Human Granulocyte Function Research Articles

Defects in Signal Recognition Particle (SRP) Components Reveal an Essential and Non-Redundant Role for Granule Biogenesis and Differentiation of Neutrophil Granulocytes

Neutrophil granulocyte play pivotal roles in inflammatory responses, immune defence, tissue remodeling, and cancer control. Studying rare patients with defects in differentiation and/or function of neutrophil granulocytes highlights genes and pathways orchestrating these important cellular functions. A previously not appreciated role of the signal recognition particle (SRP) has emerged when monoallelic mutations in SRP54 were associated with congenital neutropenia and pancreatic insufficiency. The eukaryotic SRP is composed of six distinct polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68, SRP72) bound to an RNA molecule (the 7SL RNA). SRP and its receptor (SRPRA and SRPRB) cooperatively transport nascent proteins emerging from ribosome to the endoplasmic reticulum (ER) lumen via a protein-conducting channel. We hypothesized that other constituents of the SRP may be involved in the pathophysiology of congenital neutropenia and that disturbances of the SRP may result in defective proteostasis in neutrophil granulocytes. By virtue of our international Care-for-Rare Alliance we identified three kindred with molecularly unsolved congenital neutropenia. Exome-sequencing revealed a de novo monoallelic mutation in SRPRA in one individual in one family and a homozygous splice-site mutation in SRP19 in five individuals from two families. Clinically, all patients with mutations in SRPRA and SRP19 suffered from various degrees of neutropenia and susceptibility to bacterial infections. Pancreatic insufficiency was only seen in patients with mutation in SRPRA, whereas SRP19 deficiency was not associated with any non-hematopoietic symptoms. To functionally validate these novel genetic variants, we set up an in-vitro neutrophil differentiation system allowing us to model the genetic defects in human SRP complex subunits. We introduced patient-specific mutations (SRPRA Q464E and SRP19 c.189+5G>A) into wildtype iPS cells using CRISPR-Cas9 editing. The engineered cells were differentiated into bona fide neutrophil granulocytes. In vitro generated wildtype neutrophil granulocytes had a high degree of resemblance to primary peripheral blood neutrophil granulocytes, as evidenced by phenotypic (light microscopy, cell surface markers, gene expression) and functional (phagocytosis, bacterial killing, rolling and adhesion, chemotaxis) characteristics. In striking contrast to wildtype iPS cells, both SRPRA and SRP19 mutated iPS cells had a significantly reduced capacity to differentiate into neutrophil granulocytes, as shown by colony-forming unit assays and phenotypic analysis. Furthermore, in vitro generated neutrophil granulocytes had higher susceptibility to apoptosis and evidence of increased unfolded protein responses. We went on to characterize the proteome of primary neutrophil granulocytes from patients with SRP-defects by data-independent proteomics technology. Interestingly, neutrophil granulocytes with defects in SRP constituents shared a significantly reduced abundance of the proteins Pentraxin-3 (PTX3) and Glucuronidase Beta (GUSB). These findings were confirmed in both non-hematopoietic HELA cells and promyelocytic HL60 leukemia cells engineered to express the respective SRP mutants. Upon genetic reconstitution, the aberrant protein expression was re-established. In summary, our studies define novel genetic defects causing congenital neutropenia and highlight the relevance of coordinated protein trafficking pathways and homeostasis for physiological differentiation and function of human neutrophil granulocytes. Disclosures No relevant conflicts of interest to declare.

