Quercetin regulates the inhibitory effect of monoclonal non-specific suppressor factor beta on tumor necrosis factor-alpha production in LPS-stimulated macrophages

Monoclonal non-specific suppressor factor beta (MNSFbeta) is a member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta regulates the ERK1/2-MAPK cascade in the macrophage cell line Raw 264.7. In this study, we found evidence that the flavonol quercetin regulates the effect of MNSFbeta on TNFalpha production in LPS-stimulated Raw264.7 cells. Quercetin inhibited MNSFbeta siRNA-mediated enhancement of both TNFalpha production and ERK1/2 phosphorylation in LPS-stimulated Raw264.7 cells. Quercetin decreased the expression of 33.5-kDa MNSFbeta adduct, which is important to the regulation of ERK1/2 activity, in unstimulated Raw264.7 cells. The various flavonoids tested, including other flavonols, did not affect the formation of this adduct. Collectively, MNSFbeta and quercetin might share a common pathway in regulating the ERK1/2 pathway in macrophages. This is the first report describing the involvement of flavonoids in the action of ubiquitin-like proteins.     

Alveolar macrophages regulate neutrophil recruitment in endotoxin-induced lung injury

Background

Alveolar macrophages play an important role during the development of acute inflammatory lung injury. In the present study, in vivo alveolar macrophage depletion was performed by intratracheal application of dichloromethylene diphosphonate-liposomes in order to study the role of these effector cells in the early endotoxin-induced lung injury.

Methods

Lipopolysaccharide was applied intratracheally and the inflammatory reaction was assessed 4 hours later. Neutrophil accumulation and expression of inflammatory mediators were determined. To further analyze in vivo observations, in vitro experiments with alveolar epithelial cells and alveolar macrophages were performed.

Results

A 320% increase of polymorphonuclear leukocytes in bronchoalveolar lavage fluid was observed in macrophage-depleted compared to macrophage-competent lipopolysaccharide-animals. This neutrophil recruitment was also confirmed in the interstitial space. Monocyte chemoattractant protein-1 concentration in bronchoalveolar lavage fluid was significantly increased in the absence of alveolar macrophages. This phenomenon was underlined by in vitro experiments with alveolar epithelial cells and alveolar macrophages. Neutralizing monocyte chemoattractant protein-1 in the airways diminished neutrophil accumulation.

Conclusion

These data suggest that alveolar macorphages play an important role in early endotoxin-induced lung injury. They prevent neutrophil influx by controlling monocyte chemoattractant protein-1 production through alveolar epithelial cells. Alveolar macrophages might therefore possess robust anti-inflammatory effects.     

Biological characterization of purified macrophage-derived neutrophil chemotactic factor

We have recently described the purification of a 54 kDa acidic protein, identified as macrophage-derived neutrophil chemotactic factor (MNCF). This protein causes in vitro chemotaxis as well as in vivo neutrophil migration even in animals treated with dexamethasone. This in vivo chemotactic activity of MNCF in animals pretreated with dexamethasone is an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. MNCF is released in the supernatant by macrophage monolayers stimulated with lipopolysaccharide (LPS). In the present study, we describe some biological characteristics of homogenous purified MNCF. When assayed in vitro, MNCF gave a bell-shaped dose-response curve. This in vitro activity was shown to be caused by haptotaxis. Unlike N-formyl-methionylleucyl- phenylalanine (FMLP) or interleukin 8 (IL-8), the chemotactic activity of MNCF in vivo and in vitro, was inhibited by preincubation with D-galactose but not with D-mannose. In contrast with IL-8, MNCF did not bind to heparin and antiserum against IL-8 was ineffective in inhibiting its chemotactic activity. These data indicate that MNCF induces neutrophil migration through a carbohydrate recognition property, but by a mechanism different from that of the known chemokines. It is suggested that MNCF may be an important mediator in the recruitment of neutrophils via the formation of a substrate bound chemotactic gradient (haptotaxis) in the inflamed tissues.     

Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts

Although fibroblasts are important in providing a structural framework for most tissues, they also appear to be active participants in the inflammatory process via the production of specific mediators. The production of inflammatory mediators by fibroblasts is especially important in relation to their strategic location within connective tissue as they may act as a cellular communication bridge between the interstitium and vasculature. In this paper, we demonstrate that fibroblasts may participate in these inflammatory reactions by the production of a neutrophil chemotactic factor (NCF) with characteristics similar to a recently isolated and cloned monocyte-derived NCF. Either tumor necrosis factor-alpha-, interleukin-1 alpha-, or interleukin-1 beta-stimulated fibroblasts showed both a time- and dose-dependent increase in steady-state levels of NCF mRNA and secretion of chemotactic activity. In contrast, lipopolysaccharide and interleukin-6 failed to induce fibroblast-derived NCF. The expression of fibroblast-derived NCF mRNA was first detectable by 30 min poststimulation, whereas chemotactic activity was significantly observed 3-4 h postchallenge. Heat-inactivated monokine (100 degrees C) failed to induce NCF mRNA expression, suggesting that only the active proteins are capable of inducing NCF. Gel filtration analysis using high pressure liquid chromatography indicated peak chemotactic activity with an approximate molecular mass of 8000 daltons. This peak of NCF activity was found to be relatively stable to both heat and trypsin inactivation. Specificity of the fibroblast-derived neutrophil chemotactic activity was demonstrated with inhibition of chemotaxis by the addition of neutralizing antibody directed against recombinant human neutrophil chemotactic factor. These data provide evidence that monokine-treated fibroblasts can synthesize a potent chemotactic agent with molecular and physicochemical characteristics similar to monocyte-derived NCF and that this factor may contribute to neutrophil-mediated disease processes.     

Tissue factor is not involved in the mitogenic activity of factor VIIa

The present study was undertaken to evaluate in vitro the importance of tissue factor in the mitogenic effect of factor VIIa for embryonic fibroblasts. For that purpose, embryonic fibroblasts were isolated from either wild-type or transgenic mice showing a single inactivation of the tissue factor gene or expressing a truncated form (lacking the cytosolic domain) of this protein. Factor VIIa stimulated in a dose-dependent manner the growth of the 3 types of fibroblasts, thus showing that TF is not involved in the mitogenic activity of factor VIIa. The mitogenic activity of factor VIIa disappeared in serum immunopurified in factor X and was almost totally inhibited by DX9065, a selective factor Xa inhibitor, showing that this effect of factor VIIa occurred via factor Xa generated during the incubation period. Hirudin did not show any significant effect on factor VIIa-induced fibroblast proliferation, thus showing that the effect observed for factor VIIa was selectively mediated by factor Xa and was not due to thrombin formation. Our results therefore represent the first evidence for the possible importance of factor Xa in the mitogenic effect of factor VIIa and show the negligible role of tissue factor in this process.     

Transmethylation reactions regulate affinity and functional activity of chemotactic factor receptors on macrophages

Methylation mediated by S-adenosyl-l-methionine is required for the chemotaxis of mononuclear leukocytes. We investigated whether transmethylation reactions are required for normal functioning of chemotactic factor receptors. Three chemoattracrant-mediated functions in macrophages, chemotaxis, the stimulated release of arachidonic acid from membrane phospholipids and superoxide production, are markedly depressed by agents that inhibit cellular methylation reactions. Treatment of macrophages with methylation inhibitors decreased the affinity of the N-formylated chemoattractant receptor present on these cells by a factor of 4.5, but did not significantly alter the total receptor number. These results suggest that the N-formylated chemoattractant receptor on macrophages can exist in more than one affinity state and that an ongoing methylation reaction is required for the maintenance of the receptor in its higher affinity form. Inhibition of methylation lowers the affinity of the receptor and renders it no nfunctional or “uncoupled” in its ability to produce chemotaxis, superoxide and the release of arachidonic acid from leukocyte membranes.     

Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.     

Synthesis and biological characterization of monocyte-derived neutrophil chemotactic factor

MDNCF is a human monocyte-derived, 72-residue chemotactic peptide, which has sequence similarity with members of a family of pro-inflammatory cytokines. The peptide was synthesized by the solid-phase method, and is identical to the natural peptide in amino acid composition, sequence and chemotactic potency. MDNCF forms two loops via a neighboring pair of disulfide bridges, the probable locations of which are residues 7-34 and 9-50. Reduction and alkylation eliminated chemotactic activity. MDNCF fragments 7-37, 30-72 and 17-72 were all biologically inactive. The data suggest that the region of the clustered pair of disulfide bridges is important for biological activity.     

