A novel neutrophil-activating factor produced by human mononuclear phagocytes.

The biological properties of a neutrophil-activating factor (NAF), which was recently identified as a novel peptide of approximately 6,000 mol wt, are described. NAF is produced de novo by human blood monocytes upon stimulation with LPS, PHA, and Con A. It induces two main responses in human neutrophils, i.e., exocytosis (release from specific granules in normal, and from specific and azurophil granules in cytochalasin B-treated cells) and the respiratory burst (formation of superoxide and hydrogen peroxide). The action of NAF appears to be mediated by a surface receptor as shown by the following observations. (a) NAF induces a rapid and transient rise in cytosolic free Ca2+; (b) interaction with NAF results in desensitization, since the cells do not respond to a second NAF challenge; and (c) the respiratory burst elicited by NAF is similar in onset, and time course to that induced by C5a or FMLP. The NAF receptor can be distinguished from the receptors of C5a, FMLP, platelet-activating factor, and leukotriene B4 by the lack of cross-desensitization. Unlike C5a, the other host-derived neutrophil-activating peptide, NAF is not inactivated by serum and thus presumably accumulates in inflamed tissue.

Subcellular localization and heterogeneity of neutral proteases in neutrophilic polymorphonuclear leukocytes.

The subcellular localization of elastase and of neutral proteases hydrolyzing histone and casein was determined in human and rabbit polymorphonuclear leukocytes using fractionation by isopycnic centrifugation. Granule-rich fractions obtained by this technique were extracted and analyzed by acrylamide gel electrophoresis, and proteolytic activity on the gels was demonstrated by staining with either N-acetyl-D,L-alanine alpha-naphthyl ester or naphthol AS-D acetate as substrate. In both species, all neutral proteases assayed were found to be localized exclusively in the azurophil granules. Specific activities were about 10-30 times higher in human than in rabbit preparations. In extracts of human azurophil granules up to 10 proteins exhibiting esterolytic activity could be demonstrated after electrophoretic separation. Three major and two or three minor components of these esterases were shown to possess elastase activity. Similar zymograms prepared with extracts from rabbit azurophil granules revealed only one major elastase band. The electrophoretic analysis further showed that the most strongly cationic proteins of both human and rabbit PMNs were also confined to the azurophil granules.

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.

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.

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.

Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin.

A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a baculovirus vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.

HCC-1, a novel chemokine from human plasma.

A novel CC chemokine, HCC-1, was isolated from the hemofiltrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and 29-37% with the other human CC chemokines. Unlike MIP-1 alpha and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34+ myeloid progenitor cells. It was as effective as MIP-1 alpha, but about 100-fold less potent.

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils.

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.

A novel type of cytoplasmic granule in bovine neutrophils.

We obtained cell preparations containing greater than 95% neutrophils from freshly drawn bovine blood. The cells were suspended in sucrose and disrupted in a Dounce homogenizer, and the postnuclear supernate was fractionated by zonal differential sedimentation and by isopycnic equilibration. The subcellular fractions were characterized biochemically by testing for marker enzymes and other constituents known to occur in azurophil and specific granules of other species, and by electrophoretic analysis of extracts of the particulate material. In addition, each fraction was examined by random-sampling electron microscopy. We found that bovine neutrophils contain in addition to azurophil and specific granules a third type of granule, not known to occur in neutrophils of other species. These novel granules are larger, denser, and considerably more numerous than the two other types. Except for lactoferrin, they lack the characteristic constituents of azurophil granules (peroxidase, acid hydrolases, and neutral proteinases) and of specific granules (vitamin B12-binding protein). Instead, they contain a group of highly cationic proteins not found in the other granules, and they are the exclusive stores of powerful oxygen-independent bactericidal agents. We studied the fate of the large granules in bovine neutrophils exposed to opsonized particles, the ionophore A 23187, or phorbol myristate acetate. The appearance in the cell-free media of antibacterial activity and of the characteristic highly cationic proteins as revealed by electrophoresis was monitored and compared with the release of azurophil and specific granule markers. In addition, changes of the relative size of the large granule compartment induced by phagocytosis were assessed by morphometry. The results show that exocytosis of the large granules occurs following both phagocytosis and exposure to soluble stimuli. Like the specific granules, the large granules appear to be discharged by true secretion under conditions where the azurophil granules are fully retained.

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.

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.

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.

Subcellular localization of the superoxide-forming enzyme in human neutrophils.

The subcellular distribution of the superoxide (O2-)-forming enzyme in human neutrophils was investigated. Cells were activated by phorbolmyristate acetate or by opsonized zymosan, and were then fractionated by zonal-rate sedimentation at two different speeds. At high speed, the specific granules were resolved from the azurophils and the membrane fraction, while at low speed, the azurophil granules could be separated from fast-sedimenting particle aggregates. Under both conditions, the major portion of the O-2--forming activity (60--70% of the total) was found to be associated with the membrane fraction which was characterized by the presence of alkaline phosphatase, alkaline phosphodiesterase I, and acid aryl phosphatase. No significant O-2--forming activity was found in either specific or azurophil granules. Some activity was present in the fastest sedimenting fractions which, as shown by electron microscopy, were heterogeneous and contained aggregated material which included membrane fragments. These fractionation results provide strong additional support for the current view that the activable O-2--forming system is localized in the plasma membrane of human neutrophils.