Impact of Human Granulocyte and Monocyte Isolation Procedures on Functional Studies

One of the first lines of defense against infection is the activation of the innate immune system. It is becoming clear that autoimmune diseases, such as rheumatoid arthritis and Crohn's disease, may be caused by disturbed innate immunity, and relating granulocyte and monocyte functions to the patient genotype has become an important part of contemporary research. Although it is essential to move this field forward, a systematic study comparing the efficacy and suitability for functional studies of the various available protocols for the isolation of these immune cells has not been performed. Here, we compare human granulocyte functionality under three enrichment protocols: (i) Ficoll density gradient centrifugation, (ii) anti-CD15 antibody-conjugated microbeads (positive selection), and (iii) Polymorphoprep. Primary monocytes were isolated in parallel using (i) anti-CD14 magnetic microbeads, (ii) non-monocyte depletion by antibody-conjugated magnetic microbeads (negative selection), (iii) RosetteSep antibody cocktail, and (iv) the classical adherence protocol. The best results in terms of purity and cell functionality were obtained with positive selection by magnetic microbeads for both human granulocytes and monocytes. Whereas phagocytosis of Escherichia coli bacteria was identical in all isolation procedures tested, the granulocyte respiratory burst was higher in positively selected cells. In addition, different granulocyte enrichment procedures affect cell surface receptor expression to different extents. In toto, we propose that positive selection of granulocytes and monocytes be adopted as the procedure of choice for studies of human granulocyte and monocyte functions but caution investigators to be aware of possible alterations in cell phenotypes with different isolation procedures.

Bacterial DNA promotes neutrophil adhesion to endothelial cells

Bacterial DNA can be detected in the absence of bacteria in chronic inflammatory conditions. Bacterial DNA is emerging as an important regulator of functions of human neutrophil granulocytes. Bacterial DNA contains short sequences of nonmethylated CpG dinucleotides (CpG-DNA) that are recognized by TLR-9. We investigated the impact of CpG-DNA on neutrophil adherence to human umbilical vein endothelial cells (HUVEC). HUVEC express TLR-9 and respond to CpG-DNA (0.2–20 μg/ml), but not to thymus DNA, with increased expression of E-selectin and ICAM-1 protein and mRNA. Methylation of cytosines in CpG-DNA resulted in a complete loss of activity. Neutrophil adhesion to HUVEC activated with CpG-DNA was markedly enhanced under low shear conditions. The number of adherent neutrophils was further enhanced when the adhesion assay was performed in the presence of CpG-DNA, indicating that CpG-DNA activates both neutrophils and HUVEC. The adhesive interactions were blocked by antibodies against CD18 (~70% inhibition), E-selectin and L-selectin. Combination of the three antibodies inhibited adhesion by 87%. These results identify HUVEC as a target for bacterial DNA and indicate that by enhancing neutrophil adhesion to endothelial cells bacterial DNA may contribute to maintenance and aggravation of the inflammatory response. (Supported by CIHR grant MOP-67054).

CpG motifs in bacterial DNA evokes peroxynitrite signaling in human neutrophils

Bacterial DNA is emerging as an important regulator of functions of human neutrophil granulocytes. Bacterial DNA contains short sequences of nonmethylated CpG dinucleotides (CpG-DNA) that are recognized by TLR-9. Evidence suggests that peroxynitrite (ONOO-) may function as an intracellular signal for cytokine production in neutrophils. We investigated whether CpG-DNA evokes ONOO- signaling in neutrophils. High purity (>99.9%) human neutrophils express TLR-9 and respond to CpG-DNA (0.2-10 μg/ml), but not to thymus DNA, with secretion of IL-8 and, to a considerably lesser extent, IL-6. Methylation of cytosines in CpG-DNA resulted in a complete loss of activity. CpG-DNA-induced IL-8 mRNA expression and IL-8 release were blocked by inhibitors of endosomal acidification and L-NAME. CpG-DNA evoked concomitant formation of superoxide and NO, coinciding with ONOO- formation (assessed by dihydrorhodamine oxidation and nitrotyrosine formation). RT-PCR amplified endothelial NO synthase in neutrophils cultured with or without CpG-DNA. CpG-DNA evoked rapid mobilization of NF-kB and c-Fos to the nucleus that were attenuated by L-NAME parallel with inhibition of IL-8 mRNA expression. Pharmacological blockade of NF-kB activation attenuated ~75% of CpG-DNA-evoked IL-8 release. These results provide evidence that bacterial DNA and unmethylated CpG motifs in particular activate ONOO- signaling in neutrophils, underlying IL-8 gene and protein expression through activation of NF-kB and c-Fos. (Supported by CIHR MOP-64283).

