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Biomedical subjects

B Dewald

Publications and source records attributed to B Dewald.

At least 19 recordsLinked to original sources
PubMed1992

G-protein activation by interleukin 8 and related cytokines in human neutrophil plasma membranes.

Interleukin 8 (IL-8) is a member of the rapidly growing superfamily of those cytokines which are thought to be involved in the regulation of inflammatory processes and cell proliferation. In neutrophils, IL-8 triggers a variety of cellular responses by interacting with specific cell-surface receptors. To examine whether IL-8 receptors are coupled to activation of guanine-nucleotide-binding proteins (G-proteins), we have investigated the influence of IL-8 on GTP hydrolysis by and guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to purified human neutrophil plasma membranes. IL-8 stimulated high-affinity GTPase about 2-fold at 100 nM, and half-maximal stimulation was observed at 1 nM. The peptide-stimulated GTPase was confined to plasma membranes upon subcellular fractionation, and was due to an increase in Vmax. rather than a decrease in Km. High-affinity binding of GTP[35S] to neutrophil plasma membranes was stimulated half-maximally and maximally (up to 5-fold) by IL-8 at about 10 nM and 100 nM respectively. GTP[35S] binding to the membranes was also stimulated by two IL-8-related cytokines, neutrophil-activating peptide 2 (NAP-2) and melanoma growth-stimulatory activity (gro/MGSA). Taken together, these results demonstrate that receptors for IL-8 and related cytokines are coupled to and activate G-proteins in neutrophil plasma membranes, indicating that G-protein activation is an important intermediate step in the induction of neutrophil functions by IL-8 and its congeners.

Cell Fractionation↗
PubMed1992

Glucocorticoids downregulate gene expression of GM-CSF, NAP-1/IL-8, and IL-6, but not of M-CSF in human fibroblasts.

Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), neutrophil-activating peptide-1/interleukin-8 (NAP-1/IL-8), and interleukin-6 (IL-6) are pivotal in the regulation of hematopoiesis and immune responses. In mesenchymal cells, their expression is induced by tumor necrosis factor alpha (TNF) and other agents. We now show that, while induction of cytokine expression by TNF in human lung fibroblasts was parallel, glucocorticoid hormones differentially affected their production. Dexamethasone (1 mumol/L) concordantly repressed expression of GM-CSF, NAP-1/IL-8 and IL-6. RNA and protein levels were reduced to approximately 5%, 20%, and 30% of control cells, respectively, as determined by Northern blot analyses and immunoassays. A 50% reduction of RNA levels for all three cytokines occurred in the range of 1 hour. In contrast, dexamethasone (1 mumol/L) did not decrease M-CSF RNA levels and protein release. M-CSF RNA and protein levels were maintained even when dexamethasone (1 mumol/L) was present for the whole duration of a 48-hour TNF stimulation. Further experiments showed that dexamethasone downregulates expression of GM-CSF, NAP-1/IL-8, and IL-6 mainly by decreasing the mRNA stability of these cytokines, and that the dexamethasone-mediated repression of cytokine expression depends on ongoing protein and RNA syntheses. Our study suggests that glucocorticoid hormones repress expression of a set of cytokine genes important in conditions of stress. However, they seem not to affect M-CSF expression, which is likely to be more crucial in maintaining long-term functions of myeloid cells.

Blotting, Northern↗
PubMed1992

Interleukin-8 in inflammatory rheumatic diseases: synovial fluid levels, relation to rheumatoid factors, production by mononuclear cells, and effects of gold sodium thiomalate and methotrexate.

The content of interleukin-8 (IL-8) in the synovial fluid and its production by blood and synovial fluid mononuclear cells (PBMC and SFMC) was compared in rheumatoid arthritis (RA) and various other inflammatory rheumatic disorders. The study included 125 patients and 20 healthy individuals. The highest concentrations of IL-8 were found in the synovial fluids and culture supernatants of PBMC and SFMC from patients with seropositive RA. Only PBMC from seropositive patients, and not from other rheumatic diseases, exhibited significant spontaneous release of IL-8 that correlated with serum IgM rheumatoid factor titers. Gold sodium thiomalate (GST) and methotrexate (MTX) inhibited the spontaneous and stimulated IL-8 production by PBMC by 55-86% at 50 and 10 micrograms/ml, respectively. Two main conclusions were drawn: (1) rheumatoid factors appeared to be a major cause of enhanced IL-8 production in seropositive RA, and (2) inhibition of IL-8-mediated neutrophil migration and activation could be part of the mechanism of action of GST and MTX.

Anti-Inflammatory Agents↗
PubMed1992

IP-10, a gamma-interferon-inducible protein related to interleukin-8, lacks neutrophil activating properties.

