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Biomedical subjects

B Dewald

Publications and source records attributed to B Dewald.

At least 37 records · Page 2Linked to original sources
PubMed1995

Inhibition of the arachidonic acid pathway prevents induction of IL-8 mRNA by phorbol ester and changes the release of IL-8 from HL 60 cells: differential inhibition of induced expression of IL-8, TNF-alpha, IL-1 alpha, and IL-1 beta.

The promyelocytic HL 60 cell line can be used as an in vitro model system to study hematopoetic cell differentiation and inflammatory events. We studied the signal transduction pathway of induced interleukin (IL)-8 expression and compared it with those of tumor necrosis factor alpha (TNF-alpha), IL-1 alpha, and IL-1 beta. The differentiation of HL 60 cells to macrophage-like cells by PMA resulted in a rapid and marked induction of these inflammatory cytokines. The up-regulation occurred in the absence of ongoing protein synthesis, but cycloheximide-sensitive gene products modulated their induction kinetics. Staurosporine, a potent inhibitor of protein kinases, strongly inhibited their gene expression. Phosphorylation may not act directly on latent transcription factors, since bromophenacyl bromide, an inhibitor for the release of arachidonic acid from phorbol-12 myristate 13-acetate (PMA)-stimulated HL 60 cells, markedly depressed the induced mRNAs for IL-8, TNF-alpha, and IL-1 alpha and -beta. Similarly, 5,8,11,14 eicosatetraynoic acid (ETYA), another inhibitor of the arachidonic acid pathway, blocked the induction of transcripts for TNF-alpha, and both IL-1 genes in phorbol ester-stimulated HL 60 cells. In contrast, ETYA increased the induced IL-8 RNA levels and stimulated the release for IL-8. Also, ketoconazole, an inhibitor of 5-lipoxygenase and indomethacin, an inhibitor of cyclooxygenases did not block the induction of IL-8 mRNA. However, the release of IL-8 protein was regulated by indomethacin and ketoconazole. Our results indicate that arachidonic acid metabolites are mediators in the signal transduction pathway of IL-8 expression and that the involved second messengers are different from those which are important for the induction of TNF-alpha and IL-1 beta expression.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1995

Structure-activity relationships of chemokines.

Structural analysis of chemokines has revealed that the alpha/beta structural-fold is highly conserved among both the CXC and CC chemokine classes. Although dimerization and aggregation is often observed, the chemokines function as monomers. The critical receptor binding regions are in the NH2-terminal 20 residues of the protein and are the least ordered in solution. The flexible NH2-terminal region is the most critical receptor binding site and a second site also exists in the loop that follows the two disulfides. The well-ordered regions are not directly involved in receptor binding but, along with the disulfides, they provide a scaffold that determines the conformation of the sites that are critical for receptor binding. These general requirements for function are common to all the chemokines. For the CC chemokines, receptor activation and receptor binding regions are separate within the 10 residue NH2-terminal region. This has allowed identification of high affinity analogs that do not activate the receptor and are potent antagonists.

Amino Acid Sequence↗
PubMed1995

Methotrexate action in rheumatoid arthritis: stimulation of cytokine inhibitor and inhibition of chemokine production by peripheral blood mononuclear cells.

This open label study examines whether methotrexate (MTX) treatment modulates ex vivo synthesis of interleukin-1 receptor antagonist (IL-1ra), soluble tumour necrosis factor receptors (sTNFR p55 and p75), interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by peripheral blood mononuclear cells (PBMC) and whether changes reflect clinical response. Significant stimulation of IL-1ra and sTNFR p75 as well as inhibition of IL-8 production of PBMC were associated with clinical improvement observed in patients treated with MTX. When defining the characteristics of patients at study entry retrospectively in responders and non-responders, a significantly lower ratio of IL-1ra:IL-1 beta production before and its increase upon treatment was associated with clinical response in 13 patients compared to five patients not responding to MTX. In addition, clinical improvement was associated with decreased synthesis of IL-1 beta, TNF-alpha and IL-8 induced by bacterial lipopolysaccharide, IL-1 alpha and IL-1 beta in PBMC in vitro. These findings suggest that MTX therapy reverses the inflammatory type of rheumatoid arthritis (RA) blood mononuclear cells by stimulating cytokine inhibitor production while inhibiting inflammatory cytokine release at the same time. This may explain the powerful anti-inflammatory properties of low-dose MTX as observed in most RA patients. Pretreatment determination of the IL-1ra:IL-1 beta ratio in PBMC may be predictive with regard to a favourable therapeutic response and therefore may be useful for the selection of RA patients to be treated with MTX.