Effects of CNI-1493 on human granulocyte functions

During acute bacterial infections such as sepsis and meningitis, activation of inflammatory mediators such as nitric oxide (NO) plays a crucial role in both pathogenesis and host defense. We have previously reported that CNI-1493, a macrophage deactivator, reduced mortality in infant rats infected with Haemophilus influenzae type b (Hib) with associated decrease in the number of granulocytes in the infected tissue. The aim of the present study was to investigate how CNI-1493 affects granulocytes and macrophages in vitro. Murine macrophages (RAW 264.7) pre-incubated with CNI-1493 prior to activation with lipopolysaccharide (LPS)/interferon gamma (IFN γ) had decreased NO production measured as NO 2 − /NO 3 − levels and reduction in inducible NO-synthase (iNOS) expression. Reactive oxygen species (ROS) production was increased in formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated granulocytes following CNI-1493 treatment, whereas F-actin content, motility and chemotaxis were decreased under the same conditions. The effects of CNI-1493 on both NO production in LPS/IFN γ-activated macrophages and ROS production, F-actin content, motility and chemotaxis in granulocytes, may contribute to the reduced inflammatory response and increased survival in Hib-infected animals treated with CNI-1493.

Differential expression of leukocyte-associated Ig-like receptor-1 during neutrophil differentiation and activation

Inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motifs play an important regulatory role in immune cell activation. In addition, several studies suggest that these receptors are involved in the regulation of hematopoietic cell differentiation. Here, we have investigated the expression of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), an inhibitory receptor expressed on most peripheral blood leukocytes and on CD34+ hematopoietic progenitor cells, in neutrophil differentiation and activation. We found that although LAIR-1 was expressed on peripheral blood eosinophils, cell-surface expression on mature neutrophils was low, suggesting that LAIR-1 expression is regulated during granulocyte differentiation. Indeed, the promyeloid cell line HL-60 expressed LAIR-1, but the expression decreased during chemical-induced differentiation toward neutrophils. Similarly, in bone marrow-derived neutrophil precursors, the most immature cells expressed LAIR-1, and loss of LAIR-1 expression was associated with neutrophil maturation. LAIR-1 was re-expressed rapidly on the membrane of mature neutrophils upon stimulation with tumor necrosis factor alpha, granulocyte macrophage-colony stimulating factor, or N-formyl-methionyl-leucyl-phenylalanine, indicating that LAIR-1 may also regulate neutrophil effector function. Our studies suggest that LAIR-1 may play a regulatory role in differentiation and function of human granulocytes.

Activation of TLR-9 Induces IL-8 Secretion through Peroxynitrite Signaling in Human Neutrophils

Bacterial DNA containing unmethylated CpG motifs is emerging as an important regulator of functions of human neutrophil granulocytes (polymorphonuclear leukocytes (PMN)). These motifs are recognized by TLR-9. Recent studies indicate that peroxynitrite (ONOO-) may function as an intracellular signal for the production of IL-8, one of the key regulators of leukocyte trafficking in inflammation. In this study we investigated whether bacterial DNA (CpG-DNA) could induce ONOO- signaling in human PMN. Human whole blood, isolated PMN (purity, >95%), and high purity (>99%) PMN respond to CpG-DNA, but not to calf thymus DNA, with secretion of IL-8 and, to a lesser extent, IL-6 and TNF. Methylation of cytosines in CpG-DNA resulted in a complete loss of activity. The endosomal acidification inhibitors, bafilomycin A and chloroquine, inhibited CpG-DNA-induced cytokine release from PMN. CpG-DNA-induced IL-8 mRNA expression and release was also blocked by the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester. CpG-DNA evoked concomitant increases in intracellular superoxide and NO levels, leading to enhanced ONOO- formation and, consequently, nuclear accumulation of c-Fos and NF-kappaB. Pharmacological inhibition of NF-kappaB activation attenuated approximately 75% of CpG-DNA-evoked IL-8 release. These results identify ONOO- -dependent activation of NF-kappaB and c-Fos as an important mechanism that mediates PMN responses, including IL-8 gene expression and release, to bacterial DNA and unmethylated CpG motifs in particular. Enhanced ONOO- formation represents a mechanism by which bacterial DNA may contribute to prolongation and amplification of the inflammatory response.

