Macrophage-derived Neutrophil Chemotactic Factor Research Articles

The macrophage-derived neutrophil chemotactic factor, MNCF: A lectin with TNF-α-like activities on neutrophils

The macrophage-derived neutrophil chemotactic factor (MNCF) is an α-galactoside-binding lectin, known to induce dexamethasone-insensitive neutrophil recruitment. We further characterized MNCF effects on neutrophils and showed that it shares with TNF-α the ability to delay apoptosis and to trigger degranulation. MNCF and TNF-α effects show similar kinetics and involve Src kinases and MAPKinases dependent pathways. They were, however, clearly distinguished, since the soluble TNF-receptor etanercept prevented TNF but not MNCF effects, while melibiose disaccharide inhibited MNCF but not TNF effects. Absorption of MNCF on detoxi-gel did not alter its properties, precluding an LPS contamination effect. By contrast, galectin-3 required LPS to activate neutrophils. Specific antibodies allowed to further demonstrate that MNCF and galectin-3 are two distinct molecules. Finally, MNCF- and IL-8-induced neutrophil activation differed by their kinetic and sensitivity to pertussis toxin. In conclusion, MNCF is a distinct neutrophil agonist, with pro-inflammatory activities involving its carbohydrate recognition domain.

Neutrophil haptotaxis induced by mouse MNCF: interactions with extracellular matrix glycoproteins probably contribute to overcoming the anti-inflammatory action of dexamethasone

The Macrophage-derived Neutrophil Chemotactic Factor (MNCF) has been characterized as a dexamethasone-resistant neutrophil chemotactic lectin produced by rat macrophages. This study was undertaken to evaluate different MNCF cellular sources and investigate the mechanisms by which MNCF overcomes the anti-inflammatory actions of dexamethasone. The mouse macrophage-like cell line P388D1 and thioglycollate-elicited mouse macrophages were studied regarding their capacity to release MNCF. Neutrophil migration assays were performed in vivo and in vitro, in either the presence or absence of extracellular matrix glycoproteins (ECM). Mouse and P388D1 macrophages release a lectin that reproduces the activities of rat MNCF. The ability of MNCF to induce neutrophil adhesion and haptotaxis is enhanced through its interaction with laminin and fibronectin. These properties are not inhibited by dexamethasone. Together, our results suggest that dexamethasone-resistant neutrophil migration induced by MNCF occurs probably because of its interactions with ECM.

The Macrophage-derived Lectin, MNCF, Activates Neutrophil Migration through a Pertussis Toxin-sensitive Pathway

The macrophage-derived neutrophil chemotactic factor (MNCF) is a D-galactose-binding lectin that induces neutrophil migration in vitro and in vivo. Neutrophil recruitment induced by MNCF is resistant to glucocorticoid treatment and is inhibited by the lectin-specific sugar, D-galactose. In the present study, we characterized the binding of MNCF to neutrophils and the responses triggered by this binding. Exposure to MNCF resulted in cell polarization, formation of a lamellipodium, and deep ruffles on the cell surface. By confocal microscopy, we observed that MNCF was evenly distributed on the cell surface after 30 min of incubation. The labeling intensity progressively diminished with longer incubations. Internalization kinetics showed that MNCF/ligand complexes were rapidly internalized, reaching maximum intracellular concentrations at 120 min and then decreased thereafter. The binding and internalization of MNCF were selectively inhibited by D-galactose. MNCF-induced neutrophil chemotaxis was inhibited by pertussis toxin. This fact strongly suggests that the MNCF-ligand on the neutrophil surface is a G-protein-coupled receptor (GPCR), similar to receptors for well-established neutrophil attractants. Our observations on the ability of MNCF to activate neutrophils are consistent with the increasing evidence for the participation of animal lectins in the innate immune response.

Cloning and characterization of Swine interleukin-17, preferentially expressed in the intestines.

