Beatrice Dewald

Interleukin-8 and related chemotactic cytokines--CXC and CC chemokines.

In the production of hollow bodies from a thermoplastic material, in which preforms are produced from thermoplastic material by molding, each resulting preform is transferred to a final blow-mold, and each preform is molded in a blow-mold to produce a desired final body, the production of each preform is carried out to provide the preform with a false neck having at least one positioning lug, and transfer of each preform to the blow-mold is carried out in such a manner as to cause the preform to have a predetermined angular position relative to the blow-mold by the action of its associated positioning lug.

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.

The polymorphonuclear leukocyte

Polymorphonuclear leukocytes (PMNs) are one of the main sources of enzymes responsible for tissue damage in inflammatory processes. These enzymes are stored in two types of cytoplasmic granules. Azurophil granules contain lysosomal hydrolases, neutral serine proteinases, and bactericidal elements (myeloperoxidase and lysozyme). Specific granules contain collagenase, lysozyme and lactoferrin but lack lysosomal hydrolases. PMNs store all four classes of tissue proteinases, carboxyl, thiol and serine proteinases in the azurophil granules, and metallo proteinases in the specific granules. Three serine proteinases have been identified, elastase, cathepsin G and a third enzyme, which together account for a large proportion of the protein of the azurophil granules. In the course of phagocytic events, all these enzymes are released extracellularly. The neutral proteinases degrade proteoglycans and collagen. In vitro, they stimulate B-lymphocytes, which suggests that they may have immuno-potentiating activity when they are released at sites of chronic inflammation.

Release of gelatinase from a novel secretory compartment of human neutrophils.

Gelatinase is a metallo-proteinase that acts specifically on denatured collagen. In human neutrophils, this enzyme is localized in small, morphologically still unidentified storage organelles that are resolved from the specific and the azurophil granules upon subcellular fractionation by differential sedimentation. When neutrophils isolated from freshly drawn blood are exposed to soluble stimuli such as N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, phorbol myristate acetate, or the calcium ionophore A 23187, or are induced to phagocytose opsonized zymosan, they rapidly release gelatinase in large amounts (30-70% of the cellular content in 10 min). When neutrophils from donor blood, which had been stored for 24 h at 4 degrees C are used, extensive release even occurs without added stimuli by simply warming to 37 degrees C. Gelatinase release appears to occur by secretion because it is not dependent on phagocytosis. It is paralelled by the release of specific granule contents (vitamin B(12)-binding protein), but is more rapid and much more extensive. It is, however, dissociated from the discharge of azurophil granules (as assessed by beta-glucuronidase). In addition, it was found that gelatinase release does not depend on the activation of the respiratory burst, although the two responses are often observed in parallel. Release is not due to cell damage as the cytoplasmic enzyme lactate dehydrogenase is fully retained. The distinct subcellular distribution and kinetics of release of gelatinase reported in this paper uncover a novel, truly secretory compartment of human neutrophils, which is highly responsive to stimulation. Gelatinase and possibly other enzymes stored in this secretory organelle may be involved in the early events of neutrophil mobilization, the response to chemotactic signals and diapedesis.

Inhibition of the phagocytosis-induced respiratory burst by the fungal metabolite wortmannin and some analogues☆

The sterol-like fungal metabolite wortmannin and a number of natural and chemically-derived analogues were found to block the induction of the respiratory burst during phagocytosis. 17-Hydroxy wortmannin, the most active compound tested, showed a 50% inhibition of the burst in neutrophils and mononuclear phagocytes at concentrations ranging between 0.8 and 17 nM, while wortmannin itself was about half as potent. Chemical derivation showed that a furane structure between ring A and B with adjacent carbonyl functions is essential for activity. At concentrations that entirely prevented superoxide or hydrogen peroxide production, the wortmannins were not cytotoxic and did not inhibit phagocytosis. At even higher concentrations (10 microM), 17-hydroxy wortmannin had no effect on the NADPH oxidase, once activated. This suggests that the wortmannins interfere with the signal transduction sequence initiated by the particulate stimulus and leading to the activation of the respiratory burst oxidase.

Granulocyte chemotactic protein 2 acts via both IL‐ 8 receptors, CXCR1 and CXCR2

Interleukin‐8 (IL‐γ) acts on human neutrophils via two receptors, CXCR1 and CXCR2. It shares CXCR2 with all neutrophil‐activating chemokines, which like IL‐8 have a conserved Glu‐Leu‐Arg (ELR) N‐terminal motif, but is generally considered to be the only relevant agonist for CXCR1. IL‐8 has a basic residue at the sixth position after the second cysteine, which was suggested to contribute to CXCR1 specificity. Among the other ELR chemokines, only granulocyte chemotactic protein 2 (GCP‐2) has such a basic determinant. Using Jurkat cells that stably express either CXCR1 or CXCR2, we studied receptor activation by IL‐8, GCP‐2 epithelial neutrophil‐activating protein 2 (ENA‐78) (which shares 77 % identical amino acids with GCP‐2) and growth‐regulated oncogene α (GROα). At 10 nM and higher concentrations, GCP‐2 and IL‐8 induced significant activation of CXCR1‐expressing cells, but no activity was found with GROα and ENA‐78. As expected, however, all four chemokines had similar activities on CXCR2‐expressing cells. A variant of GCP‐2 in which the basic residue, Arg20, was replaced by a glycine was synthesized. This derivative was ineffective on CXCR1, but was as active as wild‐type GCP‐2 in CXCR2‐expressing cells. GCP‐2 displaced radiolabeled IL‐8 from both receptors with low affinity, and in this respect resembled ENA‐78 and GROα. Our data show that GCP‐2 acts via both IL‐8 receptors and thus appears to be functionally more similar to IL‐8 than to the other ELR chemokines. Activation of CXCR1 appears to depend significantly on the presence of a basic binding determinant close to the second cysteine.

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.

Activation of NADPH oxidase in human neutrophils. Synergism between fMLP and the neutrophil products PAF and LTB4.

The induction of the respiratory burst in human neutrophils by combinations of fMLP and either PAF or LTB4 was studied. Pretreatment with PAF (0.0001 to 10 uM), which by itself did not elicit the burst, greatly enhanced the rate and extent of fMLP-induced superoxide production. A synergism of a different kind was observed with the reversed stimulus sequence: Pretreatment with fMLP made the neutrophils capable to respond to PAF with superoxide production. A moderate enhancement of the fMLP response was also obtained following pretreatment with LTB4. The response of the cells to LTB4, however, was not influenced by fMLP, and no synergism was observed between the two neutrophil products PAF and LTB4. The results of this study demonstrate a marked synergism between fMLP and PAF and suggest that PAF may function as an amplifier of the respiratory burst response of stimulated neutrophils.

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

SummaryThe 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 μg/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.

[8] Solubilization and polyacrylamide gel electrophoresis of me mbrane enzymes with detergents

Publisher Summary Polyacrylamide gel electrophoresis is an excellent and widely employed method for the separation and identification of soluble enzymes, but membrane proteins, including enzymes, present difficulties because of their presumed lipoprotein character. Large lipoprotein molecules or aggregates are unlikely to enter gels composed of 5–10% acrylamide that is the best compromise between firmness and large pore size, or, if they enter, fail to resolve satisfactorily. Consequently, it is necessary to solubilize or disaggregate the membrane proteins prior to electrophoresis. Disaggregation or solubilization has most often been accomplished by the method of Takayama or modifications in which membranes are treated with phenol, acetic acid, and urea, subsequent electrophoresis being accomplished in gels containing 5 M urea and 35 % acetic acid.