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In human peripheral blood, the neutrophil granulocytes (neutrophils) are the most abundant white blood cells. These professional phagocytes are rapidly recruited from the bloodstream to inflamed tissues by chemotactic factors that signal danger. Neutrophils, which express many receptors that are members of the large family of G protein-coupled receptors (GPCRs), are critical for the elimination of pathogens and inflammatory insults, as well as for the resolution of inflammation leading to tissue repair. Danger signaling molecular patterns such as the N-formylated peptides that are formed during bacterial and mitochondrial protein synthesis and recognized by formyl peptide receptors (FPRs) and free fatty acids recognized by free fatty acid receptors (FFARs) regulate neutrophil functions. Short peptides and short-chain fatty acids activate FPR1 and FFA2R, respectively, while longer peptides and fatty acids activate FPR2 and GPR84, respectively. The activation profiles of these receptors include the release of reactive oxygen species (ROS) generated by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Activation of the oxidase and the production of ROS are processes that are regulated by proinflammatory mediators, including tumor necrosis factor α and granulocyte/macrophage colony-stimulating factor. The receptors have signaling and functional similarities, although there are also important differences, not only between the two closely related neutrophil FPRs, but also between the FPRs and the FFARs. In neutrophils, these receptors never walk alone, and additional mechanistic insights into the regulation of the GPCRs and the novel regulatory mechanisms underlying the activation of NADPH oxidase advance our understanding of the role of receptor transactivation in the regulation of inflammatory reactions., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Neutrophils express many surface receptors that sense environmental changes. One such sensor is FFAR2 (free fatty acid receptor 2), a receptor that detects gut microbiota-derived short-chain fatty acids. As such, FFAR2 has been regarded as a molecular link between metabolism and inflammation. Our recent studies on FFAR2, using its endogenous agonist propionate in combination with allosteric modulators, have identified several novel aspects of FFAR2 regulation. A recent study has also identified the ketone body acetoacetate as an endogenous ligand for mouse FFAR2. Whether human FFAR2 also recognizes acetoacetate and how this recognition modulates human neutrophil functions has not been investigated. In this study, we found that acetoacetate can induce a decrease of cAMP and translocation of β-arrestin in cells overexpressing FFAR2. In addition, we show that similar to propionate, FFAR2-specific allosteric modulators enhance acetoacetate-induced transient rise in cytosolic calcium, production of reactive oxygen species, and cell migration in human neutrophils. In summary, we demonstrate that human neutrophils recognize the ketone body acetoacetate through FFAR2. Thus, our data further highlight the key role of FFAR2 in inflammation and metabolism., Competing Interests: Conflict of interest statement. Saray Gutierrez is an employee of AstraZeneca., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

The formyl peptide receptors FPR1 and FPR2 are abundantly expressed by neutrophils, in which they regulate proinflammatory tissue recruitment of inflammatory cells, the production of reactive oxygen species (ROS), and resolution of inflammatory reactions. The unique dual functionality of the FPRs makes them attractive targets to develop FPR-based therapeutics as novel anti-inflammatory treatments. The small compound RE-04-001 has earlier been identified as an inducer of ROS in differentiated HL60 cells but the precise target and the mechanism of action of the compound was has until now not been elucidated. In this study, we reveal that RE-04-001 specifically targets and activates FPR1, and the concentrations needed to activate the neutrophil NADPH-oxidase was very low (EC 50 ∼1 nM). RE-04-001 was also found to be a neutrophil chemoattractant, but when compared to the prototype FPR1 agonist N-formyl-Met-Leu-Phe (fMLF), the concentrations required were comparably high, suggesting that signaling downstream of the RE-04-001-activated-FPR1 is functionally selective. In addition, the RE-04-001-induced response was strongly biased toward the PLC-PIP 2 -Ca 2+ pathway and ERK1/2 activation but away from β-arrestin recruitment. Compared to the peptide agonist fMLF, RE-04-001 is more resistant to inactivation by the MPO-H 2 O 2 -halide system. In summary, this study describes RE-04-001 as a novel small molecule agonist specific for FPR1, which displays a biased signaling profile that leads to a functional selective activating of human neutrophils. RE-04-001 is, therefore, a useful tool, not only for further mechanistic studies of the regulatory role of FPR1 in inflammation in vitro and in vivo, but also for developing FPR1-specific drug therapeutics., (©2020 Society for Leukocyte Biology.)

