Recombinant Endotoxin-neutralizing Protein Research Articles

Determinants of the Proinflammatory Action of Ambient Particulate Matter in Immortalized Murine Macrophages

BackgroundProximity to traffic-related pollution has been associated with poor respiratory health in adults and children.ObjectivesWe wished to test the hypothesis that particulate matter (PM) from high-traffic sites would display an enhanced capacity to elicit inflammation.MethodsWe examined the inflammatory potential of coarse [2.5–10 μm in aerodynamic diameter (PM2.5–10)] and fine [0.1–2.5 μm in aerodynamic diameter (PM0.1–2.5)] PM collected from nine sites throughout Europe with contrasting traffic contributions. We incubated murine monocytic-macrophagic RAW264.7 cells with PM samples from these sites (20 or 60 μg/cm2) and quantified their capacity to stimulate the release of arachidonic acid (AA) or the production of interleukin-6 and tumor necrosis factor-α (TNFα) as measures of their inflammatory potential. Responses were then related to PM composition: metals, hydrocarbons, anions/cations, and endotoxin content.ResultsInflammatory responses to ambient PM varied markedly on an equal mass basis, with PM2.5–10 displaying the largest signals and contrasts among sites. Notably, we found no evidence of enhanced inflammatory potential at high-traffic sites and observed some of the largest responses at sites distant from traffic. Correlation analyses indicated that much of the sample-to-sample contrast in the proinflammatory response was related to the content of endotoxin and transition metals (especially iron and copper) in PM2.5–10. Use of the metal chelator diethylene triamine pentaacetic acid inhibited AA release, whereas recombinant endotoxin-neutralizing protein partially inhibited TNFα production, demonstrating that different PM components triggered inflammatory responses through separate pathways.ConclusionsWe found no evidence that PM collected from sites in close proximity to traffic sources displayed enhanced proinflammatory activity in RAW264.7 cells.

Size-partitioning of an urban aerosol to identify particle determinants involved in the proinflammatory response induced in airway epithelial cells

BackgroundThe contribution of air particles in human cardio-respiratory diseases has been enlightened by several epidemiological studies. However the respective involvement of coarse, fine and ultrafine particles in health effects is still unclear. The aim of the present study is to determine which size fraction from a chemically characterized background aerosol has the most important short term biological effect and to decipher the determinants of such a behaviour.ResultsAmbient aerosols were collected at an urban background site in Paris using four 13-stage low pressure cascade impactors running in parallel (winter and summer 2005) in order to separate four size-classes (PM0.03–0.17 (defined here as ultrafine particles), PM0.17–1 (fine), PM1–2.5(intermediate) and PM2.5–10 (coarse)). Accordingly, their chemical composition and their pro-inflammatory potential on human airway epithelial cells were investigated. Considering isomass exposures (same particle concentrations for each size fractions) the pro-inflammatory response characterized by Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) release was found to decrease with aerosol size with no seasonal dependency. When cells were exposed to isovolume of particle suspensions in order to respect the particle proportions observed in ambient air, the GM-CSF release was maximal with the fine fraction. In presence of a recombinant endotoxin neutralizing protein, the GM-CSF release induced by particles is reduced for all size-fractions, with exception of the ultra-fine fraction which response is not modified. The different aerosol size-fractions were found to display important chemical differences related to the various contributing primary and secondary sources and aerosol age. The GM-CSF release was correlated to the organic component of the aerosols and especially its water soluble fraction. Finally, Cytochrome P450 1A1 activity that reflects PAH bioavailability varied as a function of the season: it was maximal for the fine fraction in winter and for the ultrafine fraction in summer.ConclusionIn the frame of future regulations, a particular attention should thus be paid to the ultrafine/fine (here referred to as PM1) fraction due to their overwhelming anthropogenic origin and predominance in the urban aerosol and their pro-inflammatory potential.

Bacterial endotoxin as inhibitor of the enzymatic activity of human thrombin

Endotoxemia caused by bacterial lipopolysaccharides (LPS) deleteriously affects many aspects of hemostasis. Much of this effect is well characterized as being secondary to the LPS-mediated inflammatory response, but direct effects of LPS on coagulation factors may also contribute to disregulation of the hemostatic process. Spectrophotometric assays were used to investigate the effects of LPS from different bacteria on thrombin and plasmin activities. We found that enzymatic activity of purified thrombin, but not plasmin, decreases in the presence of endotoxin. LPS-mediated inhibition of thrombin activity can be reversed by plasma gelsolin and recombinant endotoxin-neutralizing protein. Preincubation of thrombin with LPS before platelet activation results in inhibition of aggregation and secretion. Additionally, a decrease of elastic shear moduli of fibrin gels was observed when their formation was induced with thrombin preincubated with LPS or when LPS was present in fibrinogen solutions during fibrin gel formation. When added to platelet-rich plasma, after activation with collagen, LPS-inhibited thrombin activity. LPS-mediated inhibition of thrombin activity may contribute to the hemostasis dysfunctions observed during endotoxemia.

