neutrophil chemoattractant production in response to LPS and C5a. Under these conditions of stimulation, activation of MAPKs (p38, p42/p44) occurs in sham neutrophils but not in CLP neutrophils, while under the same conditions phosphorylation of p38 and p42/p44 occurs in both sham and CLP alveolar macrophages. These data indicate that, under septic conditions, there is impaired signaling in neutrophils and enhanced signaling in alveolar macrophages, resulting in CXC chemokine production, and C5a appears to play a pivotal role in this process. As a result, CXC chemokines increase in lung, setting the stage for neutrophil accumulation in lung during sepsis.

Amino Acid Sequence↗
PubMed1998

Pulmonary fibrosis correlates with duration of tissue neutrophil activation.

The role of inflammatory cells such as neutrophil granulocytes in the pathogenesis of pulmonary scarring is unclear. We determined the metabolic activity of neutrophils with positron emission tomography (PET) to measure regional uptake of (18F)-2-fluoro-2-deoxy-D-glucose (18FDG) following its intravenous injection. Fibrogenic or nonfibrogenic substances were instilled into the right upper lobe of rabbit lungs. Time course and intensity of the 18FDG signal in the affected region varied markedly, depending on the stimulus. Time to peak signal (Tmax) and rate constant for its decline (k) for the test substances were, respectively: C5a 10 h (Tmax), 0.045 +/- 0.030 h-1 (k); Streptococcus pneumoniae 15 h, 0.068 +/- 0.012 h-1; bleomycin 28 h, 0.002 +/- 0.001 h-1; microcrystalline silica (microXSiO2), 90 h, 0.0012 +/- 0.0007 h-1; amorphous silica (aSiO2), no response. Response to the nonfibrogenic agents C5a, S. pneumoniae and aSiO2 was brief or nonexistent, falling to baseline values within 3 d, whereas that to the fibrogenic agents bleomycin and microXSiO2 persisted for up to 4 wk. Neutrophil numbers in the lung were proportional to the 18FDG signal following C5a and S. pneumoniae, but not bleomycin and microXSiO2. Autoradiography of lungs following administration of (3H)-deoxyglucose [(3H)-DG] showed specific localization to neutrophils in all models. Thus, 18FDG uptake provides a remarkably specific measure of neutrophil activity in situ, and the development of pulmonary fibrosis may be related to persistence of this activity.

Animals↗
PubMed1992

IL-8 production by human polymorphonuclear leukocytes. The chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway.

IL-8 is a novel chemotactic cytokine, produced by a variety of blood and tissue cells, that has marked activating effects on polymorphonuclear leukocytes (PMN). We report that IL-8 is produced and released by human PMN after stimulation with the chemotactic agonist FMLP. Release of IL-8 in response to FMLP was transient and not influenced by PMN adherence or by the absence of serum in the medium. Maximum yields were usually obtained with 10 nM FMLP within 2 h of stimulation (0.5-3.5 ng/ml/7 x 10(6) cells, range of 17 different donors). IL-8 release was dependent on FMLP-induced de novo protein synthesis because it was inhibited by cycloheximide, was paralleled by enhanced expression of IL-8 mRNA and was potentiated from two- to sixfold after preincubation of PMN with cytochalasin B. The FMLP effect was direct and not dependent on LPS or on contaminating monocytes, which showed only low responsiveness to FMLP. Pretreatment of PMN with pertussis toxin prevented FMLP-dependent IL-8 production, the effect being evident both at the level of mRNA expression and protein secretion. In addition, two other chemoattractans, platelet-activating factor and C5a, were found capable to induce release of IL-8 by PMN. The results of this study suggest that chemotactically stimulated PMN may be able to amplify the recruitment process of PMN to the inflammatory site by releasing IL-8. As a long-lived cytokine, IL-8 could markedly prolong the attractant effect.

Cells, Cultured↗
PubMed1991

C5a structural requirements for neutrophil receptor interaction.