Recruitment of neutrophils from blood to tissues is a cardinal event in inflammation during which neutrophils switch from a resting, naive state to a preactivated, primed phenotype; the priming process is characterized by alterations in the composition of cell surface adhesins, for example, shedding of l-selectin and mobilization of granule-stored integrins to the cell surface. Ligation of chemotactic receptors and interactions with the endothelial lining are established triggers of neutrophil priming and in line with this, in vivo transmigrated neutrophils obtained from tissues are typically highly primed. We here characterize the priming of neutrophils brought about by in vivo recruitment from blood to inflamed joints by the analyses of synovial fluid and blood from patients with inflammatory arthritis. For comparisons, we used controlled in vivo models of neutrophil transmigration to skin of healthy subjects. In contrast to the residing view and in vivo transmigrated neutrophils from skin models, neutrophils from synovial fluid were often surprisingly resting and phenotypically very similar to naive cells isolated from peripheral blood; synovial fluid cells often retained l-selectin and had undergone minimal up-regulation of integrin receptors. In complete agreement with our in vivo findings, cell-free synovial fluid was potently chemotactic without triggering alteration of surface receptors also in vitro. We conclude that tissue recruitment of neutrophils does not by default trigger l-selectin shedding and granule mobilization, and the chemoattractant(s) guiding neutrophils to synovial fluid apparently operate without inducing cellular priming., (©2018 Society for Leukocyte Biology.)

Acetate, an agonist for the free fatty acid receptor 2 (FFA2R/GPR43), triggers an increase in the cytosolic concentration of free Ca 2+ in neutrophils without any assembly of the superoxide generating NADPH-oxidase. We show that the phenylacetamide compound 58 (Cmp 58; (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide), lacking a direct activating effect on neutrophils, acts as a positive FFA2R modulator that turns acetate into a potent activating agonist that triggers an assembly of the NADPH-oxidase. The NADPH-oxidase activity could be further increased in neutrophils treated with the pro-inflammatory cytokine TNF-α. Many neutrophil chemoattractant receptors are stored in secretory organelles but no FFA2R mobilization was induced in neutrophils treated with TNF-α. The receptor selectivity was demonstrated through the inhibition of the neutrophil response induced by the combined action of acetate and Cmp 58 by the FFA2R antagonist CATPB. Receptor modulators that positively co-operate with natural FFA2R agonists and prime neutrophils in their response to such agonists, may serve as good tools for further unraveling the physiological functions of FFA2R and its involvement in various diseases. In this study, we show that neutrophils primed with a presumed allosteric FFA2R modulator produce increased amounts of reactive oxygen species when activated by receptor specific agonists., (©2018 Society for Leukocyte Biology.)

Formyl peptide receptor (FPR)-desensitized neutrophils display increased production/release of superoxide (O 2 - ) when activated by platelet-activating factor (PAF), a priming of the response achieved through a unique receptor crosstalk mechanism. The aim of this study was to determine the effect of an inhibitor selective for small, heterotrimeric G proteins belonging to the Gαq subclass on that receptor crosstalk. We show that signals generated by FPRs and the PAF receptor (PAFR) induce activation of the neutrophil O 2 - , producing NADPH-oxidase, and that response was sensitive to Gαq inhibition in cells activated by PAF, but no inhibition was obtained in cells activated by FPR agonists. Signaling in naive neutrophils is terminated fairly rapidly, and the receptors become homologously desensitized. The downstream sensitivity to Gαq inhibition in desensitized cells displaying increased production/release of O 2 - through the PAFR receptor crosstalk mechanism also comprised the reactivation of the FPRs, and the activation signals were redirected from the PAFR to the desensitized/reactivated FPRs. The Gαq-dependent activation signals generated by the PAFRs activate the Gαi-coupled FPRs, a receptor crosstalk that represents a novel pathway by which G protein-coupled receptors can be regulated and signaling can be turned on and off., (© Society for Leukocyte Biology.)

Testing for the presence of ANCAs in circulation is part of the clinical examinations routinely performed upon suspected autoimmune disorders, mainly vasculitis. The autoantibodies are typically directed toward neutrophil MPO or PR3. These are major granule-localized proteins, and similar to all hitherto-described ANCA antigens, they are expressed by all neutrophils, and ANCA-containing sera thus give rise to uniform reactivity toward all neutrophils in a sample. In this paper, we describe sera from 2 unrelated patients with diffuse inflammatory symptoms that gave rise to peculiar c-ANCA patterns, only reacting with a subpopulation (roughly 30%) of human neutrophils. By immunoblotting, both sera reacted to the same antigen, which was expressed in intracellular granules. The antigen could be released to the extracellular milieu through secretion but also through the formation of NETs. Neutrophils have long been considered a homogenous cell population, but it is becoming increasingly clear that distinct subpopulations, defined by the presence or absence of certain proteins, exist. One such marker that defines a neutrophil subset is the granule protein OLFM4. The unusual, subset-restricted c-ANCA sera reacted only with OLFM4-positive neutrophils, and MS analysis revealed that the autoantigen was, in fact, OLFM4. These data describe for the first time a c-ANCA pattern reactive to only a subpopulation of neutrophils and identify the granule protein OLFM4 as a novel autoantigen., (© Society for Leukocyte Biology.)

