Activation of human neutrophil NADPH oxidase by phosphatidic acid or diacylglycerol in a cell-free system. Activity of diacylglycerol is dependent on its conversion to phosphatidic acid.

The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg(2+) and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation.

Chicken antibodies to a recombinant fragment of the equine immunoglobulin epsilon heavy-chain recognising native horse IgE.

An equine immunoglobulin E (IgE) heavy-chain cDNA fragment (CH3-CH4, nucleotides 1132 to 1592) was cloned, expressed in Escherichia coli as a fusion protein with a [His]6-tag and purified over a Ni-NTA column. The recombinant protein was used to immunise hens. Testing of the raised egg yolk immunoglobulin G (IgG) in Western-blot and ELISA revealed high titres against the recombinant equine IgE fragment (reqIgEf). The reqIgEf-specific IgG was successfully affinity-purified on an unconventional affinity matrix: the [His]6-tagged recombinant IgE fragment was bound to Ni-NTA agarose and used to adsorb specific immunoglobulins. In Western-blot of ammonium sulphate precipitated horse serum and bronchoalveolar lavage fluid, separated by SDS-PAGE under denaturing-reducing conditions, the raised antibodies reacted with a protein of approximately 80 kDa. A reaction of the reqIgEf-specific IgG was seen with a 190-200 kDa band when the same horse serum or bronchoalveolar fluid (BALF) was separated under non-reducing conditions. These reactions could be inhibited by preincubation of the immune IgG with reqIgEf, while preincubation with horse IgG did not inhibit the reaction. Antibody-affinity chromatography of horse serum with the reqIgEf-specific chicken IgG resulted in an enrichment of the 80 kDa protein in denaturing Western-blot. Determination of the amino acid composition of this protein and comparison with the equine IgE heavy- chain sequence strongly indicates that the 80 kDa band corresponds to the heavy chain of the horse IgE. The reqIgEf-specific chicken IgG was further characterised in an ELISA for the detection of allergen-specific horse IgE. It was demonstrated that heating IgE positive horse sera at 54 degrees C for 10 min drastically diminished the recognition by the reqIgEf-specific chicken IgG. The reaction is inhibitable by preincubation with reqIgEf in a concentration dependent manner. In addition, preincubation with horse IgG, a nonrelevant [His]6-tagged protein or 2% equine colostrum had no influence on the reqIgEf-specific chicken IgG binding characteristic. This antibody recognising horse IgE will be useful for further studies on the pathogenesis of equine allergic diseases.

Differential regulation of inducible nitric oxide synthase production in bovine and caprine macrophages.

Inducible nitric oxide synthase (iNOS) regulation in human and murine macrophages in vitro differs considerably. In this study, expression of macrophage iNOS in ruminants was addressed. Nitric oxide (NO) output by cattle and goat macrophages was as different as that by human and mouse macrophages. Bovine macrophages activated by heated Salmonella dublin or lipopolysaccharide (LPS) expressed high levels of iNOS mRNA, protein, and enzyme activity. Analogously cultured caprine macrophages did not respond to these and other activators by NO generation and iNOS expression. The lack of response was not due to general unresponsiveness to stimuli. Caprine iNOS mRNA was induced by stimulation of caprine macrophages with LPS, as shown by reverse transcription polymerase chain reaction. The level of mRNA expression in activated goat macrophages was lower than in resting bovine macrophages. A caprine 372-bp iNOS mRNA fragment that was sequenced closely resembled the bovine counterpart. This points to species-specific iNOS gene regulation.

The caprine arthritis encephalitis virus tat gene is dispensable for efficient viral replication in vitro and in vivo.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus closely related to visna virus and more distantly to other lentiviruses, such as human immunodeficiency virus. The genomes of visna virus and CAEV contain a tat gene encoding a protein able to weakly transactivate its own long terminal repeat, suggesting that transactivation may be a dispensable function for viral replication. Three different tat gene mutants of an infectious molecular clone of CAEV were used to study their replication after transfection or infection of primary goat synovial membrane cells and of blood-derived mononuclear cells or macrophages. Our results showed no difference between replication of the wild type and either the complete tat deletion mutant or the tat stop point mutant, whereas slower growth kinetics and lower levels of expression of the partial tat deletion mutant that of the wild type were obtained in these cells. Quantitative PCR and reverse transcription-PCR analyses of the different steps of a single replicative cycle revealed an identical pattern of retrotranscription, transcription, and viral production, whereas time course analysis demonstrated that the intracellular level of viral genomic RNA was affected by the partial tat deletion at later time points. We then compared the infectious properties of the wild-type and tat mutant viruses in vivo by direct inoculation of proviral DNAs into the joints of goats. All the animals seroconverted between 27 and 70 days postinoculation. Moreover, we were able to isolate tat mutant CAEV from blood-derived macrophages that was still able to infect synovial membrane cells in vitro. This study clearly demonstrates that the tat gene of CAEV is dispensable for viral replication in vitro and in vivo.