Use of an Antimicrobial Protein for Endotoxin Detection in a Competitive Electrochemical Assay

Endotoxins, also referred to as pyrogens, are part of the cellular walls of Gram-negative bacteria and are capable of inducing fever when entering into the blood stream. Current methods of detection involve the use of either amoebocytes from horseshoe crab that clots upon exposure to endotoxin or a live assay on rabbits. In this work, the detection of endotoxins by an electrochemical competitive assay is presented. Two configurations of modified electrodes were constructed using a recombinant endotoxin neutralizing protein (ENP) as recognition element. A modified lipopolysaccharide with horseradish peroxidase was used for the competitive assay. Modified electrodes constructed by electrostatic interaction of ENP and an electroactive polymer can detect the presence of endotoxins in concentrations as low as 0.2EU mL(-1), below the limit imposed for water in injectable drugs in the American Pharmacopoeia. Modified electrodes constructed by covalent linking of ENP to a carboxymethyl dextran matrix bound to the electrode show a better dynamic range but a higher detection limit.

Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide.

Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-alpha induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a K(d) of 19 micro m, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a K(d) in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS-ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form.

Proinflammatory and cytotoxic effects of Mexico City air pollution particulate matter in vitro are dependent on particle size and composition.

Exposure to urban airborne particulate matter (PM) is associated with adverse health effects. We previously reported that the cytotoxic and proinflammatory effects of Mexico City PM10 (less than or equal to 10 micro m mean aerodynamic diameter) are determined by transition metals and endotoxins associated with these particles. However, PM2.5 (less than or equal to 2.5 micro m mean aerodynamic diameter) could be more important as a human health risk because this smaller PM has the potential to reach the distal lung after inhalation. In this study, we compared the cytotoxic and proinflammatory effects of Mexico City PM10 with those of PM2.5 using the murine monocytic J774A.1 cell line in vitro. PMs were collected from the northern zone or the southeastern zone of Mexico City. Elemental composition and bacterial endotoxin on PMs were measured. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production by J774A.1 cells was measured in the presence or absence of recombinant endotoxin-neutralizing protein (rENP). Both northern and southeastern PMs contained endotoxin and a variety of transition metals. Southeastern PM10 contained the highest endotoxin levels, 2-fold higher than that in northern PM10. Northern and southeastern PM2.5 contained the lowest endotoxin levels. Accordingly, southeastern PM10 was the most potent in causing secretion of the proinflammatory cytokines TNF-alpha and IL-6. All PM2.5 and PM10 samples caused cytotoxicity, but northern PMs were the most toxic. Cytokine secretion induced by southeastern PM10 was reduced 50-75% by rENP. These results indicate major differences in PM10 and PM2.5. PM2.5 induces cytotoxicity in vitro through an endotoxin-independent mechanism that is likely mediated by transition metals. In contrast, PM10 with relatively high levels of endotoxin induces proinflammatory cytokine release via an endotoxin-dependent mechanism.

Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification

Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies. The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 ( his4). Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x- enp strains were analyzed by Southern blot for the presence of the ENP gene(s). Isolate 3x5q, containing a 3x- enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500 mg rENP/L with an average of 5.46 mg rENP/g DCW. Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86%. Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.

Induction of the lung myofibroblast PDGF receptor system by urban ambient particles from Mexico City.

Platelet-derived growth factor (PDGF) and its receptor system regulate mesenchymal cell proliferation. We recently reported that emission-source fly-ash particles and asbestos fibers induce the PDGF alpha-receptor through a macrophage-dependent pathway, and upregulation of this receptor greatly enhances the mitogenic response of lung myofibroblasts to PDGF (Lindroos and colleagues, Am. J. Respir. Cell Mol. Biol. 1997;16:283-292). In the present study we investigated the effect of particulate matter <= 10 micrometers in size (PM10) from the southern, central, and northern regions of Mexico City on PDGF receptor induction and compared these urban, ambient particles with Mt. St. Helen's volcanic ash particles as a negative control. All Mexico City PM10 samples, but not volcanic ash, stimulated rat alveolar macrophages to secrete a soluble, upregulatory factor(s) for the PDGF alpha-receptor on early passage rat lung myofibroblasts. The macrophage-derived upregulatory activity was blocked by the interleukin (IL)-1 receptor antagonist. The ability of PM10 to stimulate IL-1beta release was blocked in part by a recombinant endotoxin neutralizing protein (rENP). Lipopolysaccharide/endotoxin (LPS) and vanadium, both constituents that were present within these PM10 samples, also stimulated macrophages to secrete factor(s) that upregulated PDGF-Ralpha on lung myofibroblasts. Direct exposure of myofibroblasts to PM10 also elicited upregulation of the PDGF alpha-receptor, and this effect was blocked by rENP and mimicked by LPS, but not vanadium. These findings suggest that PM10 particles induce expression of the PDGF receptor system through macrophage-dependent and -independent mechanisms involving endotoxin and metals.