IP-10, a small, gamma-interferon-inducible protein with structural homology to interleukin-8 (IL-8), was prepared by automated chemical synthesis and compared with synthetic IL-8, GRO alpha and neutrophil-activating peptide 2 (NAP-2) for neutrophil stimulating activity. The following functions were tested: cytosolic free calcium changes, chemotaxis in vitro, respiratory burst, exocytosis of azurophil and specific granules from cytochalasin B-pretreated cells, and competition for IL-8 receptor binding. At 1, 10, 100 and 1000 nM, IP-10 was inactive in all assays, in contrast to the reference peptides which exhibited the expected neutrophil stimulating effects. In addition, IP-10 did not induce neutrophil accumulation after intradermal injection in rats, and did not act as IL-8 antagonist.

Calcium↗
PubMed1991

Phagocytosing neutrophils produce and release high amounts of the neutrophil-activating peptide 1/interleukin 8.

After phagocytosis of yeast opsonized with IgG, neutrophil leukocytes (polymorphonuclear leukocytes [PMN]) expressed high levels of neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8) mRNA, which peaked after 3-5 h and were still elevated after 18 h. A similar but quantitatively less prominent effect was obtained with lipopolysaccharide (LPS). After phagocytosis, but not after exposure to LPS, the PMN progressively released considerable amounts of NAP-1/IL-8 into the culture medium (18.6-50 ng/ml in 18 h). The peptide released was biologically active, as indicated by the transient elevation of cytosolic-free calcium in PMN exposed to aliquots of the culture supernatants, and desensitization by prestimulation of the cells with recombinant NAP-1/IL-8. By producing NAP-1/IL-8 at sites where they phagocytose invading microorganisms, PMN could enhance the recruitment of new defense cells.

Calcium↗
PubMed1991

Human neutrophil migration into skin chambers is associated with production of NAP-1/IL8 and C5a.

Respiratory burst activity initiated by the chemoattractants fMLP, rC5a and rNAP-1/IL8 was investigated in human exudated and peripheral blood neutrophils. Exudated cells were isolated after migration into a skin chamber and the respiratory burst activity was measured as chemiluminescence amplified by luminol and horseradish peroxidase. The response to fMLP (5 x 10(-8) mol/l) was significantly enhanced (p less than 0.01) in the exudated cells but was significantly decreased after stimulation (5 x 10(-8) mol/l) with rC5a and rNAP-1/IL8 (p less than 0.05 and p less than 0.01, respectively). Analysis revealed that, in the chamber fluid, the activated complement C5a was generated during exudation (p less than 0.01). Determinations of NAP-1/IL8 showed that this substance was also produced and released into the chamber fluid (p less than 0.01). No correlation was found between the number of exudated cells and the amount of C5a or NAP-1/IL8 in the exudation fluid, thus indicating that, in vivo, the exudation process is controlled by multiple factors and not by the quantity of a single chemoattractant. The present study shows that NAP-1/IL8 and C5a are produced in humans during an aseptic inflammation, and that this occurs in parallel to the migration of neutrophils into the skin chambers. The significant desensitization of the exudated cells to NAP-1/IL8 and C5a reflects a previous exposure to these attractants. These results suggest that the novel tissue-derived cytokine NAP-1/IL8 plays a role in human neutrophil exudation in vivo.

Cell Movement↗
PubMed1991

Neutrophil-activating peptide-1/interleukin-8 detection in human urine during acute bladder inflammation caused by transurethral resection of superficial cancer and bacillus Calmette-Guérin administration.

Neutrophil-activating peptide-1/interleukin-8 (NAP-1/IL-8), secreted by monocytes, macrophages and a number of other cells, acts as a chemoattractant for neutrophil leukocytes and stimulates them to produce a series of responses such as shape change, adherence, exocytosis and respiratory burst, events that are of importance in inflammation. To study the release of NAP-1/IL-8, two human models of inflammation were chosen: transurethral resection of superficial bladder cancer and the subsequent instillation of bacillus Calmette-Guérin (BCG), performed in order to reduce the recurrence rate of papillary bladder tumors. As the secretions of the bladder wall are retained in the urine, patients' urine was collected during 4-hour periods. These urine samples were chromatographed on phosphocellulose. In the elution fractions NAP-1/IL-8 was quantified by a bioassay that measured the elastase release by human neutrophils. The neutrophil-stimulating activity was further purified by reverse phase high performance liquid chromatography. Although no NAP-1/IL-8 activity could be detected in normal individuals, formation of this inflammatory cytokine was observed in patients after transurethral resection and after BCG treatment. The significance and possible use of this secretion are discussed.