Characterization of the role of CaMKI-like kinase (CKLiK) in human granulocyte function

AbstractActivation of granulocyte effector functions, such as induction of the respiratory burst and migration, are regulated by a variety of relatively ill-defined signaling pathways. Recently, we identified a novel Ca2+/calmodulin-dependent kinase I-like kinase, CKLiK, which exhibits restricted mRNA expression to human granulocytes. Using a novel antibody generated against the C-terminus of CKLiK, CKLiK was detected in CD34+-derived neutrophils and eosinophils, as well as in mature peripheral blood granulocytes. Activation of human granulocytes by N-formyl-methionyl-leucyl-phenylalanine (fML142) and platelet-activating factor (PAF), but not the phorbol ester PMA (phorbol 12-myristate-13-acetate), resulted in induction of CKLiK activity, in parallel with a rise of intracellular Ca2+ [Ca2+]i. To study the functionality of CKLiK in human granulocytes, a cell-permeable CKLiK peptide inhibitor (CKLiK297–321) was generated which was able to inhibit kinase activity in a dose-dependent manner. The effect of this peptide was studied on specific granulocyte effector functions such as phagocytosis, respiratory burst, migration, and adhesion. Phagocytosis of Aspergillus fumigatus particles was reduced in the presence of CKLiK297–321 and fMLP-induced reactive oxygen species (ROS) production was potently inhibited by CKLiK297–321 in a dose-dependent manner. Furthermore, fMLP-induced neutrophil migration on albumin-coated surfaces was perturbed, as well as β2-integrin-mediated adhesion. These findings suggest a critical role for CKLiK in modulating chemoattractant-induced functional responses in human granulocytes.

Influence of thiamphenicol on the primary functions of human polymorphonuclear leucocytes against Streptococcus pyogenes

Current antibiotic therapy encourages the use of antibiotics that may potentiate the host’s immune defences. We therefore investigated the effect of thiamphenicol (TAP), the active principle of thiamphenicol glycinate acetylcysteinate (TGA), on human granulocyte functions, mainly phagocytosis and intracellular killing of Streptococcus pyogenes. Our findings support the use of thiamphenicol in the treatment of respiratory tract infections caused by S. pyogenes as it acts directly against the pathogen as well as in cooperation with PMNs by eliciting their intracellular killing.

Identification and characterization of CKLiK, a novel granulocyte Ca++/calmodulin-dependent kinase

Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction–based strategy was used to identify novel granulocyte-specific kinases. A novel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calcium-calmodulin–dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD34+ stem cells. CKLiK kinase activity was dependent on Ca++ and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiK- transfected cells treated with ionomycin demonstrated an induction of CRE- binding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinaseα enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca++/calmodulin-dependent PMN- specific kinase that may play a role in Ca++-mediated regulation of human granulocyte functions.

Identification and characterization of CKLiK, a novel granulocyte Ca++/calmodulin-dependent kinase

AbstractHuman granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction–based strategy was used to identify novel granulocyte-specific kinases. A novel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calcium-calmodulin–dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD34+ stem cells. CKLiK kinase activity was dependent on Ca++ and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiK- transfected cells treated with ionomycin demonstrated an induction of CRE- binding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinaseα enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca++/calmodulin-dependent PMN- specific kinase that may play a role in Ca++-mediated regulation of human granulocyte functions.