Interleukin-17 (IL-17), initially reported as CTLA-8, is a proinflammatory cytokine produced mainly by activated T cells. In the present study, the cDNA of a swine IL-17 (PoIL-17) gene was cloned from activated neonatal thymocytes, and the recombinant PoIL-17 (rPoIL-17) was biologically characterized. The complete open reading frame (ORF) of PoIL-17 contains 462-bp coding deduced 153 amino acid residues, with a calculated molecular weight of 17.3 kDa. The amino acid sequence showed 72.9%, 64.9%, 64.7%, 60.1%, and 47.4% similarities with that of human, rat, mouse, Herpesvirus saimiri ORF 13, and chicken, respectively. The six cysteine residues conserved over species including the virus were observed in PoIL-17. We successfully prepared the recombinant mature form of PoIL-17 and analyzed its biologic activities for swine splenocytes. RT-PCR analysis revealed a marked upregulation of expression of IL-1beta, IL-8, tumor necrosis factor-alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), and monocyte chemotactic protein-1 (MCP-1) mRNA expression in splenocytes treated with 100 ng/ml rPoIL-17 for 3 h. Furthermore, a swine chemokine, alveolar macrophage-derived neutrophil chemotactic factor II (AMCF-II), which was classified into the CXC subfamily was also augmented in mRNA level. This evidence indicates that recombinat PoIL-17 expressed in Escherichia coli was biologically active and exerted similar effects to those of a human (HuIL-17) and murine IL-17 (MuIL-17). The PoIL-17 mRNA is strongly expressed in the adult heart, skin, and, interestingly, intestinal tissues, including mesenteric lymph nodes but is restricted in neonatal tissues by using real-time quantitative RT-PCR. The gene sequence and biologically active recombinat protein for PoIL-17 will be useful for elucidation of the role of IL-17 in the regulation of intestinal immune responses.

Mast Cell Degranulation Induced by Lectins: Effect on Neutrophil Recruitment

The mammalian lectin macrophage-derived neutrophil chemotactic factor (MNCF) and the plant lectin KM+ were characterized for their ability to activate and degranulate mast cells. The association between mast cell activation and the induction of neutrophil migration was also investigated. Incubation of rat peritoneal mast cells with these lectins resulted in degranulation and mediator release. By confocal microscopy, both lectins were evenly distributed on the cell surface. MNCF activated RBL-2H3 mast cells only if the cells had been sensitized with IgE. KM+ was able to activate either unsensitized or IgE sensitized RBL-2H3 cells. In microplate assays MNCF, but not KM+, bound to rat IgE. In rats that were depleted of mast cells, neutrophil recruitment by MNCF and KM+ were significantly reduced indicating that mast cell activation provides an amplification loop for the neutrophil recruitment induced by these lectins. The present study supports the concept that mammalian lectins play a fundamental role in innate immunity.

Perivascular inflammation after balloon angioplasty of porcine coronary arteries.

Inflammation has been suggested to play a role in vascular lesion formation after angioplasty. Whereas previous studies have focused on inflammatory reactions in the intima and media, less attention has been paid to adventitial and perivascular responses and their potential role in vascular remodeling. Balloon overstretch injury of porcine coronary arteries was performed with standard clinical angioplasty catheters. Vessels were examined from 0.5 hour to 14 days after injury by immunohistochemistry and in situ hybridization (ISH) for neutrophil and macrophage markers, cell adhesion molecules (P-selectin, E-selectin, and vascular cell adhesion molecule-1), and neutrophil-specific CXC chemokines (alveolar macrophage-derived neutrophil chemotactic factor [AMCF]-I/interleukin-8 and AMCF-II). Neutrophils accumulated in the adventitia surrounding the injury site from 2 hours to 3 days, followed by macrophages from 1 to 7 days after angioplasty. Inflammation was associated temporally with the expression of mRNAs encoding cell adhesion molecules and chemokines. The main inflammatory and proliferative foci were not limited to the adventitia but rather extended many millimeters away from the injured vessel throughout the surrounding adipose and myocardial tissues. Inflammatory responses after angioplasty of porcine coronary arteries occurred throughout the entire perivascular tissue. We hypothesize that perivascular inflammatory cells play a role in the recruitment and/or proliferation of adventitial myofibroblasts, possibly through the release of reactive oxygen species and/or cytokines, and thus contribute to vascular remodeling associated with postangioplasty restenosis.

Antiviral effect of nitric oxide during Japanese encephalitis virus infection

The ability of Japanese encephalitis virus (JEV) and JEV-induced macrophage derived neutrophil chemotactic factor (MDF) to produce nitric oxide (NO), and the possible antiviral effect of NO during JEV infection, was investigated. Splenic macrophages of JEV infected mice produced maximum NO in vivo at day 7 post infection, and in vitro at 24 h after JEV stimulation. MDF-induced NO production was dose dependent and maximal at 60 min after MDF treatment. The response was sensitive to anti-MDF antibody treatment and the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). Pretreatment of mice with L-NMMA increased the mortality to 100% in JEV infected mice in vivo and inhibited NO production in vitro, while MDF stimulated macrophages inhibited virus replication with high levels of NO production. MDF treatment increased the survival rate of JEV infected mice. The findings thus demonstrate that MDF induces production of NO during JEV infection, which has an antiviral effect. This may be one of the important mechanisms of natural immunity in controlling the initial stages of JEV infection.