A number of C5a modifications were tested to determine effects on receptor binding to polymorphonuclear leukocyte (PMNL) membrane receptors and triggering of PMNL chemokinesis and myeloperoxidase (MPO) release. Site-directed mutagenesis was used to probe relationships of key C-terminal residues, and suggested a role for additional sites, particularly Lys19-20. A synthetic peptide based on C5a 19-30, weakly inhibited C5a binding. Potency of the C-terminal octapeptide, a full agonist, was markedly improved by a single Phe substitution for His67, and a Phe point mutation at this site was shown to enhance activity of the full recombinant protein.

Cells, Cultured↗
PubMed1984

The inhibitory effect of heparin and related glycosaminoglycans on neutrophil chemotaxis.

Clinical observations have shown that heparin has antiinflammatory activities. The effect of heparin on neutrophil chemotaxis was evaluated in vitro in the Boyden Chamber. This method enabled differentiation between the direct effects of heparin on neutrophil migration and locomotion, and its effects on chemotactic factors. Heparin inhibited both the random migration and directed locomotion of human neutrophils toward zymosan-activated serum (ZAS) and F-met-leu-phe (FMLP). Inhibition was found to be dependent on the concentrations of the heparin and of the chemotactic factors. No specific binding of heparin to the neutrophils could be demonstrated, and heparin's inhibitory effects were eliminated by simple washing of the cells. When added directly to the chamber containing chemotactic factor, heparin inhibited the chemotactic activity of ZAS but not that of FMLP, suggesting a direct inhibitory effect against C5a, the principal chemotactic factor in ZAS. Experiments performed with low-molecular-weight heparin, N-desulfated heparin, dextran sulfate, chondroitin sulfate and dextran indicated that the inhibitory effects of heparin on neutrophil chemotaxis are not related to its anticoagulant activity, but probably depend on the degree of sulfation of the heparin molecule.

Cell Movement↗
PubMed1986

Performance and biocompatibility of a new hemodialysis membrane.

Performance studies and investigations of the biocompatibility of a new hemodialysis membrane were conducted in patients being chronically hemodialyzed. The investigated polycarbonate membrane revealed a very satisfying performance with regard to clearance values and the ultrafiltration rate. The biocompatibility studies showed a significant leukopenia and an increase of C5a, platelet factor 4 and granulocyte elastase. Thrombocytes and C3d remained unchanged. Long-term studies have to confirm whether or not the new membrane is more biocompatible, a suggestion which could be advanced, as no febrile episodes were observed during any treatment, a finding which is not typical for Cuprophan hemodialysis.

Biocompatible Materials↗
PubMed1992

Recombinant human C5a induces transcription but not translation of interleukin-1 beta mRNA in human monocytes.

The effect of recombinant human C5a (rhC5a) on the synthesis of interleukin-1 beta (IL 1 beta) was investigated in highly purified human monocytes, isolated by leukapheresis and counter-current elutriation. RhC5a induced IL 1 beta mRNA synthesis in a dose- and time-dependent manner; maximal induction was achieved at 3 h with rhC5a concentrations of 200 to 500 ng/ml. The IL 1 beta mRNA induction with rhC5a was about 75% of the response observed using lipopolysaccharide (LPS) as the stimulus (300 ng/ml). On the other hand, rhC5a in optimal concentrations only weakly stimulated IL 1 beta protein synthesis, as measured by a two-site-directed enzyme-linked immunoassay. When LPS- or C5a-stimulated RNA was used to program IL 1 beta synthesis in an in vitro translation system from rabbit reticulocytes, no difference in translational efficiency was observed. Our results indicate that, in human monocytes, two signals for IL 1 beta gene expression are necessary, one signal for transcriptional activation and a second signal to induce translation of the mRNA.

Complement C5a↗
PubMed1983

5-L-hydroxy-6,8,11,14-eicosatetraenoate potentiates the human neutrophil degranulating action of platelet-activating factor.