Comparative study on the effect of cyclic nucleotides related to the function of Biomphalaria glabrata hemocytes and human granulocytes

The biochemical processes linked up to cyclic nucleotides related to the phenomenon of phagocytosis in Biomphalaria glabrata hemocytes were studied. In hemocytes the results suggest the presence of a phagocytic capacity similar to that observed in human cells related to regulation by cyclic adenylate monophosphate (cAMP), but not by cyclic guanylate monophosphate (cGMP). This similarity and differences in the metabolic process of phagocytary capacity between human phagocytary cells and hemocytes could represent an interesting model aimed at studying the phylogenetic evolution of enzymatic complexes.

Colony-Stimulating Factors Signal for Increased Transport of Vitamin C in Human Host Defense Cells

Although serum concentrations of ascorbic acid seldom exceed 150 μmol/L, mature neutrophils and mononuclear phagocytes accumulate millimolar concentrations of vitamin C. Relatively little is known about the mechanisms regulating this process. The colony-stimulating factors (CSFs), which are central modulators of the production, maturation, and function of human granulocytes and mononuclear phagocytes, are known to stimulate increased glucose uptake in target cells. We show here that vitamin C uptake in neutrophils, monocytes, and a neutrophilic HL-60 cell line is enhanced by the CSFs. Hexose uptake studies and competition analyses showed that dehydroascorbic acid is taken up by these cells through facilitative glucose transporters. Human monocytes were found to have a greater capacity to take up dehydroascorbic acid than neutrophils, related to more facilitative glucose transporters on the monocyte cell membrane. Ascorbic acid was not transported by these myeloid cells, indicating that they do not express a sodium-ascorbate cotransporter. Granulocyte (G)- and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated increased uptake of vitamin C in human neutrophils, monocytes, and HL-60 neutrophils. In HL-60 neutrophils, GM-CSF increased both the transport of dehydroascorbic acid and the intracellular accumulation of ascorbic acid. The increase in transport was related to a decrease in Km for transport of dehydroascorbic acid without a change in Vmax. Increased ascorbic acid accumulation was a secondary effect of increased transport. Triggering the neutrophils with the peptide fMetLeuPhe led to enhanced vitamin C uptake by increasing the oxidation of ascorbic acid to the transportable moiety dehydroascorbic acid, and this effect was increased by priming the cells with GM-CSF. Thus, the CSFs act at least at two distinct functional loci to increase cellular vitamin C uptake: conversion of ascorbic acid to dehydroascorbic acid by enhanced oxidation in the pericellular milieu and increased transport of DHA through the facilitative glucose transporters at the cell membrane. These results link the regulated uptake of vitamin C in human host defense cells to the action of CSFs.

Colony-Stimulating Factors Signal for Increased Transport of Vitamin C in Human Host Defense Cells

AbstractAlthough serum concentrations of ascorbic acid seldom exceed 150 μmol/L, mature neutrophils and mononuclear phagocytes accumulate millimolar concentrations of vitamin C. Relatively little is known about the mechanisms regulating this process. The colony-stimulating factors (CSFs), which are central modulators of the production, maturation, and function of human granulocytes and mononuclear phagocytes, are known to stimulate increased glucose uptake in target cells. We show here that vitamin C uptake in neutrophils, monocytes, and a neutrophilic HL-60 cell line is enhanced by the CSFs. Hexose uptake studies and competition analyses showed that dehydroascorbic acid is taken up by these cells through facilitative glucose transporters. Human monocytes were found to have a greater capacity to take up dehydroascorbic acid than neutrophils, related to more facilitative glucose transporters on the monocyte cell membrane. Ascorbic acid was not transported by these myeloid cells, indicating that they do not express a sodium-ascorbate cotransporter. Granulocyte (G)- and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated increased uptake of vitamin C in human neutrophils, monocytes, and HL-60 neutrophils. In HL-60 neutrophils, GM-CSF increased both the transport of dehydroascorbic acid and the intracellular accumulation of ascorbic acid. The increase in transport was related to a decrease in Km for transport of dehydroascorbic acid without a change in Vmax. Increased ascorbic acid accumulation was a secondary effect of increased transport. Triggering the neutrophils with the peptide fMetLeuPhe led to enhanced vitamin C uptake by increasing the oxidation of ascorbic acid to the transportable moiety dehydroascorbic acid, and this effect was increased by priming the cells with GM-CSF. Thus, the CSFs act at least at two distinct functional loci to increase cellular vitamin C uptake: conversion of ascorbic acid to dehydroascorbic acid by enhanced oxidation in the pericellular milieu and increased transport of DHA through the facilitative glucose transporters at the cell membrane. These results link the regulated uptake of vitamin C in human host defense cells to the action of CSFs.