Platelet-activating factor as a stimulus of exocytosis in human neutrophils.

The properties of platelet-activating factor (PAF-acether) 42-48ulus of exocytosis in human neutrophils have been re-investigated with particular attention to effects on cells that were not pretreated with cytochalasin B. Release of gelatinase, the most sensitive marker of exocytosis, was determined in addition to that of vitamin B-12-binding protein and beta-glucuronidase. Superoxide production was assayed as a measure of the respiratory burst. The effects of PAF-acether were compared to those of leukotriene B4 and N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). Our results show that PAF-acether elicits marked secretion in untreated human neutrophils, and refute the prevalent view that cytochalasin B treatment is required for responsiveness. PAF-acether induced abundant release of gelatinase, increasing on average from 20% at 10 nM to 35% at 1 microM. This release was very rapid, i.e., almost complete after 2 min. fMet-Leu-Phe induced the same maximum response already at 0.1 microM, but release was considerably slower. Leukotriene B4 was less potent with a maximum release of 20%. Exocytosis of gelatinase was always paralleled by liberation of smaller but significant amounts of vitamin B12-binding protein from the specific granules. In contrast to their effect on exocytosis, PAF-acether and leukotriene B4 were very weak stimuli of the respiratory burst when compared with fMet-Leu-Phe.

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.

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.

Enhanced expression of eotaxin and CCR3 in atopic dermatitis.

Chemokines are thought to play an important part in the development of inflammation in atopic dermatitis. Eotaxin, a CC chemokine, is a potent chemoattractant and activator of human eosinophils, basophils and Th2 lymphocytes which acts via the chemokine receptor CCR3. We studied the expression of eotaxin and CCR3, as well as MCP-3, MIP-1alpha and interleukin-8, in atopic dermatitis and normal skin by immunohistochemistry and nested reverse transcriptase-polymerase chain reaction. Skin biopsy specimens were obtained from nonlesional and lesional skin of patients with atopic dermatitis and of nonatopic controls. Immunoreactivity and transcripts of eotaxin and CCR3 were significantly increased in lesional skin from atopic dermatitis, but not in nonatopic controls. In nonlesional atopic dermatitis samples CCR3 expression was also significantly increased at the mRNA and protein level, whereas eotaxin was increased at the mRNA level only. No significant difference in the expression of MCP-3, MIP-1alpha, and interleukin-8 was observed between skin samples from atopic dermatitis and nonatopic controls. The enhanced local production of eotaxin may lead to the recruitment of eosinophils and T lymphocytes, which both express CCR3 and contribute to the initiation and maintenance of inflammation.

1H NMR evidence that Glu-38 interacts with the N-terminal functional domain in interleukin-8.

In order to assess the importance of the buried Glu-38 observed in the structure of interleukin-8, an analog in which Glu-38 was replaced with Ala (E38A analog) was investigated by 1H NMR spectroscopy and neutrophil activation. Detailed analysis of the NMR NOESY data showed that the solution structure of the E38A analog is essentially the same as that for the native protein. Also, the neutrophil elastase activity of the E38A analog was similar to that of the native protein. However, the Gln-8 and Cys-9 amide proton chemical shifts, which are significantly downfield-shifted in the native protein, exhibit more 'normal' values. This observation indicates that in the native protein, Glu-38 side-chain carboxylate interacts with Gln-8 and Cys-9 amide protons. Although the N-terminal residues are critical for function, this interaction is not essential for neutrophil activation.

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.

2,3-Dihydrobenzofuran-2-ones: a new class of highly potent antiinflammatory agents.

A series of 2,3-dihydrobenzofuran-2-one analogues of the mold metabolite wortmannin, which is a powerful antiinflammatory compound, was synthesized. Most of these compounds were tested for their ability to inhibit the carrageenin paw edema and the adjuvant-induced arthritis of the rat and for their ability to inhibit prostaglandin synthesis in vitro. Indomethacin and diclofenac were used as references. The results show that compounds bearing an alkyl or aryl group in position 6 and an additional substituent, preferably chlorine, in position 5 are very powerful antiinflammatory agents and inhibitors of prostaglandin synthesis. The most active among these compounds, 5-chloro-6-cyclohexyl-2,3-dihydrobenzofuran-2-one, was significantly more potent than diclofenac in all testing models, more powerful than indomethacin in inhibiting acute inflammation and prostaglandin synthesis, and somewhat less potent than the latter compound in the adjuvant arthritis model.