Tumor necrosis factor primes neutrophils to kill Staphylococcus aureus by an oxygen-dependent mechanism and Plasmodium falciparum by an oxygen-independent mechanism.

The cytokine tumor necrosis factor (TNF) plays the important role of priming neutrophils for increased antimicrobial activity. We now demonstrate that human neutrophils which lack the ability to generate oxygen radicals, from patients with chronic granulomatous disease, show TNF-induced enhancement of killing of intraerythrocytic stages of Plasmodium falciparum but not of Staphylococcus aureus.

Tumor necrosis factor priming of peripheral blood neutrophils from rheumatoid arthritis patients.

Recently it was shown that tumor necrosis factor-alpha (TNF) receptors on neutrophils may be down-regulated after stimulation with proinflammatory mediators. Since in rheumatoid arthritis neutrophils are likely to encounter these mediators in the circulation, we tested the hypothesis that rheumatoid arthritis neutrophil TNF receptors are down-regulated. Peripheral blood neutrophils from patients with rheumatoid arthritis and healthy subjects were compared with respect to their TNF binding activity and ability to be primed by TNF. There were no differences between rheumatoid arthritis and control neutrophils in receptor-mediated TNF binding, superoxide release in response to agonist, and TNF priming of this respiratory burst or in the ability to degrade cartilage in vitro and TNF priming for increased cartilage damage. It is evident that rheumatoid arthritis blood neutrophils retain the ability to bind TNF and can be primed by TNF for increased oxygen radical production and augmented cartilage damage. These findings further implicate the role of neutrophils in the pathogenesis of arthritis.

Adhesion and TNF priming in neutrophil-mediated cartilage damage.

Neutrophils predominate in the acute stages of rheumatoid arthritis and are implicated in the cartilage damage which is characteristic of this disease. In vitro neutrophils can be primed for increased ability to damage host tissues by a number of cytokines including tumor necrosis factor-alpha (TNF). The role of adherence in this process was investigated. Opsonization of cartilage with aggregated IgG (HAGG) and complement promoted neutrophil damage to cartilage. Adherence was increased by HAGG and TNF. Separation of neutrophils from cartilage markedly reduced the neutrophil-mediated injury and abolished the priming effect of TNF. Inactivation of complement or antibodies against CD11a or CD11b reduced neutrophil-mediated cartilage damage and markedly reduced TNF-priming of this damage and yet did not alter adherence of control or TNF-primed cells. These results suggest that neutrophil damage to cartilage is promoted by agents that favor adherence. The failure to block adhesion of neutrophils by complement inactivation or antibodies to CD11a or CD11b suggests that neutrophil adherence to cartilage occurs simultaneously through several different receptors. The massive reduction of TNF enhancement of neutrophil damage to cartilage by preventing adhesion suggests that adherence is required for this action of TNF.

Interaction of Staphylococcus aureus with human neutrophils and the down-regulation of TNF receptors.

We have shown previously that pre-exposure of neutrophils to TNF significantly enhanced their killing of opsonized Staphylococcus aureus. We now demonstrate that the ability of TNF to enhance the bactericidal activity is dependent on preincubation time; enhancement was still evident when TNF and bacteria were added simultaneously to neutrophils but if TNF addition was delayed by 5 min, no enhancement was seen. Evidence is presented that suggests that this could be related to a down-regulation of TNF receptors by the bacteria, but in addition, the release of TNF receptor fragments may contribute to the inhibition observed. Scatchard analyses demonstrated a decrease from approximately 3000 TNF receptor (receptor binding) sites per cell to 450 following treatment with S. aureus, but essentially no change in receptor affinity. Using mAb directed against the type A (75 kDa) receptor (utr-1) and the type B (55 kDa) receptor (htr-9), it was found that the expression of both receptors was decreased following treatment with the bacteria. The time course of loss of these receptors showed that the surface expression of both molecules was markedly decreased by 5 min which correlated with the loss in ability of TNF to enhance the bactericidal activity. In contrast to changes seen in the binding of TNF, similarly treated neutrophils showed essentially no change in the binding of radiolabeled tripeptide FMLP and, if anything, an increase in the expression of the CD11b Ag (CR3 receptor). When another phagocytic stimulus was used, opsonized fungi (Torulopsis glabrata), a similar depression of TNF binding was also found, but opsonized sheep erythrocytes had no effect on the TNF binding, suggesting that the effects on the TNF receptor cannot be explained simply on the basis of particle phagocytosis.