The intravenous administration of tumor necrosis factor alpha, interleukin 8 and macrophage‐derived neutrophil chemotactic factor inhibits neutrophil migration by stimulating nitric oxide production

1. The i.v. administration of tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site. 2. Pretreatment of mice with the NO synthase antagonist, NG-monomethyl-L-arginine (L-NMMA, 15-60 mg kg(-1)), but not the inactive enantiomer D-NMMA (30 mg kg(-1)), prevented in a dose-dependent manner the TNF-alpha, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities. 3. Treatment of the neutrophils with TNFalpha (10(-7) M), IL-8 (10(-7) M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50-200 microM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-alpha or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis. 4. Preincubating the neutrophils with L-NMMA (200 microM) significantly increased the TNF-alpha (10(-7) M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10(-7) M)-mediated adhesion. 5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-alpha and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-alpha-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.

Macrophage‐derived neutrophil chemotactic factor is involved in the neutrophil recruitment inhibitory activity present in the supernatants of LPS‐stimulated macrophages

In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-α and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal−) and bound (D-gal+) fractions, with MNCF being found in the D-gal+ fraction. Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal− fraction. Furthermore, the incubation of the D-gal− fraction with anti-TNF-α plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal+ activity. Overall, these results suggest that the D-gal− inhibitory effect is partially mediated by TNF-α and IL-8, and that MNCF accounts for the inhibition of neutrophil migration in vivo by the D-gal+ fraction.

Partial characterization of the RNA from LPS-stimulated macrophages that induces the release of chemotactic cytokines by resident macrophages.

It is well established that exogenous RNA is incorporated into eukaryotic cells and is able to exert various biological responses. Little, however, is known about the effects of such RNA on macrophages. In this study, we demonstrate that RNA extracted from macrophages stimulated with Escherichia coli lipopolysaccharide (LPS), referred to as L-RNA, in contrast to RNA from non-stimulated macrophages (N-RNA), induces the release of a macrophage-derived neutrophil chemotactic factor (MNCF) and interleukin-8 (IL-8) from macrophage monolayers. The effect of L-RNA was dependent of the integrity of the polynucleotide chain and was not due to LPS contamination since its ability to induce MNCF and IL-8 release was strongly reduced by RNase but was not affected by DNase or polymyxin B. The poly A(+)L-RNA and poly A(-)L-RNA fractions were able to induce the release of MNCF and IL-8, indicating that the L-RNA could be acting at transcriptional and translational levels. The demonstration that actinomycin-D and cycloheximide inhibited the release of MNCF and IL-8 by L-RNA-stimulated macrophages confirms this assumption. Fractionation of the total L-RNA by centrifugation on a 5-20% sucrose gradient showed that the L-RNA which sediments in the 4-5S region of the gradient is the only fraction capable of inducing the release of MNCF from naive macrophages. We have previously shown that macrophage monolayers stimulated with interleukin-1 beta or LPS release a low molecular RNA which also sediments in the same 4-5S region. Taken together, these results support our proposal that resident macrophages, when activated by injurious stimuli, in addition to secreting cytokines, also release a low molecular weight (4-5S) RNA which may act on the surrounding macrophages to further stimulate the release of cytokines. This process would amplify the inflammatory response and would increase the mechanisms involved in the defense response or tissue injury.

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.

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.

Regulation of vascular permeability by macrophage-derived chemotactic factor produced in Japanese encephalitis.

The vascular effects of Japanese encephalitis virus (JEV)-stimulated splenic macrophage-derived neutrophil chemotactic factor (MDF) were evaluated in mice. Intraperitoneal injection of MDF in mice resulted in a rapid increase in capillary permeability in a dose-dependent manner as assessed by leakage of intravenously injected radiolabelled albumin ([125I]-albumin) or Evans blue dye. Intradermal inoculation of MDF in rabbits caused [51Cr]-labelled neutrophil emigration and accumulation into injected sites. Peak plasma leakage and neutrophil infiltration were observed at 1 h following MDF inoculation, and plasma leakage was restored by 2.5 h. The increase in capillary permeability was sensitive to pretreatment of mice with avil and ranitidine (H1 and H2 histamine receptor blockers, respectively), resulting in abrogation of the response; indomethacin, a prostaglandin synthetase inhibitor, did not have any effect.

Regulation of interleukin-8 expression in porcine alveolar macrophages by bacterial lipopolysaccharide.