Neutrophils respond to microbial invasion or injury by transmigration from blood to tissue. Transmigration involves cellular activation and degranulation, resulting in altered levels of surface receptors and changed responsiveness to certain stimuli. Thus, fundamental functional changes are associated with neutrophil transmigration from blood to tissue. Neutrophils isolated from peripheral blood spontaneously enter apoptosis, a process that can be accelerated or delayed by different pro- or antiapoptotic factors. How tissue neutrophils that have transmigrated in vivo regulate cell death is poorly understood. In this study, in vivo-transmigrated neutrophils (tissue neutrophils) were collected using a skin chamber technique and compared with blood neutrophils from the same donors with respect to regulation of cell death. Skin chamber fluid contained a variety of cytokines known to activate neutrophils and regulate their lifespan. Freshly prepared tissue neutrophils had elevated activity of caspase 3/7 but were fully viable; spontaneous cell death after in vitro culture was also similar between blood and tissue neutrophils. Whereas apoptosis of cultured blood neutrophils was delayed by soluble antiapoptotic factors (e.g., TLR ligands), tissue neutrophils were completely resistant to antiapoptotic stimulation, even though receptors were present and functional. In vitro transmigration of blood neutrophils into skin chamber fluid did not fully confer resistance to antiapoptotic stimulation, indicating that a block of antiapoptotic signaling occurs specifically during in vivo transmigration. We describe a novel, functional alteration that takes place during in vivo transmigration and highlights the fact that life and death of neutrophils may be regulated differently in blood and tissue.

Eosinophils express the chemoattractant receptors CCR3 and FPR. CCR3 binds several agonists such as eotaxin-1, -2, and -3 and RANTES, whereas the FPR binds the formylated tripeptide fMLP and a host of other ligands. The aim of this study was to investigate if there is interplay between these two receptors regarding the elicitation of migration and respiratory burst in human blood-derived eosinophils. Inhibition of the FPR with the antagonists CyH and boc-MLP abrogated the migration of eosinophils toward all of the CCR3 agonists. Similar results were seen when the FPR was desensitized with its cognate ligand, fMLP. In contrast, the respiratory burst triggered by eotaxin-1 was not inhibited by CyH. Thus, signals evoked via the FPR caused unidirectional down-regulation of CCR3-mediated chemotaxis but not respiratory burst in human eosinophils. The underlying mechanism was neither reduced ability of the CCR3 ligand eotaxin-1 to bind to CCR3 nor down-regulation of CCR3 from the cell surface. Finally, confocal microscopy and adFRET analysis ruled out homo- or heterodimer formation between FPR and/or CCR3 as an explanation for the reduction in chemotaxis via CCR3. Pharmacologic inhibition of signal transduction molecules showed that the release of free oxygen radicals in response to eotaxin-1 compared with fMLP is relatively more dependent on the p38 MAPK pathway.

Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that approximately 35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site.

Serum amyloid A (SAA) is one of the acute-phase reactants, a group of plasma proteins that increases immensely in concentration during microbial infections and inflammatory conditions, and a close relationship between SAA levels and disease activity in rheumatoid arthritis (RA) has been observed. RA is an inflammatory disease, where neutrophils play important roles, and SAA is thought to participate in the inflammatory reaction by being a neutrophil chemoattractant and inducer of proinflammatory cytokines. The biological effects of SAA are reportedly mediated mainly through formyl peptide receptor like-1 (FPRL1), a G protein-coupled receptor (GPCR) belonging to the formyl peptide receptor family. Here, we confirmed the affinity of SAA for FPRL1 by showing that stably transfected HL-60 cells expressing FPRL1 were activated by SAA and that the response was inhibited by the use of the FPRL1-specific antagonist WRWWWW (WRW4). We also show that SAA activates the neutrophil NADPH-oxidase and that a reserve pool of receptors is present in storage organelles mobilized by priming agents such as TNF-alpha and LPS from Gram-negative bacteria. The induced activity was inhibited by pertussis toxin, indicating the involvement of a GPCR. However, based on FPRL1-specific desensitization and use of FPRL1 antagonist WRW4, we found the SAA-mediated effects in neutrophils to be independent of FPRL1. Based on these findings, we conclude that SAA signaling in neutrophils is mediated through a GPCR, distinct from FPRL1. Future identification and characterization of the SAA receptor could lead to development of novel, therapeutic targets for treatment of RA.