Adult↗
PubMed1994

Further characterization of the gelatinase-containing particles of human neutrophils.

In addition to azurophil and specific granules, a third storage compartment is known to exist in the neutrophils. This compartment which consists of morphologically heterogeneous particles is characterized by a high specific activity in gelatinase. A gelatinase enriched fraction was prepared by subcellular fractionation of neutrophil homogenates using rate zonal centrifugation. This fraction was enriched in diamine oxidase. Among the proteins released from the neutrophils upon stimulation by formyl peptides, those belonging to the gelatinase enriched fraction were determined after removal of the proteins from specific and azurophil granules by selective immunoadsorption. Gelatinase was recovered together with tetranectin, beta 2-microglobulin and diamine oxidase in the same fraction. Differences in the kinetics of release of gelatinase and diamine oxidase versus vitamin B-12-binding protein suggest that the proteins belong to distinct subcellular structures.

Amine Oxidase (Copper-Containing)↗
PubMed1994

Structural requirements for interleukin-8 function identified by design of analogs and CXC chemokine hybrids.

Structure-activity relationships of human interleukin-8 (IL-8) were probed using chemically synthesized analogs with single or double amino acid substitutions, as well as hybrids derived by substituting IL-8 regions into IP10, a related protein that lacks IL-8 activity. The analogs were tested for functional activity by measuring induction of elastase release from human neutrophils and competition for binding of radiolabeled IL-8. The hybrid studies indicated that Gly31 and Pro32, as well as the NH2-terminal region from IL-8 are required to convert IP10 into a fully functional protein, suggesting that these elements are critical for IL-8 activity. Both disulfide bridges, linking residue 7 to 34 and residue 9 to 50, were critical for function, as shown by substituting the cysteine pairs with alpha-aminobutyric acid. Single conservative substitutions were generally accepted into the 10-22 region of IL-8, which contrasts with the ELR motif (residues 4-6), previously shown to be essential for activity. The importance of residues within the 10-15 region and the 17-22 region was demonstrated with hybrids. In addition, some of the 4-22 residues have structural roles that may be important; for example, Tyr13, Phe17, and Phe21 are involved in aromatic interactions in the IL-8 structure, and are also moderately sensitive to modification. Except for Cys50, the results argue against a role for the 36-72 region, including the COOH-terminal alpha-helix, in receptor binding. We conclude that the disulfide bridges and 30-35 turn provide a structural scaffold for the NH2-terminal region which includes the primary receptor-binding site (the ELR motif) and secondary binding and conformational determinants between residues 10 and 22.

Amino Acid Sequence↗
PubMed1994

Neutrophil activation by monomeric interleukin-8.

Interleukin-8 (IL-8), a pro-inflammatory protein, has been shown by nuclear magnetic resonance (NMR) and x-ray techniques to exist as a homodimer. An IL-8 analog was chemically synthesized, with the amide nitrogen of leucine-25 methylated to selectivity block formation of hydrogen bonds between monomers and thereby prevent dimerization. This analog was shown to be a monomer, as assessed by analytical ultracentrifugation and NMR. Nevertheless, it was equivalent to IL-8 in assays of neutrophil activation, which indicates that the monomer is a functional form of IL-8.

Calcium↗
PubMed1994

Monoclonal antibody based enzyme-linked and chemiluminescent assays for the human interleukin-1 receptor antagonist. Application to measure hIL-1ra levels in monocyte cultures and synovial fluids.

Interleukin-1 receptor antagonist (IL-1ra) has the potential to counteract at least part of the biological effects of interleukin-1. The outcome of an inflammatory reaction may therefore be determined by the balance between IL-1 and IL-1ra, rather than by IL-1 alone. We have developed an immunoassay to address this issue as well as to assess the effects of anti-inflammatory agents on the expression of IL-1 and IL-1ra in vitro or in body fluids. Recombinant human IL-1ra was expressed in an E. coli system, purified to homogeneity, and used to derive monoclonal antibodies in mice as well as polyclonal antibodies in rabbits. A sandwich ELISA was constructed with F(ab')2 fragments of a high affinity monoclonal antibody and the rabbit serum as a source of secondary antibody. The assay required no sample treatment to avoid interference by rheumatoid factor. The measuring range was 0.020-2 ng/ml. By labelling a second monoclonal antibody with an acridinium ester, a chemiluminescence assay with a wider measuring range (0.050-15 ng/ml) was generated. In accord with published data, we found that IL-1ra was secreted by human monocytes stimulated with LPS, Zymosan, IL-1 alpha, or human IgG. After an induction phase of ca. 4 hours and depending on the stimulus, IL-1ra accumulated linearly for periods up to 96 h. IL-1ra levels in synovial fluids of 19 patients suffering from various inflammatory joint diseases were compared with the cytokine levels of IL-1 beta, IL-6, IL-8, and TNF-alpha. Highest positive correlations were found with IL-8 and IL-1 beta. In normal blood donors IL-1ra serum levels were 150-800 pg/ml (Median: 387 pg/ml). Owing to its sensitivity and large measuring range the newly developed assays appear to be suitable for measuring IL-1ra in cell cultures as well as in biological fluids.