Platelet-activating factor (AAGPC) and two of its structural analogues degranulated human neutrophils with respective potencies that were increased up to 100 to 1000-fold by 16 nM to 5 microM of 5-L-hydroxyeicosatetraenoate (5-L-HETE). 5-rac-HETE had similar actions but 8-rac-HETE was without effect. Furthermore, 5-L-HETE did not influence the degranulating actions of C5a, A23187 or a formalated oligopeptide chemotactic factor and none of the HETEs, by themselves, caused degranulation. Thus, 5-L-HETE and AAGPC selectively interact to induce degranulation. Since these products rapidly form in stimulated PMNs, they may serve as potentiator and agonist, respectively, to transduce biological signals into cell function.

Arachidonic Acids↗
PubMed1996

Inhibition of monocyte chemotaxis to C-C chemokines by antisense oligonucleotide for cytosolic phospholipase A2.

Monocyte chemotactic protein (MCP)-1, a member of the C-C (or beta) branch of the chemokine superfamily, at chemotactic concentrations, induced a rapid release of [3H]arachidonic acid but not of [14C]oleic acid from prelabeled human monocytes. This effect was associated with an increase in the intensity of the immunoreactive band corresponding to the phosphorylated form of cytosolic phospholipase A2, (cPLA2). To address the role of cPLA2 in the induction of monocyte chemotaxis, cells were treated with a specific antisense oligonucleotide. Monocytes cultured in the presence of 10 microM antisense oligonucleotide for 48 h showed a marked decrease (57 +/- 5%; n = 4) of cPLA2 expression, as evaluated by Western blot analysis and a nearly complete inhibition (81.8 +/- 4.2%; n = 3) of [3H]arachidonic acid release in MCP-1-stimulated cells. Monocyte chemotaxis in response to MCP-l also was inhibited in a concentration-dependent manner by cPLA2 antisense oligonucleotide (IC50 = 1.9 +/- 1.1 microM; n = 3), with complete inhibition observed between 3 and 10 microM. No inhibition of chemotactic response was observed in monocytes treated with a control oligonucleotide. Monocyte migration in response to MCP-3, RANTES (regulated on activation normal T cells expressed and secreted), and MIP-1 alpha/LD78 also was inhibited (>70%) in antisense oligonucleotide-treated cells. On the contrary, the chemotactic response elicited by formyl-methionyl-leucyl-phenylalanine and C5a, two "classical" chemotactic agonists, was minimally affected (<20%) by antisense oligonucleotide treatment. These data show that cPLA2 plays a major role in [3H]arachidonic acid release by MCP-1 in human monocytes and provide direct evidence for the involvement of cPLA2 in C-C chemokine-induced monocyte chemotaxis.

Arachidonic Acid↗
PubMed2004

Upregulation of fibronectin but not of entactin, collagen IV and smooth muscle actin by anaphylatoxin C5a in rat hepatic stellate cells.

Rat Kupffer cells (KC), hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) all express the C5a receptor (C5aR) constitutively in contrast to hepatocytes (HC). HSC showed an unexpectedly high level of expression of the C5aR. As these cells are known to play a key role in the induction of liver fibrosis we hypothesized that C5a may possibly induce fibrogenetic proteins in these cells. HSC are known to express the extracellular matrix (ECM) proteins collagen IV, fibronectin, entactin and the structure protein smooth muscle actin (SMA) which is regarded as a marker for the fibrotic conversion of HSC to myofibroblast-like cells. We investigated the effect of recombinant rat C5a (rrC5a) on the upregulation of these ECM-proteins and of SMA, all of which are known to be expressed by HSC. The profibrotic cytokine TGF-beta1 (2 ng/ml), which was used as a control, clearly upregulated the three matrix proteins but not SMA. In the absence of any stimulus HSC upregulated the three ECM-proteins as well as SMA during their conversion into myofibroblast-like cells. This resulted in a high stimulus-independent plateau of the mRNA expressions for all four proteins after four to five days of culture. Readouts were therefore taken at 72 h after the isolation of the HSC when the investigated mRNA levels had not yet reached their maxima due to the conversion of the cells. The first 24 h of culture were performed without stimulus and the following 48 h in the presence of 100 nM rrC5a (1 micro g/ml) or TGF-beta1 (2 ng/ml). Only fibronectin-specific mRNA was clearly upregulated by C5a whereas entactin, collagen IV and SMA were not affected by C5a. By competitive-quantitative PCR the upregulation of fibronectin-specific mRNA was determined to be about five-fold. As TGF-beta1 upregulated all of the three investigated ECM-proteins but not SMA it was checked as to whether C5a might act indirectly by upregulating the expression of TGF-beta1 in KC and HSC, as both cell types are known to be sources of this profibrotic cytokine. However, using RT-PCR, such an effect was not detectable in either cell type after 3, 10 or 24 h.