Mature human neutrophils contain small amounts of interleukin-8 [CXC chemokine ligand 8 (CXCL-8)], which upon proinflammatory activation, increases significantly. It has been suggested that the CXCL-8 content of resting human neutrophils is stored in the secretory vesicles. Here, we have used a fractionation technique, which allows isolation of these vesicles, and we find that CXCL-8 neither colocalizes with the secretory vesicles nor with markers of any of the classical neutrophil granules. To increase resolution in the system, we induced CXCL-8 production by lipopolysaccharide. After 8 h of stimulation, CXCL-8 was visualized within the cell using immunoelectron microscopy. The images revealed CXCL-8-containing stuctures resembling neutrophil granules, and these were distinct from all known neutrophil organelles, as shown by double immunostaining. Further, the CXCL-8 organelle was present in nonstimulated neutrophil cytoplasts, entities lacking all other known granules and secretory vesicles. Upon fractionation of the cytoplasts, CXCL-8 was found to partly cofractionate with calnexin, a marker for endoplasmic reticulum (ER). Thus, part of CXCL-8 may be localized to the ER or ER-like structures in the neutrophil.

Aberrant activation of the translation initiation machinery is a common property of malignant cells, and is essential for breast carcinoma cells to manifest a malignant phenotype. How does sustained activation of the rate limiting step in protein synthesis so fundamentally alter a cell? In this report, we test the post transcriptional operon theory as a possible mechanism, employing a model system in which apoptosis resistance is conferred on NIH 3T3 cells by ectopic expression of eIF4E. We show (i) there is a set of 255 transcripts that manifest an increase in translational efficiency during eIF4E-mediated escape from apoptosis; (ii) there is a novel prototype 55 nt RNA consensus hairpin structure that is overrepresented in the 5'-untranslated region of translationally activated transcripts; (iii) the identified consensus hairpin structure is sufficient to target a reporter mRNA for translational activation under pro-apoptotic stress, but only when eIF4E is deregulated; and (iv) that osteopontin, one of the translationally activated transcripts harboring the identified consensus hairpin structure functions as one mediator of the apoptosis resistance seen in our model. Our findings offer genome-wide insights into the mechanism of eIF4E-mediated apoptosis resistance and provide a paradigm for the systematic study of posttranscriptional control in normal biology and disease.

Truncation of the N-terminal part of the calcium-regulated and phospholipid-binding protein annexin AI has been shown to change the functional properties of the protein and to generate immunoregulatory peptides. Proinflammatory as well as anti-inflammatory signals are triggered by these peptides, and the two formyl peptide receptor (FPR) family members expressed in neutrophils, FPR and FPR-like 1 (FPRL1), have been suggested to transduce these signals. We now report that an annexin AI peptide (Ac9-25) activates, as well as inhibits, the neutrophil release of superoxide anions. Results obtained from experiments with receptor antagonists/inhibitors, desensitized cells, and transfected cells reveal that the Ac9-25 peptide activates the neutrophil reduced nicotinamide adenine dinucleotide phosphate oxidase through FPR but not through FPRL1. The Ac9-25 peptide also inhibits the oxidase activity in neutrophils triggered, not only by the FPR-specific agonist N-formyl-Met-Leu-Phe but also by several other agonists operating through different G protein-coupled receptors. Our data show that the two signals generated by the Ac9-25 peptide are transmitted through different receptors, the inhibitory signal being transduced by a not-yet identified receptor distinct from FPR and FPRL1.

The persistent infection caused by hepatitis C virus (HCV) is presumably explained by a deficient immune response to the infection, but the basis for the inefficiency of immune-mediated virus eradication is not known in detail. This study addresses mechanisms of relevance to dysfunction of cytotoxic lymphocytes in HCV infection, with a focus on the role of phagocyte-derived oxygen radicals. We show that NS3, a nonstructural, HCV-encoded protein, induces a prolonged release of oxygen radicals from mononuclear and polymorphnuclear phagocytes by activating a key enzyme in radical formation, the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The NS3-activated phagocytes, in turn, induced dysfunction and/or apoptosis in three major subsets of lymphocytes of relevance to defense against HCV infection: CD3+/56- T cells, CD3-/56+ natural killer (NK) cells, and CD3+/56+ NKT cells. Two inhibitors of the NADPH oxidase, histamine and diphenylene iodonium, suppressed the NS3-induced oxygen radical production and efficiently protected lymphocytes against NS3-induced apoptosis and dysfunction. In conclusion, we propose that NS3, by triggering oxygen radical formation in phagocytes, may contribute to the dysfunction of antiviral lymphocytes in HCV-infected liver tissue and that strategies to circumvent oxidative stress may be useful in preventing HCV-associated carcinogenesis and facilitating lymphocyte-mediated clearance of infected cells.