Neutrophil function and glutathione-peroxidase (GSH-px) activity in healthy individuals after treatment with N-acetyl-L-cysteine

The objective of this study was to evaluate the effect of N-acetyl-L-cysteine (NAC) on neutrophilic functions and as an antioxidant. NAC, 600 mg daily, given orally to healthy individuals for a period of 2 weeks, affected some functions of human neutrophilic granulocytes when tested in vitro. NAC treatment caused a decrease in the production of superoxide anions by stimulated neutrophils and the improvement of their phagocytic capacity although it did not affect their random or chemotactic migration. The level of glutathione peroxidase (GSH-px) in thrombocytes of the NAC-treated individuals was increased in comparison with the activity before treatment. These results suggest that NAC might act as a scavenger of oxygen-derived free radicals released by stimulated neutrophils and thereby protect the tissue against the radical caused injury as well as optimize phagocytosis.

Recombinant interferon-gamma preserves human granulocyte bactericidal and chemoluminescence activities.

The effects of recombinant human interferon (IFN)-gamma on functions of human granulocytes (PMNL) were investigated. Incubation with IFN-gamma for up to 24 h enhanced the chemoluminescent (CL) response of polymorphonuclear leukocytes (PMNL) to Staphylococcus aureus, FMLP, opsonized zymosan, and PMA in a dose-dependent fashion for concentrations of 1-1000 units/mL. Short exposures to tumor necrosis factor (TNF)-alpha also enhanced the CL responses of PMNL, but the effects IFN-gamma and TNF alpha together, at the concentrations used, were less than additive. Incubation of PMNL for 8 h with IFN-gamma preserved their bactericidal capabilities relative to control PMNL incubated for the same length of time without IFN-gamma. Preservation of neutrophil function may be a subtle but important role for IFN-gamma.

Immunochemical Characterization and Biological Properties of a Cell Surface Antigen Extracted from Encapsulated Staphylococcus epidermidis Strain SE-l0

Protection inducing antigen (PIA) was mechanically extracted from Staphylococcus epidermidis (encapsulated strain SE-10) and purified by DEAE-Sephadex A 25 (C1- form) ion exchange chromatography. Major carbohydrate constituents of PIA were galactose, glucose, and N-acetylglucosamine at the molar ratio 1.00:0.96:0.78. Antigenic activity was considerably reduced after sodium metaperiodate oxidation, however, pronase digestion was not effective. N-acetylglucosamine residues were shown to be closely related to the antigenic determinant. No cross reactivity to PIAs from other encapsulated strains of S. epidermidis was found which indicates type specificity. Protection of mice after immunization and enhancement of human granulocyte function suggests that PIA might be considered to be a biological response modifying substance.

Effect of Staphylococcus aureus delta-toxin on human granulocyte functions and platelet-activating-factor metabolism

