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Biomedical subjects

M Baggiolini

Publications and source records attributed to M Baggiolini.

At least 127 records · Page 7Linked to original sources
PubMed1992

IL-8 production by human polymorphonuclear leukocytes. The chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway.

IL-8 is a novel chemotactic cytokine, produced by a variety of blood and tissue cells, that has marked activating effects on polymorphonuclear leukocytes (PMN). We report that IL-8 is produced and released by human PMN after stimulation with the chemotactic agonist FMLP. Release of IL-8 in response to FMLP was transient and not influenced by PMN adherence or by the absence of serum in the medium. Maximum yields were usually obtained with 10 nM FMLP within 2 h of stimulation (0.5-3.5 ng/ml/7 x 10(6) cells, range of 17 different donors). IL-8 release was dependent on FMLP-induced de novo protein synthesis because it was inhibited by cycloheximide, was paralleled by enhanced expression of IL-8 mRNA and was potentiated from two- to sixfold after preincubation of PMN with cytochalasin B. The FMLP effect was direct and not dependent on LPS or on contaminating monocytes, which showed only low responsiveness to FMLP. Pretreatment of PMN with pertussis toxin prevented FMLP-dependent IL-8 production, the effect being evident both at the level of mRNA expression and protein secretion. In addition, two other chemoattractans, platelet-activating factor and C5a, were found capable to induce release of IL-8 by PMN. The results of this study suggest that chemotactically stimulated PMN may be able to amplify the recruitment process of PMN to the inflammatory site by releasing IL-8. As a long-lived cytokine, IL-8 could markedly prolong the attractant effect.

Cells, Cultured↗
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↗
PubMed1992

Interleukin-8 immunoreactivity in the skin of healthy subjects and patients with palmoplantar pustulosis and psoriasis.

Previous studies have shown that neutrophil-activating peptide 1/interleukin-8 (IL-8) is present in psoriatic scales and to a lesser extent in normal human epidermis. A panel of monoclonal antibodies and polyclonal antisera raised against IL-8 was used to localize IL-8 with immunoperoxidase techniques in non-lesional and lesional skin of patients with psoriasis and palmo-plantar pustulosis (PPP), and in corresponding sites from healthy subjects. Intracellular IL-8 immunoreactivity was found in all epidermal cell layers in biopsies of healthy subjects and in non-lesional and lesional skin in both PPP and psoriasis. The most intense immunolabeling was regularly found in the basal cell layer. Intercellular epidermal IL-8 immunolabeling was regularly detected in lesional biopsies in PPP and psoriasis, but not in healthy subjects or non-lesional skin in PPP and psoriasis. No intercellular immunolabeling was detected after successful treatment of lesional skin. The majority of cells along the eccrine sweat glands, dermal mononuclear cell infiltrates, and endothelial cells were IL-8 immunoreactive in all biopsies studied. The present study suggests that IL-8, its precursor form, or, alternatively, a degradation product is present in normal human epidermis.

Humans↗
PubMed1991

Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis.

Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN.

Actins↗
PubMed1991

Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities.

Interleukin-8 (IL-8) is an inflammatory mediator that stimulates neutrophil migration and functional activation. Analogs of human IL-8 were chemically synthesized, purified, and compared with the full-length 72-residue synthetic IL-8 for their ability to stimulate neutrophil chemotaxis and exocytosis as measured by assaying for release of elastase, as well as their binding to specific receptors in competition assays. Analogs corresponding to the less abundant natural forms, 3-72, 4-72, and 77-residue IL-8, were evaluated and the 3-72 and 4-72 had 2-5-fold higher potencies, whereas the 77-residue IL-8 was 2-fold less potent. A major finding was that NH2-terminal residues 4, 5, and 6 were absolutely essential for IL-8 activity and receptor binding. Quantitative dissociation of elastase release and chemotaxis activity was detected with 5-72, which compared with 1-72, was 80-fold less potent in the elastase assay, but was only slightly less potent in stimulating chemotaxis. IL-8 6-72 lacked all the biological activities tested but had detectable receptor binding activity. The NH2-terminal peptide, AVLPRSAKEL, lacked activity and receptor binding, suggesting that the NH2-terminal region alone is not sufficient for function. Comparison of analogs shortened at the COOH terminus showed that potency was progressively reduced as the COOH-terminal residues were excluded. However activity was retained in an analog (1-51) with the entire COOH-terminal alpha helix and beta turn missing. A peptide corresponding to the COOH-terminal 22 residues, although inactive alone, synergized with the 1-51 analog in stimulating elastase release. The results suggest that the NH2-terminal residues 4, 5, and 6, which are disordered in the IL-8 solution structure, are directly involved in receptor binding, but the COOH-terminal alpha helix is probably important for stabilizing the three-dimensional structure. Other regions within residues 7-51 are also functionally important.