The vif gene is essential for efficient replication of caprine arthritis encephalitis virus in goat synovial membrane cells and affects the late steps of the virus replication cycle.

Complex retrovirus genomes contain a variable number of accessory genes, among which is the vif gene. We investigated in vitro the role of the vif gene of caprine arthritis encephalitis virus (CAEV) by studying the phenotype of five vif mutants after infection of primary goat synovial membrane (GSM) cells and blood-derived monocytes/macrophages. Any deletion introduced into the vif gene resulted in slow and low viral replication and production of virions with an infectious titer lower than that of wild-type viral particles. The wild-type phenotype could be restored by the trans expression of the vif gene in a complementation assay. Quantitative PCR and reverse transcription-PCR analyses were performed in order to determine which stage of the replicative cycle was impaired by the vif deletion. Our results demonstrated that CAEV Vif did not act at the level of reverse transcription or transcription but rather at the late stage of virus formation and/or release, as lower amounts of virus were produced after a single replicative cycle. The vif-deleted CAEV produced after 24 h of infection was still able to infect GSM cells, indicating that the vif gene is not essential for virus infectivity but is required for efficient virus production.

Absolute requirement for GTP in activation of human neutrophil NADPH oxidase in a cell-free system: role of ATP in regenerating GTP.

Guanine and/or adenine nucleotides appear to be involved in the activation of the superoxide-generating NADPH oxidase of phagocytic cells. Their precise roles, however, are unclear, as much of the evidence for their involvement comes from experiments in which nucleotides have been added to complex systems already rich in both endogenous nucleotides and enzymes capable of interconverting them. To circumvent this problem we have examined the role of nucleotides in neutrophil NADPH oxidase activation by using a cell-free system in which adenine and guanine nucleotide concentrations were carefully controlled and monitored by (i) depletion of endogenous nucleotides by extensive dialysis and charcoal treatment; (ii) reconstitution of the depleted system with reagents analyzed for purity; and (iii) measurement of nucleotide levels in cytosol preparations and in oxidase reaction mixtures by HPLC analysis. In contrast to previous reports that have demonstrated only a several-fold enhancement of oxidase activity by GTP or its analogs, we have shown that oxidase activation was absolutely dependent upon GTP in reactions containing dialyzed cytosol in which the total endogenous nucleotide levels were reduced by greater than 99.5%. Kinetic studies revealed that GTP is required at or before the rate-limiting step in oxidase activation. Two nonhydrolyzable analogs of GTP, guanosine 5'-(gamma-thio)triphosphate and guanylyl imidodiphosphate, were even more active than GTP, suggesting the involvement of one or more GTP-binding proteins. In contrast, ATP was neither necessary nor sufficient for oxidase activation. If reaction mixtures were contaminated with GDP and/or GMP, however, ATP (but not its nonhydrolyzable analog adenylyl imidodiphosphate) could indirectly support oxidase activation by means of endogenous enzymes that catalyze the ATP-dependent conversion of GMP and GDP to GTP.

Monocyte-derived neutrophil chemotactic factor/interleukin-8 is a potential mediator of crystal-induced inflammation.