Actins↗
PubMed1986

A rapid method for monocyte isolation and chemotaxis.

A rapid method for isolating human peripheral blood monocytes and studying chemotaxis is described. The cells are isolated at a mean purity of 60% (+/- 10%) using Dextran. T500 sedimentation of whole blood, followed by centrifugation of the leucocyte-rich plasma obtained on a discontinuous Percoll gradient (density 1.067 g/ml). Chemotaxis was performed using a 48-well chamber and the chemoattractants activated serum (C5a) and FMLP and was proven by checkerboard analysis. Using this technique pooled serum was found to be activated by the PVP-coated polycarbonate filter in use, producing the attractant C5a. At high concentrations this produced a chemotactic response identical to zymosan-activated serum (ZAS). This technique requires small volumes of blood (15 ml) and enables monocyte isolation and chemotaxis to be completed in approximately 4 hr using leucocyte preparations in which the monocyte is the predominant cell. It therefore facilitates the sequential investigation of monocyte function in patients.

Cell Separation↗
PubMed1985

Effect of leukocyte activation on the formation of heterotypic tumor-cell aggregates in vitro.

Walker tumor cell (1 x 10(6) cells/ml) were incubated at 37 degrees C in a stirred cuvette with rat peritoneal exudate cells (9 x 10(6) cells/ml) with or without the synthetic leukocyte chemo-attractant fMLP (2 x 10(6) M) or biologically active concentrations of the major endogenous chemo-attractant, C5a. Aggregation, induced by the chemo-attractants, was detected after 3 min by a platelet aggregometer and by studying cytocentrifuge preparations. The response was amplified in the presence of cytochalasin B (5 micrograms/ml). Tumor cells could be identified in the aggregates by their morphology or by autoradiography after labeling with 3H-thymidine. Although tumor cells were incorporated into the leukocyte aggregates, there was no appreciable change in the number of aggregates formed between tumor cells themselves. Levine III human breast tumor cells (1 x 10(6)/ml) in heparinized human blood were incorporated into leukocyte aggregates within 30 min of adding 50 U cobra venom factor to activate complement. Aggregation correlated with a decrease in complement hemolytic activity (CH50). The aggregation reaction was not cytotoxic to tumor cells when evaluated by Trypan blue exclusion or by 51Cr release. We conclude that local tumor cells can be incorporated into aggregates formed when leukocytes are stimulated by chemo-attractants. We postulate that intravascular activation of neutrophils might affect the localization of circulating tumor cells by incorporating them into microembolic cell aggregates and by causing damage to the pulmonary endothelium.

Animals↗
PubMed2000

Surface expression of lactoferrin by resting neutrophils.

We examined the surface expression of lactoferrin by human neutrophils. Western blot analysis with anti-lactoferrin antibodies demonstrated the presence of a 78- to 79-kDa band in plasma membranes isolated from resting neutrophils that corresponded to the 78- to 79-kDa protein in neutrophil secondary granules. Flow cytometry using FITC-conjugated anti-lactoferrin antibodies confirmed that lactoferrin is expressed on the neutrophil surface. Preincubating the neutrophils in acidic (pH 3.9) buffer did not alter staining of the cells by the antibodies. Surface expression of lactoferrin was also detected on neutrophils in whole blood. Neutrophil activation by C5a or the calcium ionophore A23187 did not increase the surface expression of lactoferrin. Instead, the level of lactoferrin expression detected with one of two monoclonal antibodies was diminished after neutrophil activation, suggesting a possible conformational change in the lactoferrin. The surface-expressed lactoferrin may provide a mechanism for the interaction between lactoferrin-binding microorganisms and neutrophils.