Cytokines in the host response to mycotic agents.

In summary, different approaches have been taken to understand cytokine responses to different fungal infections. Singer-Vermes and co-investigators indirectly examined cytokine responses to paracoccidioidomycosis by studying the types of cellular and humoral immune responses that were induced in resistant and susceptible mouse strains. Their results implicated Th1 cell responses in the resistant mouse strain and Th2 cell responses in the mouse strain susceptible to paracoccidioidomycosis. By measuring cytokine production and through cytokine depletion experiments, Wu-Hsieh showed that besides IFN gamma, TNF alpha was important in host defences against the intracellular pathogen, H. capsulatum. Both cytokines play important roles in the regulation of other cytokines. In histoplasmosis, the dynamics of the complex interactions amongst cytokines govern the efficiency of host clearance of the fungus from tissues. Ferrante and collaborators, examining TNF alpha and TNF alpha receptors on neutrophils presented data showing that TNF alpha plays an important role in the activation of neutrophils for anti-Candida activity. Through the detection of cytokine mRNAs with RT-PCR, Moser and co-workers found that cytokine mRNAs of macrophage origin were produced preferentially in the lungs of mice infected with Histoplasma or Blastomyces. A great challenge still lies ahead of us. It is well understood that the interactions of cytokines are extremely complex at the levels of the induction and expression of the immune responses as well as on effects on natural cellular defences. Work accomplished thus far has laid the ground work for future studies in the effort to dissect host cytokine responses and to understand the roles of cytokines in protection against fungal infections.

Hepatocyte growth factor stimulates neutrophil degranulation but not respiratory burst.

Neutrophil function is regulated in part by cytokines with growth factor activities for different cell types. Hepatocyte growth factor (HGF) is a cytokine produced during injury to the liver and other organs. Neutrophils are numerous in such tissue injury sites and may be influenced by HGF. In the present study the effect of HGF on neutrophils was investigated. The data show that HGF at 1-10 ng/ml increased lysosomal enzyme release from both specific and azurophilic granules of cytochalasin-B treated neutrophils. The release of specific granule contents in response to N-formyl-methionyl-leucylphenylalanine was also increased by HGF. In contrast there were no significant effects of HGF on neutrophil respiratory burst, adherence or locomotion. It is concluded that HGF modulates neutrophil granule exocytosis.

Platelet-activating factor inhibits proteoglycan synthesis and enhances neutrophil-mediated proteoglycan degradation in cartilage explants.

OBJECTIVE: Platelet-activating factor (PAF), which stimulates the release of tissue-destructive enzymes and reactive oxygen metabolites from neutrophils, was investigated for its role in neutrophil-mediated cartilage breakdown. METHODS: Bovine cartilage explants were incubated with or without human neutrophils, PAF, and other reagents. Cartilage damage was measured as either proteoglycan degradation (percent release of 35S-labeled proteoglycan from 35S-labeled cartilage) or inhibition of proteoglycan synthesis (rate of incorporation of 35S into proteoglycan). RESULTS: PAF increased neutrophil-mediated proteoglycan degradation in the 2-20 microM range. Three specific PAF-receptor antagonists, WEB2086, CV3988, and CV6209, reversed this effect of PAF. These antagonists also reduced the enhancement of neutrophil-mediated cartilage damage caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF alpha). The results suggest that there may be a positive feedback mechanism whereby cytokine-primed neutrophils produce PAF, which amplifies the release of other tissue-damaging substances from neutrophils. In the absence of neutrophils, PAF (2-20 microM) inhibited the synthesis of proteoglycan by bovine cartilage. Neutrophils also inhibit proteoglycan synthesis, but PAF probably is not involved in this effect of neutrophils because the PAF receptor antagonists had no consistent effect. CONCLUSION: PAF increases neutrophil-mediated cartilage proteoglycan degradation in vitro. GM-CSF and TNF alpha enhancement of neutrophil damage to cartilage is partly due to PAF. PAF alone inhibits cartilage proteoglycan synthesis.