Animals↗
PubMed1994

Production of interleukin-1 receptor antagonist, inflammatory chemotactic proteins, and prostaglandin E by rheumatoid and osteoarthritic synoviocytes--regulation by IFN-gamma and IL-4.

This study analyzes the effects of the T cell cytokines IL-4 and IFN-gamma on the spontaneous and stimulated production of IL-8, MCP-1, IL-1 receptor antagonist (IL-1ra), and PGE by synoviocytes from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Cells from both sources constitutively released IL-8 and MCP-1, but no IL-1ra or PGE. Stimulation with IL-1 beta or TNF-alpha massively increased chemokine production and induced the generation of PGE and low amounts of IL-1ra. The constitutive or cytokine-stimulated release of IL-8 was inhibited by IFN-gamma, but not by IL-4. The constitutive or IL-1 beta-stimulated release of MCP-1, by contrast, was markedly enhanced by IL-4 and IFN-gamma. Both cytokines, however, had only borderline effects on the release stimulated by TNF-alpha. The yield of IL-1ra was strongly enhanced by IFN-gamma in all cases, whereas the effect of IL-4 was pronounced only in IL-1 beta-stimulated OA synoviocytes. IL-4, on the other hand, markedly decreased the release of PGE, which was less susceptible to IFN-gamma. The observed effects on chemokines, IL-1ra expression, and PGE release by synoviocytes suggest that IFN-gamma and IL-4 are important regulatory elements in the inflamed synovium and may exert anti-inflammatory effects.

Arthritis, Rheumatoid↗
PubMed1994

Monocyte chemoattractant protein 1 and interleukin 8 production by rheumatoid synoviocytes. Effects of anti-rheumatic drugs.

Activated synoviocytes are major effector cells in the pathogenesis of rheumatoid arthritis (RA) because of their capacity to secrete a variety of inflammatory mediators. Among these mediators, the chemotactic proteins monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-8) are likely to contribute to the recruitment of inflammatory cells into the arthritic joint. We examined the effects of anti-rheumatic drugs on the MCP-1 and IL-8 production by cultured RA synoviocytes exposed to pro-inflammatory agonists. Both chemotactic cytokines were quantified by specific enzyme-linked immunosorbent assays (ELISA), and found to accumulate in the culture supernatants. Although the time course of formation was similar, the yield of IL-8 was three to 10-fold higher than that of MCP-1. Non-steroidal anti-inflammatory drugs inhibited the synthesis of prostaglandins, but did not influence the production and release of both chemotactic cytokines. Of three disease-modifying drugs tested, dexamethasone and gold sodium thiomalate (GST) inhibited the production of IL-8 and MCP-1, while methotrexate (MTX) was inactive. Dexamethasone reduced the production of MCP-1 and IL-8 by 20-65% and 60-80%, respectively, whilst GST inhibited MCP-1 and IL-8 synthesis in suboptimally, but not in optimally stimulated synoviocytes. Taken together, these results show that the production of MCP-1 and IL-8 is similarly affected by anti-rheumatic drugs and that dexamethasone is the most potent inhibitor suggesting that part of the anti-rheumatic action of glucocorticoids is due to prevention of accumulation of chemotactic cytokines acting on neutrophils and monocytes.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1993

Constitutive expression of interleukin-8 and its receptor in human myeloid and lymphoid leukemia.

Interleukin-8 (IL-8), a member of the family of small inducible cytokines, is mainly known for its striking neutrophil-activating properties. Constitutive IL-8 production is negligible in normal leukocytes. We examined expression of IL-8 and its receptor in purified leukemic cells from patients with untreated acute myeloblastic leukemia (AML) and lymphoid leukemias. In the majority of cases (18 of 26 AML, 8 of 15 lymphoid leukemias), the cells constitutively expressed IL-8 mRNA transcripts. In all but 3 of these cases, IL-8 mRNA-expressing cells secreted biologically active IL-8 protein. Immunocytochemical analysis showed intracellular IL-8 (5% to 90% of total cells), demonstrating that the leukemic cells themselves rather than contaminants (monocytes or lymphocytes) were the source of IL-8. Ten of 25 AML samples expressed IL-8 receptor mRNA and, with 1 exception, the IL-8 receptor expressing cells also produced its ligand. In contrast, all lymphoid leukemias were negative. Furthermore, frequent coexpression of IL-8 and IL-1 beta transcripts was seen in both AML and lymphoid leukemia samples, whereas fewer cases coexpressed IL-8 and either macrophage colony-stimulating factor or granulocyte-macrophage colony-stimulating factor. In leukemic cells expressing the IL-8 receptor, IL-8 induced cytosolic free calcium changes, indicating activation of the classical signaling pathway. These results suggest that IL-8 may have biologic activities in hematopoiesis.