Administration, Intravesical↗
PubMed1991

Enhanced production of neutrophil-activating peptide-1/interleukin-8 in rheumatoid arthritis.

Production of the neutrophil-activating peptide (NAP)-1/IL-8 by mononuclear phagocytes from patients with RA and from control subjects was studied under various conditions. Mononuclear cells from bone marrow (BMMC), PBMC, and synovial fluid (SFMC) were cultured for up to 48 h in the absence or presence of Escherichia coli LPS, different interleukins, interferon-gamma, zymosan, or immune complexes, and the neutrophil-stimulating activity released into the culture medium was determined. As shown by neutralization with an antiserum raised against human recombinant NAP-1/IL-8, over 90% of this activity could be attributed to NAP-1/IL-8. In unstimulated mononuclear cells from control individuals and BMMC from RA patients, the production of NAP-1/IL-8 was very low and was enhanced moderately by stimulation with LPS. By contrast, the spontaneous production of NAP-1/IL-8 was 3- to 10-fold higher in PBMC and even much higher in SFMC from RA patients. In all instances, the yield of NAP-1/IL-8 could be enhanced by stimulation in culture. In addition to LPS, rheumatoid factor-containing immune complexes, zymosan, and IL-1 were highly effective in inducing NAP-1/IL-8 production, while IL-3, GM-CSF, tumor necrosis factor (TNF), and IL-2 were somewhat less potent. An inhibitory effect was obtained with IFN-gamma, which significantly decreased the spontaneous NAP-1/IL-8 release from SFMC and the IL-1- and LPS-induced NAP-1/IL-8 from RA and control PBMC. Inhibition was also observed with glucocorticoids. The production of NAP-1/IL-8 was markedly reduced by dexamethasone in phagocytosis-stimulated PBMC, and almost totally inhibited in SFMC obtained from joints after intraarticular administration of betamethasone. By contrast, the cyclooxygenase inhibitor, indomethacin, tended to increase the NAP-1/IL-8 yield from PBMC in culture.

Arthritis, Rheumatoid↗
PubMed1991

[Purification of gelatinase secreted by human polynuclear neutrophils].

Gelatinase was purified from DFP treated human neutrophils which have been stimulated by the chemotactic peptide, N-formyl-methionyl-leucylphenylalanine. The secreted gelatinase was purified in two major steps: a gelatinase enriched fraction was recovered after a specific immunoadsorption of contaminant proteins from cytosol and immunoadsorption of proteins from specific or azurophilic granules; then gelatinase was isolated by affinity chromatography and FPLC gel filtration. Some kinetic properties of the Mr 94,000 purified enzyme were investigated.

Catalysis↗
PubMed1989

Staurosporine inhibits the respiratory burst and induces exocytosis in human neutrophils.

The protein kinase C inhibitor staurosporine influenced in different ways the functions of human neutrophils. Staurosporine prevented the enhanced protein phosphorylation in phorbol ester- and N-formylmethyionyl-leucylphenylalanine (fMLP)-stimulated cells, and was a powerful inhibitor of the respiratory burst induced by phorbol myristate acetate [IC50 (concentration causing 50% inhibition) 17 nM] and the chemotactic peptides fMLP and C5a (IC50 24 nM). It did not alter, however, the superoxide production by cell-free preparations of NADPH oxidase. Staurosporine had no effect on agonist-dependent changes in cytosolic free Ca2+ and exocytosis of specific and azurophil granules, and showed only a slight inhibition of the release of vitamin B12-binding protein induced by phorbol myristate acetate (decreased by 40% at 200 nM). On the other hand, staurosporine also exhibited neutrophil-activating properties: it induced the release of gelatinase (from secretory vesicles) and vitamin-B12-binding protein (from specific granules). These effects were protracted, concentration-dependent, insensitive to Ca2+ depletion, and strongly enhanced by cytochalasin B. Staurosporine, however, did not induce the release of beta-glucuronidase or elastase (from azurophil granules). Except for the sensitivity to cytochalasin B, these properties suggest a similarity between the exocytosis-inducing actions of staurosporine and PMA. The results obtained with staurosporine provide further evidence that different signal-transduction processes are involved in neutrophil activation, and suggest that protein phosphorylation is required for the induction of the respiratory burst, but not for exocytosis.

Alkaloids↗
PubMed1989

Effects of the neutrophil-activating peptide NAP-2, platelet basic protein, connective tissue-activating peptide III and platelet factor 4 on human neutrophils.