The production of delta-toxin is supposed to be responsible for various pathophysiological effects during infection with Staphylococcus aureus. We compared the effects of delta-toxin with the structurally related bee venom toxin melittin on granulocyte functions and inflammatory mediator release. Delta-toxin and melittin induced a rapid Ca2+ influx, as was shown by fluorescence detection. Furthermore, oxygen radical production, as determined by luminol-enhanced chemiluminescence, was triggered by delta-toxin (0.15 to 15 micrograms/ml), whereas melittin showed only marginal effects. Release of lysozyme and beta-glucuronidase was observed only at high concentrations of 15 micrograms of melittin and delta-toxin per ml. Preincubation (15 min) of neutrophils with both toxins resulted in the formation of 3H-platelet-activating factor (3H-PAF) from 3H-lyso-PAF. After 5 min of incubation, the exogenously added lyso-PAF was converted to PAF (delta-toxin, 80 +/- 2%; melittin, 27 +/- 12% of total radioactivity; n = 3, mean +/- standard error of the mean) and 1-O-alkyl-2-acyl-glycerophosphorylcholine (alkyl-acyl-GPC) (corresponding values, 20 +/- 3% and 51 +/- 14% of total radioactivity). The newly generated PAF was rapidly metabolized to lyso-PAF and alkyl-acyl-GPC during the subsequent incubation period of 60 min. In the absence of any toxin, no formation of PAF from lyso-PAF was observed. Further studies indicated that the metabolism of PAF into lyso-PAF and alkyl-acyl-GPC was inhibited in the presence of delta-toxin. Melittin had no significant effects on PAF metabolism. Neither delta-toxin nor melittin modulated the uptake of PAF and lyso-PAF significantly. Our data provide evidence that delta-toxin has an effect on the activity of neutrophil granulocytes with regard to its proinflammatory capacity.

Lipid peroxidation of human granulocytes (PMN) and monocytes by iron complexes.

Because the phagocytic function of non-stimulated human polymorphonuclear granulocytes (PMN) is impaired after incubation with either polynuclear Fe(III) or Fe(II), we decided to study lipid peroxidation of PMN and monocytes by these iron complexes. Lipid peroxidation was assessed by measuring thiobarbituric acid reactive substances and fluorescent compounds. In this study we report that monocytes, isolated in the standard way (EDTA-mediated detachment), release significantly more thiobarbituric acid reactive substances after incubation with iron than PMN. Monocytes, however, isolated without EDTA release the same amount after incubation with iron as PMN. The iron complexes shown to impair phagocyte function also stimulated the peroxidation of membrane lipids. Ascorbic acid in high concentrations enhanced iron-induced lipid peroxidation. Lipid peroxidation induced by ferrous ascorbate (1:20) could be inhibited by catalase, the iron chelators deferoxamine and transferrin, and the hydroxyl radical scavenger thiourea. Mononuclear complexes (ferric citrate 1:20) did not impair granulocyte function and did not induce lipid peroxidation. Our results suggest that impaired phagocyte function in patients with iron overload may be due to non-transferrin bound iron-mediated peroxidation of membrane lipids. However, the lowest amount of ferrous ascorbate (1:20) capable of inducing lipid peroxidation (50 microM) was significantly higher than that which impaired phagocyte function (10 microM).

In vitro impairment of human granulocyte functions by lead.

Chemotaxis and receptor independent phagocytosis of human polymorphonuclear leukocytes (PMNs) exposed to lead in vitro (concentrations between 1.2 microM and 115 microM) were studied. Chemotaxis was measured in Boyden chambers and phagocytosis was investigated using latex beads. Additional methods were also applied. Superoxide anion formation from PMNs activated with preopsonized zymosan was quantified as superoxide dismutase-inhibitable reduction of ferricytochrome c. Steady state fluorescence polarization was performed using trimethylammonium diphenylexatriene (TMA-DPH). Lead concentrations were highly correlated both with decreased chemotactic activity (r = 0.70 p less than 0.01) and with decreased phagocytosis (r = 0.68 p less than 0.01). Ferricytochrome c reduction was not significantly affected. An increase in fluorescence polarization was recorded at the highest concentration of lead used, i.e. 57.6 microM and 115 microM, both in unstimulated PMNs and in PMNs activated with N-formyl-methionyl-leucyl-phenylalanine chemotactic peptide (n-FMLP). Moreover, an increase in the fluorescence polarization was observed in PMNs pretreated with a microtubule disrupting drug, exposed to lead concentrations of 14.4 microM and 57.6 microM and then activated with n-FMLP; no increase was recorded at the lowest lead concentrations used, i.e. 1.2 microM and 3.6 microM. The possible interaction of lead with the membrane-cytoskeleton apparatus is discussed.