Natural killer (NK) cells are functionally suppressed and induced to apoptosis by reactive oxygen species (ROS) produced by mononuclear phagocytes (MPs). These inhibitory events are reversed by the biogenic amine serotonin. MPs generate hydrogen peroxide (H(2)O(2)), which is processed further by myeloperoxidase (MPO) to even more toxic compounds. Earlier studies suggest that serotonin scavenges MP-derived oxygen radicals generated by the MPO-H(2)O(2) system. These findings led us to explore the capability of MPO-deficient MPs to induce NK cell dysfunction. We show that MPs recovered from subjects with MPO deficiency trigger inhibition of NK cells. In addition, MPs recovered from healthy subjects conveyed suppression of NK cells in the presence of the MPO inhibitor ceruloplasmin. We conclude that ROS-dependent inhibition of NK cell function is unrestricted by the availability of MPO-derived oxygen radicals and that the protecting properties of serotonin may operate in the absence of functional MPO. Our data suggest a complex mechanism of MP-induced NK cell inhibition, which comprises the generation of interchangeable oxygen radicals.

Whereas prokaryotes use L- and D-isomers of amino acids in their protein synthesis, eukaryotic proteins as a rule incorporate only L-isomers. Hence, D-isomers may constitute danger signals to the innate immune system. A D-methionine-containing peptide, Trp-Lys-Tyr-Met-Val-D-Met-NH(2) (WKYMVm), has been shown to be a stronger activator of neutrophils than f-Met-Leu-Phe. The aim of this study was to compare the responsiveness of eosinophils to WKYMVm with that of neutrophils. The peptide was found to induce chemotaxis and respiratory burst in eosinophils. However, it did not mobilize granule constituents, as evidenced by a lack of eosinophil cationic protein, eosinophil peroxidase, and interleukin-5 in the supernatants of stimulated eosinophils. In contrast, WKYMVm caused the release of complement receptor 3 from secretory vesicles in neutrophils. Different members of the formyl peptide receptor family were preferentially engaged by the peptide in the two classes of granulocytes: the formyl peptide receptor itself in eosinophils and formyl peptide receptor-like 1 in neutrophils.

High concentrations of the neurotransmitter serotonin can be found in inflamed and ischemic peripheral tissues, but the role of serotonin in immunoregulation is largely unknown. Here we report that serotonin protected human natural-killer (NK) cells from oxidatively induced inhibition inflicted by autologous monocytes in vitro. Serotonin protected NK cells from monocyte-mediated apoptosis and suppression of cytotoxicity and maintained the activation of NK cells induced by interleukin-2 despite the presence of inhibitory monocytes. A detailed analysis of these protective effects revealed that serotonin scavenged reactive oxygen species (ROS) derived from the H(2)O(2)-myeloperoxidase (-MPO) system. Serotonin shared this scavenger activity with its precursor, 5-hydroxytryptophan (5-HTP); however, serotonin was >10-fold more potent than 5-HTP in protecting NK cells against functional inhibition and apoptosis. We propose that serotonin, by scavenging peroxidase-derived ROS, may serve to protect NK cells from oxidative damage at inflammatory sites.

The subcellular localization of GPI-80, a novel, adhesion-regulating protein, was investigated in human neutrophils. Surface expression of GPI-80 was determined by FACS analysis as well as by the ability for phospholipase C to cleave the protein from the cell surface. Increasing amounts of GPI-80 were exposed on the cell surface after weak stimulation with the chemoattractant fMLF, suggesting that the protein can be translocated to the plasma membrane from intracellular stores. By subcellular fractionation of the neutrophils, GPI-80 was defined as a component of a light membrane fraction, containing secretory vesicles and plasma membranes, and it was absent from the neutrophil granule fractions. Separation of the plasma membranes from the secretory vesicles by flotation gradient fractionation confirmed that the GPI-80 was localized in the mobilizable secretory vesicles by approximately 50%, and the rest was plasma membrane-bound. Thus, we identify secretory vesicles as the reservoir of GPI-80 from which it may translocate to the plasma membrane after weak stimulation of the cells.