Blotting, Southern↗
PubMed1993

The interleukin-8-related chemotactic cytokines GRO alpha, GRO beta, and GRO gamma activate human neutrophil and basophil leukocytes.

GRO alpha, a protein structurally related to interleukin-8 (IL-8) and originally described as a melanoma growth stimulatory factor, possesses potent neutrophil-stimulating activity. Recently, two closely related genes, gro beta and gro gamma, were identified. In the present work, the three GRO proteins were chemically synthesized, and their biological activities on human neutrophils and other leukocytes were compared. GRO alpha, GRO beta, and GRO gamma, like IL-8, induced chemotaxis, shape change, a rise in intracellular free calcium levels, exocytosis, and the respiratory burst in neutrophils. The GRO proteins were also active toward basophils as shown by chemotaxis and intracellular calcium concentration changes. The order of potency in neutrophils and basophils was IL-8 > GRO alpha > or = GRO gamma > GRO beta. Of the two IL-8 receptors expressed on human neutrophils, one binds GRO alpha with high and the other with low affinity. Competition binding experiments using radiolabeled IL-8 and GRO alpha revealed the same characteristics for GRO beta and GRO gamma. Similarly, cross-desensitization, as assessed by the stimulus-dependent changes in intracellular calcium concentration, indicated that all three GRO proteins interact with common receptors. From these results, it can be concluded that GRO alpha, GRO beta, and GRO gamma have the same pattern of activity toward human granulocytes and that the differences in amino acid sequence among these proteins have only minor effects on biological activity.

Amino Acid Sequence↗
PubMed1993

Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg.

Amino acid deletion and mutagenesis experiments have indicated that the sequences Glu-Leu-Arg (ELR) preceding the first cysteine at the N terminus of interleukin 8 (IL-8) is required for receptor binding and neutrophil activation. Platelet factor 4 (PF4) is structurally related to IL-8 (35% sequence identity) but lacks the N-terminal ELR sequence and comparable effects on neutrophils. We introduced the ELR sequence at the N terminus of PF4 and found that the modified protein was a potent neutrophil activator and attractant. On the other hand, when the ELR sequence was introduced into the corresponding positions of two other proteins related to IL-8, gamma-interferon-inducible protein IP10 and monocyte chemoattractant protein 1, neither of them acquired neutrophil-activating properties, indicating that besides ELR additional structural determinants of IL-8 and PF4 are important for binding to IL-8 receptors. The conservation of these binding determinants suggests that PF4 may have evolved from a neutrophil activating protein.

Amino Acid Sequence↗
PubMed1993

Interleukin-8 antagonists generated by N-terminal modification.

We have previously shown that the residues Glu4-Leu5-Arg6 (ELR) preceding the first cysteine at the N terminus of the 72-residue form of interleukin-8 (IL-8) are essential for receptor binding and neutrophil activation (Clark-Lewis, I., Schumacher, C., Baggiolini, M., and Moser, B. (1991) J. Biol. Chem. 266, 23128-23134). We have now synthesized a series of analogs of IL-8(4-72), the truncated form of IL-8 with the N-terminal sequence ELRC, as potential IL-8 antagonists. Among 26 analogs with deletions or amino acid replacements in the ELR region several inhibited IL-8 function. The most potent were IL-8(6-72), with Arg6 at the N terminus, and IL-8,AAR(7-72) with N-terminal Ala4-Ala5 instead of Glu4-Leu5. They inhibited IL-8 receptor binding, exocytosis (IC50 0.3 microM), as well as chemotaxis and the respiratory burst. Inhibition was restricted to responses elicited by IL-8, GRO alpha, or NAP-2, and no effect was observed when the unrelated agonists fMet-Leu-Phe or C5a were used as stimuli. These results demonstrate that selective antagonists that prevent or attenuate the action of IL-8 and its related chemotactic cytokines are obtained by modification of the ELR sequence at the N terminus.

Amino Acid Sequence↗
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↗