Interferon-gamma increases human neutrophil-mediated cartilage proteoglycan degradation.

Interferon gamma (IFN-gamma) is known to activate neutrophils and is produced by the synoviocytes, macrophages, T cells and other inflammatory cells in rheumatoid arthritis. Neutrophils participate in articular cartilage destruction and predominate in the synovial fluid of inflamed joints during the early stages and acute exacerbations of the disease. In this study we investigated the effect of recombinant human IFN-gamma on human neutrophil damage to bovine articular cartilage explants which had been coated with heat-aggregated IgG (HAGG). Cartilage proteoglycan degradation by neutrophils was augmented by IFN-gamma at 10(2)-10(4) U/ml. IFN-gamma also increased neutrophil-mediated proteoglycan degradation when the cells were preincubated with IFN-gamma and washed before addition to cartilage. This activity of IFN-gamma was abolished by boiling and was unaffected by polymixin B, indicating that the effects of IFN-gamma were not due to endotoxin contamination. In the absence of neutrophils IFN-gamma had no effect on proteoglycan metabolism, and the ability of IFN-gamma to increase neutrophil-mediated proteoglycan degradation did not require living chondrocytes. Adherence of neutrophils to HAGG-coated and uncoated cartilage explants was enhanced by IFN-gamma. Opsonisation of the cartilage with HAGG also increased the adhesion of neutrophils. In contrast to the enhancement of neutrophil-mediated proteoglycan degradation, IFN-gamma had no effect on the inhibition of proteoglycan synthesis by neutrophils.

Mechanisms of host tissue damage by cytokine-activated neutrophils.

Although extensive investigations into the role of neutrophils and their products in tissue injury have been conducted, very little work has been carried out on the role of polypeptide cytokines in the regulation of neutrophil-mediated tissue damage. A range of cytokines are known to promote a wide variety of functions of the neutrophil, including neutrophil adhesion, surface receptor expression, ODRS production, and release of lysosomal constituents. Evidence has been presented to show that these products, either singly or in combination, cause tissue injury. At this stage, TNF alpha and TNF beta are cytokines which have been shown to regulate neutrophil tissue injury. From this work it is suggested that TNFs promote neutrophil-mediated tissue damage.

Granulocyte-macrophage colony-stimulating factor augments neutrophil-mediated cartilage degradation and neutrophil adherence.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced in large quantities by synoviocytes in the inflamed arthritic joint and is known to be a neutrophil activator. Neutrophils predominate during acute flares of arthritis and are important mediators of cartilage destruction. In this investigation, we show that treatment of neutrophils with 10-1,000 units/ml of GM-CSF augments their ability to degrade cartilage proteoglycan in vitro. This was associated with increased neutrophil adherence to cartilage and increased release of oxygen-derived reactive species and granule enzymes in response to cartilage. Coating the cartilage with heat-aggregated human immunoglobulin G (AHG) enhanced both neutrophil adherence to the tissue and tissue degradation. GM-CSF, however, augmented these neutrophil effects independently of the presence of AHG. In contrast, neutrophil-mediated inhibition of proteoglycan synthesis was unaffected by GM-CSF.

Granulocyte-macrophage colony-stimulating factor augments neutrophil killing of Torulopsis glabrata and stimulates neutrophil respiratory burst and degranulation.

The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the interaction between the fungus Torulopsis glabrata and human neutrophils was examined. Pre-incubation of neutrophils with GM-CSF increased the neutrophil fungal killing. The cytokine also increased the oxygen-dependent respiratory burst in response to opsonized fungi, measured by the lucigenin-dependent chemiluminescence assay and superoxide release. Under the same conditions the cytokine augmented release of constituents from both specific and azurophilic granules. Besides these priming effects, GM-CSF was a weak stimulus of the neutrophil respiratory burst and degranulation. The priming and stimulatory effects of GM-CSF were observed at 10-1000 U/ml with an optimal concentration of 100 U/ml.

Degradation of human cartilage by monocytes.

The effect of human peripheral blood monocytes on the degradation of human articular cartilage was studied in vitro using a radiometric assay to detect proteoglycan breakdown. The results showed that proteoglycan breakdown was increased by 60% after a 20 h exposure to monocytes (p less than 0.001).