Antibodies, Monoclonal↗
PubMed1990

Chemotactic competence of neutrophils from neonatal calves. Functional comparison with neutrophils from adult cattle.

Neonates demonstrate an increased susceptibility to infection. Defects in locomotory functions of newborn neutrophils may play a crucial role in this context. We therefore compared the migratory response of newborn (N-PMN) and adult (A-PMN) bovine neutrophils in a microwell filter assay. Stimulation with four different endotoxins (E. coli O128B:4 and O55B:5; S. abortus equi; S. typhimurium), with zymosan-activated plasma (ZAP) and with C5a induced dose-dependent migration of A-PMNs and N-PMNs. Migration of unstimulated cells and of cells stimulated with diluted ZAP or C5a was higher (P less than 0.05) in N-PMNs. Migration of A- and N-PMNs towards C5a was inhibited (P less than 0.001) by preincubation with either a steroidal (122 microM flumethasone) or nonsteroidal (3.3 microM phenylbutazone) antiinflammatory drug. Migratory responses of N-PMNs were inhibited less by SAIDs than were responses of A-PMNs (P less than 0.05); indeed dexamethasone slightly enhanced N-PMN responses towards C5a, and 510 microM flunixin meglumine enhanced C5a-induced migration in both age groups. Endotoxins from E. coli O55:B4, S. abortus equi, and S. typhimurium induced a higher rate of migration (P less than 0.05) in N-PMNs. In contrast to the above findings, measurement of the maximal distance of migration by the leading-front method did not reveal age-related differences. Migration speed of PMNs was lower after stimulation with C5a than with ZAP, but could be restored partly by adding human vitamin D-binding protein (Gc-globulin). The demonstrated hyperirritability of bovine N-PMNs represents a major functional difference to neonatal neutrophils from other species, including man. It may additionally be related to altered PMN functions and neonatal disease susceptibility.

Aging↗
PubMed1996

Interleukin-8 regulation of the Ras/Raf/mitogen-activated protein kinase pathway in human neutrophils.

Interleukin-8 (IL-8), the prototypic member of the CXC subfamily of chemokines, induces in neutrophils chemotaxis, the respiratory burst, granule release, and increased cell adhesion. The IL-8 receptor is a seven-transmembrane spanning receptor coupled to specific heterotrimeric G proteins including Gi and G16. IL-8 stimulation of its receptor on neutrophils activates Ras GTP loading and the mitogen-activated protein kinase (MAPK) pathway including Raf-1 and B-Raf. The properties of IL-8 stimulation of the MAPK pathway differ from those observed for chemoattractants such as C5a. Even though Ras GTP loading is similar for IL-8 and C5a, the maximal activation of Raf-1 and B-Raf is approximately 2-fold and 3-7-fold, respectively, less for IL-8 than that observed for C5a. Raf-1 activation is rapid but transient, returning to near basal levels by 10 min. B-Raf activation is slower in onset and does not return to basal levels for nearly 30 min. IL-8 activation of MAPK follows a time course suggesting an involvement of both Raf-1 and B-Raf. Surprisingly, wortmannin, at low concentrations, inhibits Raf-1, B-Raf, and MAPK activation in response to IL-8 and C5a demonstrating a role for phosphatidylinositol 3-kinase in the activation of Raf kinases in G protein-coupled receptor systems in human neutrophils. Furthermore, wortmannin inhibits IL-8 stimulated granule release and neutrophil adherence. These findings demonstrate the control of Raf kinases, the MAPK pathway and specific neutrophil functions by phosphatidylinositol 3-kinase enzymes.

Androstadienes↗