Amino Acid Sequence↗
PubMed1991

Structure and neutrophil-activating properties of a novel inflammatory peptide (ENA-78) with homology to interleukin 8.

A new neutrophil-activating peptide, termed ENA-78, was identified in the conditioned media of stimulated human type II epithelial cell line A549. In response to stimulation with either interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF-alpha), ENA-78 was produced and secreted concomitantly with IL-8, GRO alpha, and GRO gamma. ENA-78 consists of 78 amino acids [sequence; see text] and has a molecular weight of 8,357. It has four cysteines positioned identically to those of IL-8 and analogues, and thus belongs to the CXC family of peptides. ENA-78 is related to neutrophil-activating peptide 2 (NAP-2) and GRO alpha (sequence identity, 53% and 52%, respectively) and IL-8 (22% identity). Like NAP-2 and GRO alpha, ENA-78 stimulates neutrophils, inducing chemotaxis, a rise in intracellular free calcium and exocytosis. Cross-desensitization experiments indicate that ENA-78 acts through the same type of receptors as IL-8, NAP-2, and GRO alpha.

Amino Acid Sequence↗
PubMed1991

Interleukin 8-inhibitor and inducer of histamine and leukotriene release in human basophils.

We have shown previously that interleukin 8 (IL-8) induces histamine and leukotriene release in human basophils exposed to interleukin 3 (IL-3). We now found that pretreatment with low concentrations of IL-8 selectively inhibits this response. Inhibition was significant at 0.01 nM IL-8 and virtually complete at 1 nM, which is about 100-fold lower than the concentration required for induction of mediator release. IL-8 dependent responses were also inhibited, albeit to a lesser extent, by preincubation with neutrophil-activating peptide 2 (NAP-2), but not with connective tissue-activating peptide III (CTAP-III) or platelet factor 4 (PF4). Release induced by C5a, fMet-Leu-Phe, or anti-IgE antibody, by contrast, was not affected.

Basophils↗
PubMed1991

Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes.

The JE gene was first described as a platelet-derived growth factor (PDGF)-inducible gene in mouse 3T3 cells. The human homologue of JE encodes a protein whose predicted amino acid sequence is identical to that of the monocyte chemoattractant MCP-1 (also called MCAF and SMC-CF), which belongs to a recently identified family of small secretory proteins with cytokine properties. We purified recombinant human MCP-1/JE (hMCP-1/JE) produced in COS cells and demonstrated that it is chemotactic for human monocytes with a specific activity similar to natural MCP-1. In addition, pure recombinant hMCP-1/JE stimulates monocytes, inducing an increase in cytosolic free calcium and the respiratory burst, but is completely inactive on human neutrophils. These results help to define functionally a well-known growth factor-inducible gene and a member of a new family of cytokines.

Animals↗
PubMed1991

Neutrophil-activating peptide 2 and gro/melanoma growth-stimulatory activity interact with neutrophil-activating peptide 1/interleukin 8 receptors on human neutrophils.

Neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8), neutrophil-activating peptide 2 (NAP-2), and gro/melanoma growth-stimulatory activity (gro/MGSA) are potent inflammatory cytokines with homologous structure and similar neutrophil-activating properties. Receptors on human neutrophils that interact with these peptides were studied. Analysis of 125I-NAP-1/IL-8 binding at 0-4 degrees C revealed 64,500 +/- 14,000 receptors/cell with an apparent Kd of 0.18 +/- 0.07 nM (mean +/- S.D. of six independent experiments). Unlabeled NAP-1/IL-8, NAP-2, and gro/MGSA competed with 125I-NAP-1/IL-8 for binding to human neutrophils. Competition with increasing concentrations of unlabeled NAP-2 and gro/MGSA resolved two classes of NAP-1/IL-8 binding sites: about 70% of them bound NAP-2 and gro/MGSA with high affinity (Kd: 0.34 +/- 0.2 and 0.14 +/- 0.02), while 30% were of low affinity (Kd: 100 +/- 20 and 130 +/- 10 nM). Different binding sites, however, were not apparent upon competition with unlabeled NAP-1/IL-8, suggesting that both classes of receptors have similar affinities for NAP-1/IL-8. The existence of two receptors was also suggested by ligand cross-linking and cross-desensitization experiments. Two neutrophil membrane proteins with apparent Mr of 66,000-74,000 and 42,000-46,000 became cross-linked to 125I-NAP-1/IL-8, and the labeling was decreased when excess NAP-1/IL-8, NAP-2, or gro/MGSA was present. Stimulation of neutrophils with NAP-1/IL-8 resulted in desensitization toward a subsequent challenge with NAP-2 or gro/MGSA as shown by the rise in cytosolic free calcium. By contrast, following primary stimulation with NAP-2 or gro/MGSA, responses to NAP-1/IL-8 were only moderately attenuated, supporting the existence of NAP-1/IL-8 receptors which bind NAP-2 or gro/MGSA with low affinity. In conclusion, our results demonstrate that NAP-2 and gro/MGSA act upon human neutrophils by directly interacting with two classes of receptors for NAP-1/IL-8.

Amino Acid Sequence↗
PubMed1991

Receptor-operated activation of polymorphonuclear leukocytes: different effects of NAP-1/IL-8 and fMet-Leu-Phe or C5a.

We examined the production of PAF and LTB4 by PMN in response to NAP1/IL-8 alone, or after preincubation with GM-CSF, which has been shown to enhance PMN responsiveness and to prime PMN for production of those bioactive lipids. NAP-1/IL-8 does not induce the synthesis of PAF and LTB4 from endogenous phospholipid precursors, even after preincubation with GM-CSF. In addition and again in contrast to fMLP and C5a, NAP-1/IL-8 fails to induce an enhanced oxidative burst in GM-CSF primed PMN. Exogenously added PAF or LTB4 can mimic the priming effect of GM-CSF for an enhanced oxidative burst in response to all examined chemotactic peptides including NAP1/IL-8. Our data reveal a possible autocrine role of PAF and LTB4 in the enhanced responsiveness of GM-CSF primed PMN towards fMLP or C5a, but not NAP-1/IL-8.

Arachidonate 5-Lipoxygenase↗
PubMed1991

Formation of neutrophil-activating peptide 2 from platelet-derived connective-tissue-activating peptide III by different tissue proteinases.

Neutrophil-activating peptide 2 (NAP-2) is generated by cleavage of two inactive precursors, connective-tissue-activating peptide III (CTAP-III) and platelet basic protein (PBP), which are stored in the alpha-granules of blood platelets. Using highly purified CTAP-III as the substrate we studied the generation of NAP-2 by several neutral tissue proteinases. CTAP-III was rapidly cleaved by chymotrypsin, cathepsin G and trypsin, yielding products with neutrophil-stimulating activity. This activity remained unchanged for 24 h in the presence of chymotrypsin, decreased only slowly in the presence of cathepsin G, but was rapidly destroyed by trypsin. CTAP-III was also degraded by human neutrophil elastase and porcine pancreatic elastase, but no active fragments were obtained. By contrast, no degradation of CTAP-III was observed with thrombin, plasmin or 'granzymes' from cytolytic T-lymphocyte granules. Two active fragments of CTAP-III, generated by chymotrypsin or cathepsin G, were purified and partially sequenced, and were found to have the same N-terminal sequence as NAP-2. These results indicate that both proteinases cleave preferentially the bond between amino acids 15 (Tyr) and 16 (Ala) of CTAP-III. We conclude that chymotrypsin-like proteolytic activity in the vicinity of activated platelets may generate NAP-2 intravascularly. Due to its presence in the primary granules of neutrophils and monocytes cathepsin G is likely to be involved in this process.

Amino Acid Sequence↗
PubMed1991

Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide.

Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
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↗