Augmentation of the human monocyte/macrophage chemiluminescence response during short-term exposure to interferon-gamma and tumour necrosis factor-alpha.

The effects of short-term (30 min) pre-incubation of human monocytes and macrophages (3-day cultured monocytes) with leucocyte-derived human interferon-gamma (IFN-gamma) and recombinant human tumour necrosis factor-alpha (rTNF-alpha) were examined. Pre-incubation of either monocytes or macrophages with rTNF-alpha or IFN-gamma (100 U/5 x 10(5) cells) augmented their respiratory burst to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), measured by the luminol- and lucigenin-dependent chemiluminescence assay. In addition, both cell types showed a burst of respiratory activity in the presence of rTNF-alpha or IFN-gamma only. The effects of IFN-gamma were removed by adsorption with an anti-IFN-gamma monoclonal antibody and those of rTNF-alpha were abolished by heating at 100 degrees C, or by the addition of anti-TNF-alpha monoclonal antibody. The results demonstrate that both IFN-gamma and rTNF-alpha are stimulators of monocytes and macrophages, and rapidly alter the capacity of the cells to respond to fMLP, which binds to cell surface receptors.

Neutrophil-mediated cartilage injury in vitro is enhanced by tumour necrosis factor alpha.

Neutrophil functions relevant to tissue damage are altered by cytokines such as tumour necrosis factor alpha (cachectin, TNF alpha), known to be present in inflammatory foci. In this study we examined the effect of TNF alpha on neutrophil-mediated cartilage damage in vitro. Human neutrophils were able to injure both human and bovine articular cartilage slices by degrading proteoglycan and inhibiting its synthesis. Recombinant human TNF alpha enhanced neutrophil-mediated degradation of proteoglycan, even when neutrophils were preincubated with TNF alpha and washed before incubating with cartilage. TNF alpha alone degraded proteoglycan and inhibited its synthesis. Neutrophil-mediated inhibition of proteoglycan biosynthesis was increased after incubating cartilage together with neutrophils and TNF alpha, but was unaltered when neutrophils were preincubated with TNF alpha. We conclude that TNF alpha enhances neutrophil injury to articular cartilage.

Mechanisms of human neutrophil-mediated cartilage damage in vitro: the role of lysosomal enzymes, hydrogen peroxide and hypochlorous acid.

Cartilage is a focal point of attack by cellular and molecular elements of the inflammatory response which occurs in arthritic diseases. Neutrophils damage articular cartilage by degrading matrix components and inhibiting their synthesis. The aim of this study was to elucidate mechanisms of this damage. Human neutrophils were isolated from blood by centrifuging through Ficoll-Hypaque and granule extract prepared from them. Articular cartilage from adult humans and cattle was maintained in organ culture. Cartilage degradation (release of 35S-labelled proteoglycan) or synthesis (incorporation of 35S into proteoglycan) was determined after various treatments. Human neutrophils and neutrophil granule extract degraded proteoglycan and inhibited proteoglycan synthesis. The specific leucocyte elastase inhibitor N-methoxysuccinyl-(ala)2-pro-val-chloromethylketone (MAAPVCMK) partially reversed these effects. H2O2, a product of the neutrophil respiratory burst, when added directly at 10(-6)mol/L, or generated by glucose oxidase (GO)/glucose inhibited proteoglycan synthesis but had no effect on degradation. Hypochlorous acid (OHCl), a product of the myeloperoxidase (MPO)/H2O2/Cl system at 50 mumol/L degraded proteoglycan and inhibited its synthesis. OHCl produced by granule extract (as a source of MPO) + GO-generated H2O2 + Cl- degraded proteoglycan. The results indicate that neutrophil-mediated proteoglycan degradation and inhibition of synthesis is largely attributable to elastase and secondarily to OHCl, whereas H2O2 impairs synthesis without affecting degradation of proteoglycan.

Tumour necrosis factor-beta modulates human neutrophil-mediated cartilage damage.