Tumor necrosis factor, interleukin-1 and interleukin-8 mediate the nociceptive activity of the supernatant of LPS-stimulated macrophages

It has been suggested that the supernatant of LPSstimulated macrophages (macrophage nociceptive factor, MNF) promotes nociception in mice. Intraperitoneal administration of MNF induced dose-related writhing, which reached a plateau between 18 and 26 min after injection and decreased within 60 min. The release of MNF was inhibited by the pretreatment of the macrophages with cycloheximide, a protein synthesis inhibitor, or with the glucocorticoid dexamethasone. Cyclooxygenase inhibitors, such as indomethacin or paracetamol, had no effect. The MNF-induced nociception was inhibited in a dose-related manner by pretreatment of the animals with indomethacin, paracetamol or dexamethasone. Pretreatment of the animals with the sympatholytics guanethidine and atenolol partially reduced the MNF nociception, which was abolished by the combination of guanethidine or atenolol with indomethacin. The preincubation of MNF with antisera against TNF-alpha, IL-1 or IL-8 partially inhibited its nociceptive effect. Intraperitoneal injection of a mixture of the recombinants cytokines TNF-alpha, IL-1 and IL-8 mimicked MNF nociception. The individual injection of these cytokines was unable to induce the nociceptive effect. In conclusion, our data suggest that the nociceptive activity of the supernatant of LPSstimulated macrophages is explained by the presence of TNF-alpha, IL-1 and IL-8, the nociceptive activity of which (in mice) seems to be due to the release of cyclooxygenase and sympathetic metabolites.     

Identification of two neutrophil chemotactic peptides produced by porcine alveolar macrophages.

We have purified to homogeneity two distinct 10-kDa proteins with potent chemotactic activity for neutrophils from porcine alveolar macrophages incubated for 24 h with Escherichia coli endotoxin (lipopolysaccharide (LPS), 10 micrograms/ml). Neutrophil chemotactic activity in alveolar macrophage supernatants was concentrated by adsorption to SP-Sephadex, and purified by cation exchange and reversed phase high performance liquid chromatography. The first peptide, alveolar macrophage chemotactic factor (AMCF)-I, had chemotactic activity for both porcine and human neutrophils. The chemotactic activity for porcine neutrophils was detectable at 3 x 10(-10) M, peaked at 3 x 10(-8) M, and was comparable to that of zymosan-activated porcine serum. Segmental instillation of AMCF-I into porcine lungs caused marked neutrophil accumulation at 4 h in both bronchoalveolar lavage fluid and in lung tissue. The second peptide, AMCF-II, was active at 1.4 x 10(-9) M for porcine neutrophils, but it was less active for human polymorphonuclear neutrophils than was AMCF-I. Oligonucleotide probes to regions of the N-terminal sequences of AMCF-I and AMCF-II hybridized to mRNA recovered from LPS-stimulated alveolar macrophages. The N-terminal sequences and amino acid compositions indicate that AMCF-I and AMCF-II are distinct proteins, but that both have homologies with a family of peptide chemoattractants produced by human blood monocytes and platelets. Thus, alveolar macrophages stimulated with LPS produce two distinct 10-kDa cytokines with potent chemotactic activity for neutrophils. This indicates that there are two different peptide pathways by which alveolar macrophages can recruit neutrophils into the lung.     

A protein kinase C inhibitory activity is present in rat brain homogenate

The partial purification and characterization of (a) factor(s) from rat brain which inhibit(s) the activity of calcium and phospholipid-dependent protein kinase from the same tissue is described. This factor, present in 100 000 X g rat brain homogenate supernatant, is inactivated upon treatment by trypsin and pepsin and is therefore assumed to be a protein. It was partially purified by ion-exchange chromatography on DEAE-cellulose, ammonium sulfate precipitation and gel filtration. This inhibitor is not stable to heating at 70 degrees C for 10 min, however partial renaturation of the inhibitory activity can be observed after incubation of the denatured inhibitor for 24 h at 4 degrees C. It is precipitable by 10% trichloroacetic acid and by 2 M ammonium sulfate. It exhibits a Stokes radius of 20 A by gel exclusion chromatography, corresponding to a molecular mass of 20 kDa assuming a globular shape. Kinetic analysis of the inhibition of calcium-phospholipid-dependent histone kinase activity indicates that the inhibitor is competitive with respect to the protein substrate. No change was observed in the kinetic values of the kinase for ATP, Ca2+ and phospholipids.     