The physical interaction of particulates with resident mononuclear phagocytes is a consistent feature in certain forms of crystal-induced inflammation. In this study, we observed that monosodium urate crystals stimulated the rapid release of neutrophil chemotactic activity from monocytes, and that this activity steadily increased over 24 hours. Because the release of monocyte-derived neutrophil chemotactic activity was markedly diminished by pretreatment of the monocytes with cycloheximide, and was completely removed from conditioned media by adsorption to heparin-agarose, we addressed the possibility that monocyte-derived neutrophil chemotactic factor/interleukin-8 (IL-8), a heparin-binding neutrophil-activating polypeptide, might modulate these activities. Urate crystal-induced IL-8 secretion from monocytes was verified by radioimmunoassay. In addition, an IL-8-specific antibody markedly inhibited the neutrophil-activating capacity of the conditioned media from monocytes activated by urate crystals, as well as by inflammatory silica crystals. Last, IL-8 was significantly increased in gouty synovial fluids (range 3.0-16.8 ng/ml, mean 8.4 ng/ml, n = 6) relative to osteoarthritic synovial fluids (range 1.1-1.7 ng/ml, mean 1.5 ng/ml, n = 6) (P = 0.006). We conclude that microcrystal-induced secretion of IL-8 by mononuclear phagocytes may mediate a number of forms of crystal-induced inflammation.

The p67-phox cytosolic peptide of the respiratory burst oxidase from human neutrophils. Functional aspects.

Most cases of cytosol-defective chronic granulomatous disease are due to the deficiency of a 47-kD protein (p47-phox) whose phosphorylation normally accompanies the activation of the respiratory burst oxidase. Recently, a form of chronic granulomatous disease was described in which the failure of O2- production was associated with the absence of a 67-kD polypeptide (p67-phox) from the cytosol of affected neutrophils. Using neutrophils obtained from a patient with this form of the disease, we examined the function of p67-phox in the activation of the oxidase. Our studies showed that in whole p67-phox-deficient neutrophils, p47-phox was phosphorylated in a normal fashion. In the cell-free oxidase-activating system, the ability of the p67-phox-deficient cytosol to support oxidase activation was partly restored by the addition of p47-phox-deficient cytosol; the p67-phox-deficient cytosol, however, was not complemented by cytosol inactivated with NADPH dialdehyde, an affinity label previously found to block the NADPH-binding component of the oxidase. Despite these differences, the kinetic properties of the p67-phox-deficient cytosol closely resembled those of the p47-phox-deficient cytosol. Taken together with earlier findings, these results suggest that (a) in the neutrophil cytosol, p67-phox is at least partly complexed to p47-phox; (b) it is in the form of this complex that p67-phox participates in oxidase activation; and (c) p47-phox appears to be translocated from the cytosol to the plasma membrane during oxidase activation, but complexation to p67-phox is not necessary for this translocation, nor for the accompanying extra protein phosphorylation.

Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor.

The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharide-stimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino- and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.

Mechanism of neutrophil activation by NAF, a novel monocyte-derived peptide agonist.

The rise in cytosolic free Ca2+, shape change, superoxide formation, and granule exocytosis induced in human neutrophils by N-formyl-Met-Leu-Phe (fMLP) and by a newly discovered activating peptide, neutrophil-activating factor, termed NAF, were compared. NAF was effective in the concentration range of 0.1-10 nM and was 10- to 100-fold more potent than fMLP. In qualitative terms, the single responses to either stimulus were remarkably similar: they showed virtually identical onset and initial kinetics, and were all inhibited by pretreatment of the neutrophils with Bordetella pertussis toxin. In addition, the respiratory burst elicited by either stimulus was inhibited by 17-hydroxywortmannin and staurosporine. Two conclusions are drawn from these results: 1) neutrophil activation by NAF (as by fMLP) is dependent on a GTP-binding protein and on protein kinase C; 2) a similar, or even identical, mechanism of signal transduction must be assumed on stimulation of human neutrophils with NAF, fMLP, and other chemotactic agonists. Human monocytes, lymphocytes, and platelets did not show cytosolic free Ca2+ changes when exposed to NAF, which suggests that NAF is selective for the neutrophils.

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.

Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes.

Human blood mononuclear cells were cultured for 24 h in the presence of LPS (100 ng per 5 x 10(6) cells), and a monocyte-derived neutrophil-activating factor (NAF) was purified to apparent homogeneity from the conditioned media. The purification consisted of ammonium sulphate precipitation, gel filtration, chromatography on phosphocellulose followed by hydroxylapatite, and reversed-phase HPLC on C4 and CN-propyl columns. Amino acid sequence analysis (32 of 50 presumed residues) shows that NAF is a novel peptide with little homology to known ones. Crude and pure NAF stimulated human neutrophils to release granule enzymes and to produce superoxide and H2O2.