Interleukin (IL)-8 is a macrophage-derived neutrophil chemotactic factor that plays an important role in the recruitment of neutrophils to inflammatory loci. Hence, expression of IL-8 by alveolar macrophages may be a significant factor in host defense in the lung and in the pathogenesis of pneumonia in swine. To initiate molecular studies of IL-8 regulation in pigs, we cloned IL-8 cDNA and examined the regulation of its mRNA in alveolar macrophages. The porcine IL-8 cDNA consists of 1491 base pairs including a coding region of 309 base pairs. The deduced amino acid sequence was 75 and 81% similar to human and rabbit IL-8, respectively. Resting macrophages contained low levels of IL-8 mRNA, which increased markedly after exposure to bacterial lipopolysaccharide (LPS). LPS induction of IL-8 was direct, not mediated through elevation of tumor necrosis factor or interleukin-1. The effect of LPS on IL-8 expression was dose dependent, and induction was observed at a concentration of 10 pg/ml. IL-8 mRNA expression was detectable within 0.5 h after stimulation with LPS, peaked at 3-6 h at about 30-fold higher levels than in resting cells, and was maintained for 24 h. Secreted IL-8, measured by neutrophil chemotaxis, was induced within 4 h by LPS, and accumulated in the media throughout the 24-h period. The mechanism of induction of IL-8 mRNA appeared to involve transcription and RNA processing. Nuclear run-on analysis showed that the IL-8 gene was actively transcribed in noninduced cells; upon stimulation with LPS, the rate of IL-8 transcription was increased about 4-fold. A single mature mRNA species was detected by primer extension analysis. The half-life of IL-8 mRNA transcripts in aveolar macrophages was approximately 2 h and did not change after LPS stimulation. The ability of LPS to induce IL-8 expression was suppressed by recombinant human IL-4 and dexamethasone in a concentration-dependent manner. These observations indicate that the expression of IL-8 is an early event in the sequelae to bacterial infection in the lung.

Role of resident macrophages in canatoxin-induced in vivo neutrophil migration.

Canatoxin (Cntx), a toxic protein purified from Canavalia ensiformis seeds, was shown to have lipoxygenase-mediated effects either in vivo or in vitro. Data here show that Cntx induced a dose-dependent migration of neutrophils and mononuclear cells when injected into rat peritoneal cavities. Furthermore, Cntx was able to induce neutrophil migration into pleural cavities and into air pouches. These effects were inhibited by dexamethasone but not by inhibitors of arachidonic acid metabolism (indomethacin, NDGA, and BW-755c) or by a PAF antagonist (BN 52021). In the peritoneal cavity Cntx caused an increase in vascular permeability inhibited by dexamethasone and BW-755c. Neutrophil migration induced by this toxin was dependent on the number of resident macrophages, since the migratory effect was enhanced by increasing the peritoneal macrophage population with thioglycollate pretreatment and was diminished when this population was reduced by peritoneal wash. It was also observed that Cntx induced release of a chemotactic factor from macrophage monolayers in vitro. Dexamethasone blocked this release but did not affect in vivo neutrophil recruitment induced by that factor. These data suggest that Cntx-induced neutrophil migration may be mediated by the same macrophage-derived neutrophil chemotactic factor released by other stimuli such as LPS, IL-1, and INF-gamma.

Asbestos-Induced Release of a Human Alveolar Macrophage-Derived Neutrophil Chemotactic Factor

Neutrophils accumulate in the alveoli of asbestos-exposed individuals. In determining whether asbestos fibers induce the release of neutrophil chemotactic factor (NCF) from human alveolar macrophages, alveolar macrophages (10(6) cell/mL) obtained by bronchoalveolar lavage from six non-asbestos-exposed control subjects were exposed to crocidolite (0.1 and 1 mg/mL), chrysotile (1 mg/mL), or medium alone for 4 h, and NCF activity was measured in the supernatants in a 48-well microchemotaxis chamber with polycarbonate membrane filters (pore size, 3 microns) and purified human neutrophils. Alveolar macrophages in medium alone released negligible amounts of NCF (4 +/- 1 neutrophils per high-power field [N/HPF]). When macrophages were exposed to crocidolite (0.1 and 1 mg/mL), significant NCF release occurred (43 +/- 9 and 105 +/- 32 N/HPF, respectively; p less than 0.01 for each amount compare to alveolar macrophages cultured in medium alone). Chrysotile (1 mg/mL) induced similar NCF release (96 +/- 14 N/HPF; p less than 0.01 compared to unstimulated alveolar macrophages). Partial characterization of the NCF by Sephadex G-25 fine gel filtration demonstrated a molecular size of less than 1,000 daltons. These results show that human alveolar macrophages release NCF after exposure to asbestos. Release of NCF by alveolar macrophages in asbestos-exposed individuals may play a central role in the pathogenesis of asbestosis.