Isolation and partial chemical characterization of macrophage-derived neutrophil chemotactic factor

Macrophages stimulated with lipopolysaccharide (LPS) release a factor (MNCF; macrophage-derived neutrophil chemotactic factor) which induces neutrophil migration in vivo and in vitro. The in vivo chemotactic activity of crude MNCF is not affected by pretreating the animals with dexamethasone, an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. We purified MNCF by affinity chromatography of the supernatant from LPS-stimulated macrophages on immobilized D-galactose, followed by gel filtration of the sugar-binding material on Superdex 75. The activity was eluted in the volume corresponding to a MW of 54 kDa. SDS-PAGE of this preparation revealed a single band, also corresponding to a 54 kDa protein. MNCF is an acidic protein (pI < 4) as shown by chromatofocussing. Like the crude MNCF, the homogeneous protein induced neutrophil migration in vitro as well as in vivo. This was not modified by dexamethasone pretreatment.     

Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages

Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl(2)) and examined their apoptosis by DNA fragmentation assay, 4',6-diamidino-2'-phenylindol dihydrochloride staining, and Western blot analysis. CoCl(2) induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl(2) inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl(2) released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.     

Effects of leukocyte inhibitory factor (LIF) on neutrophil phagocytosis and bactericidal activity

Human leukocyte inhibitory factor (LIF) is a lymphokine initially defined by its ability to inhibit the random migration of neutrophils. We have recently demonstrated that LIF also potentiates a number of f-met-leu-phe-mediated functions as well as enhancing one Fc receptor-mediated function (antibody-dependent cellular cytotoxicity). In this paper, we have extended our studies involving the effects of LIF on the neutrophil, specifically its effect on phagocytosis and bactericidal activity. We demonstrate that LIF (2 U/ml) potentiates phagocytosis of opsonized heat-killed Staphylococcus aureus (up to 57.2%) and sheep erythrocytes (124.4%) as well as unopsonized latex particles (59.9%). Phagocytosis of opsonized sheep erythrocytes was inhibited by an anti-neutrophil Fc receptor antibody with control PMN but not using the LIF-treated PMN. LIF (1/2 to 1 U) also potentiates the killing of S. aureus by up to 51.6%. Higher concentrations of LIF (greater than or equal to 4 U) inhibits killing. These effects were shown not to be associated with an increase in Fc receptor availability. It is therefore possible that potentiation of these neutrophil activities by LIF may occur either as a result of increased receptor turnover or, more likely, secondary to an increase in nonspecific neutrophil adherence. These studies further support the concept that LIF may have an important role in vivo in inflammation and immunity.     

Evidence that glutamine is involved in neutrophil function

Phosphate-dependent glutaminase (PDG) activity, a key enzyme of glutamine metabolism, was determined in neutrophils obtained from the intra-peritoneal cavity (PC) or bronchoalveolar space (BAS) after administration of 1 ml or 100 microl, respectively of saline, glycogen solution (1%) or lipopolysaccharide (LPS 0.1 mg (100 microl)(-1)). Neutrophils were obtained by lavage of both sites with 20 ml saline 24 h after the administration of the stimuli. Glycogen and LPS, depending on the site the cells were obtained from, differently modulated PDG activity. Cells from BAS stimulated by glycogen or LPS had raised PDG activity to 30.5 +/- 5.2 and 42.7 +/- 12.1 nmol min(-1) mg(-1) protein, respectively, when compared with saline (9.1 +/- 0.9 nmol min(-1) mg(-1) protein); mean +/- SEM. On the other hand, cells from PC showed different PDG activity: 52.0 +/- 12.6 nmol min(-1) mg(-1) for saline, 36.5 +/- 9.5 nmol min(-1) mg(-1) for glycogen, and 76.6 +/- 11.2 nmol min(-1) mg(-1) for LPS; mean +/- SEM. Therefore, PDG activity varies with the site from which neutrophils are obtained and the stimulus imposed. The effect of glutamine on nitric oxide (NO) and tumour necrosis factor (TNF) production by peritoneal neutrophils, obtained after glycogen administration, cultured in the presence of LPS (0.5 microg ml(-1)) was also examined. The addition of glutamine at concentrations varying from 2 to 20 mM did not markedly affect NO production. Glutamine alone at 2 mM did not modify the production of TNF but in the presence of LPS caused a significant decrease. So, glutamine may preserve the function of neutrophils during infections and injuries.     