The interaction between mycobacterial phenolic glycolipids (PGLs) and phagocytes was studied. Human neutrophils were allowed to interact with each of four purified mycobacterial PGLs and the neutrophil production of reactive oxygen metabolites was followed kinetically by luminol-/isoluminol-amplified chemiluminescence. The PGLs from Mycobacterium tuberculosis and Mycobacterium kansasii, respectively, were shown to stimulate the production of oxygen metabolites, while PGLs from Mycobacterium marinum and Mycobacterium bovis BCG, respectively, were unable to induce an oxidative response. Periodate treatment of the M. tuberculosis PGL decreased the production of oxygen radicals, showing the importance of the PGL carbohydrate moiety for the interaction. The activation, however, could not be inhibited by rhamnose or fucose, indicating a complex interaction which probably involves more than one saccharide unit. This is in line with the fact that the activating PGLs from M. tuberculosis and M. kansasii contain tri- and tetrasaccharides, respectively, while the nonactivating PGLs from M. marinum and M. bovis BCG each contain a monosaccharide. The complement receptor 3 (CR3) has earlier been shown to be of importance for the phagocyte binding of mycobacteria, but did not appear to be involved in the activation of neutrophils by PGLs. The subcellular localization of the reactive oxygen metabolites formed was related to the way in which the glycolipids were presented to the cells.

The effects of the non-toxic drug 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline (B220) on the generation of reactive oxygen species froin human neutrophils were investigated. The data show that B220 inhibits neutrophil release of reactive oxygen species, as well as intracellular generation of reactive oxygen species. The inhibition is not achieved through direct oxygen radical scavenger activity of B220, and the drug has no immediate effects on the activity of the assembled oxidase. Radical production and release were inhibited by all agonists tested [i.e. the protein kinase C-activating phorbol ester phobol myristate acetate; the receptor-specific agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP); and serum-opsonized yeast particles] in the presence of B220. The drug also inhibits phagocytosis and fMLP-induced mobilization of granules. However, based on the fact that the effects of B220 on phagocytosis and granule mobilization are much less significant than its effect on radical production, we suggest that the signal(s) affected by B220 is (are) located mainly downstream of the point at which the signals are generated to promote oxidase activation and phagocytosis or granule secretion, respectively.

Hydrophilic and hydrophobic glass surfaces precoated with human albumin, fibrinogen, or IgG were investigated with respect to their ability to activate the neutrophil NADPH-oxidase. We found that IgG-coated surfaces induced a substantial and prolonged neutrophil production of reactive oxygen species (ROS). When a hydrophilic surface was used to support protein binding, a somewhat lower neutrophil response (around 35%) was obtained, compared with the response induced by IgG on a hydrophobic surface. The production of ROS was completely eliminated when cytochalasin B was added to the measuring system, suggesting the involvement of the cell cytoskeleton in the activation process. The relation between the intra- and extracellular generation of ROS was further assessed, and we found that most of the ROS produced were released from the cells, in agreement with a model in which the activating surfaces induce a 'frustrated' phagocytic response. Serum totally inhibited 'frustrated' phagocytosis provided that the IgG molecules were sticking to a hydrophilic surface.

The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) interacts with neutrophils, generating signals that induce activation of the superoxide anion/hydrogen peroxide-producing NADPH-oxidase. Low temperature binding of fMLP to its neutrophil surface receptors is associated with a desensitization of the cells with respect to activation of the oxidase. Other stimuli can still activate the oxidase (in fact even induce a primed response), indicating that the observed phenomenon is stimulus specific and could not be accounted for by an effect on the oxidase itself. Furthermore, no desensitization is obtained in the presence of cytochalasin B, suggesting that the cytoskeleton is involved in the process leading to desensitization. Okadaic acid is a toxin produced by dinoflagellates and exerts its effects by an inhibition of cellular phosphatases. To investigate the role of phosphorylation/dephosphorylation events in the desensitization process we used okadaic acid as a scientific tool. We show that neutrophils treated with okadaic acid are primed with respect to the fMLP-induced production of superoxide anion, and that no desensitization is obtained in toxin-treated cells. Because the recovery of ligand-receptor complexes in a Triton X-100-insoluble fraction is very low in the cells treated with okadaic acid, we suggest that protein dephosphorylation is required to obtain binding to the cytoskeleton of occupied fMLP receptors; binding of the occupied receptors to the cell cytoskeleton being the mechanism behind desensitization.