Human neutrophils, when cultured with human articular cartilage coated with heat-aggregated immunoglobulin G, degraded proteoglycan and inhibited its synthesis. Neutrophil-mediated degradation of cartilage was potentiated by recombinant human tumour necrosis factor-beta (TNF beta), although TNF beta alone did not alter proteoglycan degradation. This effect was seen when TNF beta, neutrophils, and cartilage were incubated together, and also when neutrophils were preincubated with TNF beta and washed before being added to cartilage. Similar results were obtained with living and killed cartilage. In contrast, pretreatment of neutrophils with TNF beta abrogated the neutrophil-mediated inhibition of proteoglycan biosynthesis. There was no effect of TNF beta alone on synthesis of proteoglycan.

Effects of tumour necrosis factor alpha and interleukin-1 alpha and beta on human neutrophil migration, respiratory burst and degranulation.

Recombinant human tumour necrosis factor alpha (rHuTNF alpha) was shown to inhibit human neutrophil migration in the presence or absence of a chemotactic gradient generated with the tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), at doses of 20-100 U/10(6) cells. In contrast, neither recombinant human interleukin-1 alpha (rHuIL-1 alpha), rHuIL-1 beta, human leucocyte-derived IL-1 alpha (1HuIL-1 alpha) nor 1HuIL-1 beta contained neutrophil migration inhibition properties. However, both the interleukins (1HuIL-1 alpha, 1HuIL-1 beta and rHuIL-1 alpha) and rHuTNF alpha stimulated a neutrophil respiratory burst and significantly elevated the neutrophil respiratory response to fMLP (measured as chemiluminescence and H2O2 production). The stimulatory effects were observed at doses of between 5 and 100 U/5 x 10(5) cells. A characteristic feature of the effects of the cytokines was the range of variation observed in neutrophil responses from different individuals. However, a concentration-related effect was observed with each experiment, delineating suboptimal, optimal and supra-optimal cytokine concentrations. Neutrophils treated with rHuTNF alpha and rHuIL-1 alpha and washed free of exogenous cytokine retained the capacity to show an enhanced response to fMLP. Pretreatment of cells with cytochalasin B enhanced their response to fMLP, and this response was further increased if the cells had also been pretreated with the cytokines. The response to phorbol myristate acetate was also enhanced by rHuTNF alpha and rHuIL-1 alpha. The effects of these cytokines on neutrophils could be abolished by boiling the preparation but not by treating it with polymixin B, suggesting that bacterial lipopolysaccharide was not responsible for the activity of these preparations. The rHuIL-1 alpha increased the release of lysozyme, beta-glucuronidase and myeloperoxidase initiated by cytochalasin B/fMLP, while rHuTNF alpha only increased lysozyme release.

Conditioned medium from stimulated mononuclear leucocytes potentiates the ability of human neutrophils to damage human articular cartilage.

Human neutrophils were able to degrade proteoglycan and inhibit its synthesis when incubated with human articular cartilage coated with heat aggregated immunoglobulin G. These effects were potentiated when culture medium conditioned by mononuclear leucocytes stimulated with killed Staphylococcus aureus was also present during the incubations. Neutrophils preincubated with this conditioned medium and washed before incubation with cartilage also showed an increased ability to degrade proteoglycan and inhibit its synthesis. The percentage of neutrophils binding to cartilage was significantly increased in the presence of this conditioned medium.

Stimulation of neutrophil respiratory burst and lysosomal enzyme release by human interferon-gamma.

Highly purified human interferon-gamma (IFN-gamma) induced a respiratory burst and lysosomal enzyme release in human neutrophils. The lymphokine augmented the respiratory burst induced by phorbol myristate acetate (PMA), or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), and increased the release of specific granule enzymes induced by opsonized zymosan. Viability and locomotion of neutrophils were unaffected by IFN-gamma.

Interleukin 2 inhibits migration and stimulates respiratory burst and degranulation of human neutrophils in vitro.

Interleukin 2 (IL2) is a lymphocyte product recognised for its role as a T lymphocyte growth factor. Since some other lymphokines and monokines can modulate the function of granulocytes we examined the effects of natural and recombinant human IL2 on neutrophil locomotion, respiratory burst and degranulation. Purified T cell-derived IL2 inhibited both random and chemotactically-directed migration of neutrophils. IL2 induced a respiratory burst and release of lysosomal enzymes in neutrophils and increased these responses in classically stimulated neutrophils. Recombinant IL2 was also effective in altering neutrophil functions. We conclude that IL2 modulates neutrophil function.