Purification and characterization of a neutrophil chemotactic factor from Dirofilaria immitis

A neutrophil chemotactic factor (NCF-Di) was purified from a crude extract of Dirofilaria immitis adult worm by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S-200. NCF-Di showed a single protein band by both polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. The molecular weight of NCF-Di was estimated to be 17,000 by gel filtration on Sephadex G-150, and 14,000 by SDS-PAGE. NCF-Di was an acidic protein with isoelectric point of 4.5. NCF-Di was absorbed neither to lentil lectin-Sepharose nor to concanavalin A-Sepharose. The chemotactic activity of NCF-Di was heat labile (56 C, 1 hr), but was resistant to periodate oxidation. These results suggest that NCF-Di is a simple peptide which has few or no sugar chains. These physicochemical properties of NCF-Di were compared to previously reported parasite-derived chemoattractants or purified allergen of D. immitis.     

Monokine-induced gene expression of a human endothelial cell-derived neutrophil chemotactic factor

Monokines have been increasingly recognized as communication signals that interact with both immune and non-immune cells during inflammation. Specifically, interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) possess potent effector activities on various cell types. We present novel data demonstrating that human endothelial cells are a major source of a neutrophil chemotactic factor (NCF) synthesized upon stimulation with either IL-1 alpha, IL-1 beta, or TNF-alpha; but not with interleukin-6 (IL-6). Northern blot analysis demonstrated that 20 ng/ml of either IL-1 or TNF-alpha could induce endothelial cells to express significant levels of NCF mRNA, while IL-6 was not active in this system. These data demonstrate that monokines play an important role in mediating acute inflammation via induction of an endothelial cell-derived NCF.     

Evaluation of inhibitory activities of plant extracts on production of LPS-stimulated pro-inflammatory mediators in J774 murine macrophages

Whole plant methanolic extracts of 14 traditionally used medicinal herbs were evaluated for their anti-inflammatory activity. Extracts of Grindelia robusta, Salix nigra, Arnica montana, and Quassia amara showed up to 4.5-fold inhibition of nitric oxide (NO) production in the J774 murine macrophage cells challenged with LPS without cytotoxicity. These four selected extracts significantly reduced the protein levels of inducible NO synthase (iNOS) and the cyclooxygenase-2 (COX-2) as observed by Western blot analysis. Culture supernatants from cells treated with these extracts indicated 3–5-fold reduction of tumor necrosis factor-α (TNF-α). However, only G. robusta and Q. amara extracts significantly inhibited (by 50%) IL-1β and IL-12 secretions. Furthermore, all these plant extracts were shown to prevent the LPS-mediated nuclear translocation of nuclear factor-κB (NF-κB). All the above observations indicate the anti-inflammatory potential of these plant extracts.     

Identification of a ubiquitin family protein as a novel neutrophil chemotactic factor

Neutrophil chemotaxis is a process that is essential for the recruitment of neutrophils to an inflamed site. In the present study, we found a remarkable increase in neutrophil chemotactic activity in the lysate of red blood cells (RBC) of mice infected with murine malaria, Plasmodium yoelii. A neutrophil chemotactic factor with an apparent molecular weight of 17 kDa (IP17) was isolated from RBC by a combination of anion-exchange chromatography on DE52 and cation-exchange chromatography on Mono S. A comprehensive GenBank database search of N-terminal amino acid sequences and MALDI-TOF mass analysis of IP17 revealed that IP17 is identical to a murine homologue of ISG15/UCRP, a member of the ubiquitin family of proteins that are inducible by interferon-beta. Recombinant mouse ISG15 showed neutrophil chemotactic activity comparable to that of natural IP17. IP17 showed specific chemotactic activity forward neutrophils and activated neutrophils to induce the release of eosinophil chemotactic factors. These results suggest that the ubiquitin family protein ISG15/UCRP has novel functions in neutrophil-mediated immune mechanisms.