The leucocytes in the secretion on the tonsillar surface have been regarded as inactivated and dying cells with no essential function in the defence of the underlying mucosa. In recent studies, we showed that in acute tonsillitis there are very few, if any, bacteria in the parenchyma, whereas in the secretion on the tonsillar surface there are huge numbers of bacteria and neutrophils, and furthermore, extensive phagocytosis. The present study was performed to elucidate the functional capacity of the neutrophils in the secretion on the tonsillar surface. Secretion samples were taken from the tonsillar surface of healthy volunteers with an imprint technique which allows transfer of secretion from the mucosa to a glass slide with maintained topographic position of the cells. The capacity of the neutrophils to respond to chemotactic stimuli and to phagocytize and further process labelled yeast particles was studied in this in vitro system. The results show that the neutrophils in the secretion have the same properties as in tissue, and also when having arrived in the secretion they can identify and attack new prey.

The effects of the phosphatase inhibitor okadaic acid (OA) on human neutrophil phagocytic activity were investigated. The chemiluminescence response was found to be greatly reduced in OA-treated neutrophils during phagocytosis of serum opsonized yeast particles in comparison to control cells. However, the OA-treated neutrophils phagocytosed yeast particles to the same extent as control cells and the engulfment of the prey was accompanied by phagolysosomal formation in both OA-treated and nontreated cells. We thus found that the OA effect was selective in the sense that it inhibits the NADPH-oxidase activity in neutrophils phagocytosing yeast particles but not uptake of the prey or phagolysosomal formation. Based on the fact that the NADPH-oxidase activity induced by the chemoattractant formyl-methionyl-leucyl-phenylalanine was not reduced but elevated in OA-treated neutrophils, we conclude that the state of phosphorylation has no direct effect on the oxidase, but is of importance for the NADPH-oxidase activating signal(s) generated during phagocytosis of the yeast particles.

The paradigm for activation of the neutrophil NADPH oxidase with the protein kinase C activator phorbol myristate acetate (PMA) states that the oxidase assembles the plasma membrane and that the metabolites generated are released extracellularly. This paradigm is challenged by the results presented. Most of the PMA-induced oxidase activity measured as chemiluminescence and dichlorofluorescein fluorescence was insensitive to scavengers of superoxide anion and hydrogen peroxide. This indicates that oxidase activity also takes place in a cellular compartment that the scavengers cannot reach. From the results obtained with granule-deficient HL-60 cells and cord blood neutrophils, we suggest that the scavenger-insensitive part of the NADPH oxidase activity in normal neutrophils resides in an intracellular compartment identical to or originating from granules. Our results also indicate that specific and azurophil granules have to be in very close contact to allow the generated oxygen metabolites to reach and react with myeloperoxidase.

We have investigated the interaction between quartz and granulocytes with respect to complement receptor expression. When N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated leukocytes were exposed to quartz at 37 degrees C, CR1 was down-regulated but CR3 was not affected. This was a direct effect on granulocytes because it occurred in a similar fashion when mixed leukocyte suspensions and isolated granulocyte populations were used as targets for quartz. The observed down-regulation by quartz was not affected by the microfilament-disrupting agent cytochalasin B and the total detectable pool of CR1 was reduced after quartz exposure. When protease inhibitors, such as aprotinin or phenylmethanesulfonyl fluoride, were present during quartz exposure, the down-regulation of CR1 was less pronounced, but this was not the case not when protease inhibitors such as EDTA-Na2 and pepstatin were present. Exposure to quartz was not accompanied by a pronounced release of beta-glucuronidase (marker for the primary granules) or vitamin B12 binding protein (marker for the secondary granules). In contrast to quartz, exposure to alumina did not affect the expression of CR1 and CR3. The spontaneous mobilization of CR1 at 37 degrees C was reduced when quartz was present but the CR3 mobilization was unaffected. Our results indicate that quartz induces a granule protease-dependent selective shedding of CR1 but not CR3 despite a low degree of degranulation.