Platelet basic protein (PBP), connective tissue-activating peptide III (CTAP-III), and platelet factor 4 (PF-4) were purified from human platelet release supernatants by heparin-Sepharose ion-exchange and reversed-phase HPLC, and their neutrophil-activating effects were compared with those of NAP-2, a peptide of 70 amino acids corresponding to part of the sequence of PBP (1) and with sequence homology to NAF/NAP-1. NAP-2-induced elastase release and a rise in cytosolic free Ca2+ at concentrations between 0.3 and 100 nM, and neutrophil chemotaxis at concentrations between 0.03 and 10 nM. It was half as potent as NAF/NAP-1 in inducing exocytosis but showed the same activity in the other responses. By contrast, only minimal if any effects were obtained with PBP, CTAP-III, and PF-4 up to 100 nM. NAP-2 thus appears to behave like a typical chemotactic receptor agonist. It could be generated from PBP and/or CTAP-III released from activated platelets and lead to the accumulation of neutrophils in platelet aggregates.

Amino Acid Sequence↗
PubMed1989

The neutrophil-activating peptide NAF/NAP-1 induces histamine and leukotriene release by interleukin 3-primed basophils.

IgE-independent mediator release from basophils is considered an important event in inflammation, particularly in nonallergic immediate hypersensitivity and in allergic late-phase reactions. This study demonstrates that after exposure to IL-3, basophils release histamine and leukotrienes in response to the neutrophil-activating peptide NAF/NAP-1. Thus, the sequential action of two pure cytokines can promote basophils mediator release. In the presence of IL-3, NAF/NAP-1 functions like a "histamine-releasing factor" and may therefore not only induce cellular infiltration but also provoke symptoms of hypersensitivity reactions.

Basophils↗
PubMed1989

Histamine and sulfidoleukotriene release from human basophils: different effects of antigen, anti-IgE, C5a, f-Met-Leu-Phe and the novel neutrophil-activating peptide NAF.

Human blood leukocytes were challenged by IgE-dependent stimuli (antigens or anti-IgE antibodies) and the chemotactic agonists C5a, N-formyl-Met-Leu-Phe (fMLP) or NAF, a novel neutrophil-activating peptide produced by stimulated human monocytes. IgE-dependent stimuli induced abundant release of histamine and sulfidoleukotrienes (LTC, LTD and LTE). The effects of the chemotactic peptides differed considerably: C5a induced high percentages of histamine release already at concentrations as low as 10(-9) M, but no leukotriene formation: NAF was completely inactive up to 10(-6)M, and fMLP induced moderate release of both products at relatively high concentration only (1-2.5 x 10(-6) M). All three chemotactic peptides, however, induced a concentration-dependent release of elastase from cytochalasin-B-treated human neutrophils. The EC50 was approximately 0.3, 2 and 5 x 10(-9) M for C5a, NAF and fMLP, respectively. NAF showed activity over a wider concentration range and its dose-response curve was less steep than that of the two other agonists. Our results suggest that NAF, that may be generated at the sites of hypersensitivity reactions, is likely to induced the immigration of neutrophils, but not the direct activation of basophils and mast cells. In this respect, NAF differs from less selective chemotactic peptides like the complement product C5a or the N-formyl-methionyl peptides.

Antibodies, Anti-Idiotypic↗
PubMed1989

In vivo inflammatory activity of neutrophil-activating factor, a novel chemotactic peptide derived from human monocytes.

Neutrophil-activating factor (NAF), a 72-amino acid peptide produced by human monocytes, induced plasma leakage and neutrophil accumulation after intradermal injection in rabbits (10(-11) to 10(-9) mol/site). NAF was about three times more potent than fMet-Leu-Phe, but considerably less potent than endotoxin. The response to NAF was not inhibited by the endotoxin inhibitor polymyxin B or the protein synthesis inhibitor actinomycin D. Histology of NAF-induced lesions showed large numbers of neutrophils, but no monocytes, eosinophils, basophils, or lymphocytes were observed. Intravascular neutrophil accumulation, aggregate formation, and venular wall damage were also apparent. In vitro, NAF stimulated rabbit neutrophils as shown by the release of N-acetyl-beta-glucosaminidase. This study demonstrates that NAF elicits a rapid inflammatory response in vivo with massive neutrophil emigration, which is qualitatively similar to that observed with other chemotactic agonists.

Animals↗
PubMed1989

[Properties and activation mechanism of neutrophilic leukocytes].

The mechanism of neutrophil activation by chemotactic receptor agonists was studied by monitoring stimulus-induced changes in the cytosolic free calcium concentration and hydrogen peroxide formation during the respiratory burst. Two receptor-dependent signal transduction sequences were identified. One sequence depends on calcium, phospholipase C and protein kinase C, and is rate limiting, while the other is comparatively fast and appears to be calcium-independent. The present studies indicate that both sequences must act in concert to transduce receptor-mediated signals and to activate the NADPH oxidase.

Animals↗