Several studies indicate that the actin filament system is not only involved in cell motility, but also in the regulation of other neutrophil functions. The aim of the present investigation was to examine the mechanisms by which actin filament formation participates in the control of the respiratory burst and degranulation in human neutrophils. The approach taken was to use both an inhibitor (cytochalasin B) and a potentiator (tetracaine) of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe)-induced actin polymerization. The total inhibition of fMet-Leu-Phe-induced actin polymerization in cytochalasin B-treated cells was accompanied by an impressive potentiation of both oxidase activity and degranulation (azurophilic and specific granules). However, preincubation with tetracaine, which causes an enhanced accumulation of F-actin in the periphery of fMet-Leu-Phe-stimulated cells, also augmented the rate and duration of peptide-induced superoxide anion production, but inhibited degranulation (specific granules). A likely explanation for the potentiating effects of cytochalasin B and tetracaine is provided by our observation that both of these substances reduced the acid-resistant binding of fMet-Leu-Phe (interpreted as a decrease in the internalization of fMet-Leu-Phe-receptor-complexes), resulting in an enhanced formation of second messengers (diacylglycerol). The findings that tetracaine potentiated the activity of the oxidase, whereas it inhibited degranulation (specific granules), suggest that actin polymerization per se plays a role in the latter process. Consequently, this study argues against the idea of a direct inhibitory effect of F-actin on chemotactic factor-induced oxidase activation, but supports an active role of actin filaments in the translocation and release of granule components.

In the presence of dimethyl sulfoxide (DMSO), the leukemic promyelocytic cell line HL-60 will differentiate into mature polymorphonuclear granulocytes. In the present report, we compare chemotactic factor binding and function in HL-60 cells with that of normal human granulocytes using the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) as ligand. The cellular response measured as CL was changed as a result of storage or conditioning of normal peripheral blood cells. With these cells, a conditioning procedure at room temperature resulted in a pronounced increase in the CL response. The increase of the CL response was probably a result of increased expression of cryptic receptors, since the changes of the oxidative response to FMLP was accompanied by increased binding of the peptide to the cell surface. Scatchard analysis revealed that the increased binding was due to an increased number of receptors. In differentiated HL-60 cells, conditioning neither led to increased production of oxidative metabolites, nor to any increased binding of the peptide. The data thus indicate that many FMLP receptors could reside in a cryptic site that is not accessible to extracellular ligands, and that conditioning results in an increased exposure of these receptors, followed by an increased oxidative response to the ligand in normal cells but not in mature HL-60 cells.

Both the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and the calcium ionophore ionomycin induced a metabolic response in normal neutrophils. However, the presence of azide, a potent inhibitor of the hydrogen peroxide-consuming enzymes catalase and myeloperoxidase, was required to detect any release of hydrogen peroxide induced by ionomycin. In differentiated HL-60 cells, only FMLP stimulation was associated with any notable metabolic activation. The response to FMLP proceeds with a rate and time course similar to that seen in normal cells. The use of ionomycin as a stimulating agent did not result in any detectable activation of the system that generates reactive oxygen metabolites, even if azide was present in the measuring system. Raising the concentration of cytoplasmic free Ca2+ is therefore not sufficient to activate the system responsible for the generation of reactive oxygen metabolites in HL-60 cells. However, preincubation with ionomycin primed HL-60 cells to an increased response during stimulation with the chemotactic peptide FMLP and the phorbol ester PMA. Since HL-60 cells lack specific granules but have an intact ligand-receptor coupling mechanism, a role for the subcellular granule is proposed, in the generation of reactive oxygen species in normal granulocytes, and analysis of the data presented leads to two conclusions: 1) FMLP, which acts through cells surface receptors, causes the cells to produce oxygen radicals, which to a large extent are released from the cells, a process that is not dependent on the specific granule content of the cells, whereas 2) ionomycin, which bypasses cell-surface receptors, is also capable of stimulating an oxygen-radical formation that is granule dependent and retained inside the cells. Furthermore, the results suggest that an increase in intracellular Ca2+ is not sufficient to initiate activation of the plasma membrane-bound system that generates reactive oxygen metabolites, but the results support a role for Ca2+ in the priming event.

Increased production of oxidative metabolites following interaction between mononuclear phagocytes and soluble stimuli can be measured as luminol-amplified chemiluminescence (CL). The effects of superoxide dismutase (SOD), catalase, and azide on the monocyte CL response were investigated. Azide, a myeloperoxidase (MPO) inhibitor, reduced the CL reaction by more than 80%, which indicates that the CL reaction is dependent on the granule enzyme MPO. Because SOD and catalase only partly inhibited the monocyte CL response, the authors propose that part of the monocyte CL response is of intracellular origin. This conclusion is further supported by the effects on the CL response obtained by adding extra peroxidase and the lack of correlation with techniques measuring only extracellular generated metabolites. However, it should be pointed out that the relation between extracellular and intracellular activity is stimulus dependent. Furthermore, even if quantitative differences exist between monocyte and granulocyte CL, the mechanism for the light-generating reaction seems to be the same in both cell types.