High-dose recombinant endotoxin neutralizing protein improves survival in rabbits with Escherichia coli sepsis

To assess the benefit of a recombinant endotoxin neutralizing protein from Limulus polyphemus in treating Gram-negative bacterial sepsis in rabbits. Prospective, blinded, controlled, laboratory trial. Animal research laboratory. New Zealand White rabbits. We established a rabbit model of Escherichia coli peritonitis and bacteremia, with high mortality rate, despite treatment with gentamicin and ceftriaxone. Twenty-five pairs of male New Zealand White rabbits were challenged intraperitoneally with E. coli O18ac K1 in 5% porcine mucin (mean 7 x 10(1) colony-forming units). All animals were treated with intravenous gentamicin (2.5 mg/kg) and ceftriaxone (100 mg/kg), and with either intravenous endotoxin neutralizing protein (50 mg/kg) or saline 1 hr after E. coli challenge. All animals were bacteremic 1 hr after challenge (mean 3.6 x 10(5) colony-forming units/mL). Animals in both groups developed tachycardia, hypotension, and acidosis (NS). Geometric mean serum endotoxin and tumor necrosis factor (TNF) concentrations were significantly ( p < .001) higher 1 hr after challenge compared with baseline prechallenge concentrations in both groups. From 1 to 2 hrs after challenge, endotoxin concentrations increased 2.5-fold in control animals (95% confidence interval = 13.1 to 32.9 endotoxin units/mL, p = .024), whereas endotoxin concentrations increased only 1.2-fold in endotoxin neutralizing protein-treated animals (95% confidence interval = 20.4 to 23.6 endotoxin units/mL, NS). TNF concentrations increased significantly (p < .001) in both groups from 1 to 2 hrs after challenge. Eighteen (72%) of 25 endotoxin neutralizing protein-treated animals vs. 11 (44%) of 25 controls survived 24 hrs (p = .032). Treatment with endotoxin neutralizing protein had the following effects: a) the increase in serum endotoxin was blunted, but not TNF concentrations measured 1 hr after antibiotic treatment; and b) survival in rabbits with E. Coli sepsis was improved.

Inhibition of Endotoxin-Induced Activation of the Coagulation and Fibrinolytic Pathways Using a Recombinant Endotoxin-Binding Protein (rBPI23)

A recombinant endotoxin-neutralizing protein, rBPI23, was shown to partially prevent endotoxin-induced activation of the fibrinolytic and coagulation systems in experimental endotoxemia in humans. In a placebo-controlled, blinded crossover study, eight volunteers were challenged twice with an intravenous bolus injection of endotoxin (40 EU/kg of body weight) and concurrently received either rBPI23 (1 mg/kg) or placebo (human serum albumin, 0.2 mg/kg). rBPI23 treatment significantly lowered the endotoxin-induced fibrinolytic response, ie, reduced the release of tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor antigen, and complex formation of plasmin alpha 2-antiplasmin (P = .0078 for each). Plasminogen activator inhibitor activity was also reduced, but not significantly according to the Hochberg method (P = .0304). The endotoxin-induced activation of the procoagulant state as reflected by increase in F1 + 2 fragments and TAT complexes was blunted by rBPI23 infusion (P = .0391 [not significant according to the Hochberg method] and .0078, respectively). These results indicate that rBPI23 is capable of reducing both the activation of the fibrinolytic and the coagulation systems after low-dose endotoxin infusion in humans.

Recombinant endotoxin neutralizing protein improves survival from Escherichia coli sepsis in rats.

A recombinant endotoxin neutralizing protein was evaluated for its ability to ameliorate the effects of Escherichia coli sepsis in rats. Prospective, controlled animal trial. Hospital animal research laboratory. Wistar rats, treated with gentamicin 1 hr after challenge with intraperitoneal E. coli O18ac. The animals received a recombinant endotoxin neutralizing protein, in doses of 5, 25, or 50 mg/kg, either 30 or 60 mins after challenge; controls received saline. Geometric mean serum endotoxin concentrations in endotoxin neutralizing protein-treated animals did not differ from control animals. Tumor necrosis factor concentrations in animals treated with endotoxin neutralizing protein 30 mins after challenge were significantly lower than controls. Animals treated with 25 or 50 mg/kg of endotoxin neutralizing protein 30 mins after E. coli challenge had significant improvements in survival compared with controls. Animals treated with 50 mg/kg of endotoxin neutralizing protein 60 mins after E. coli challenge had significant improvements in survival compared with controls. Endotoxin neutralizing protein significantly reduces mortality from Gram-negative sepsis in an antibiotic-treatment model of E. coli peritonitis and bacteremia in rats, mediated by a neutralization of the biological effects of endotoxin.

Effect of a recombinant endotoxin-neutralizing protein on endotoxin shock in rabbits.

Limulus anti-lipopolysaccharide factor, an 11.8-kilodalton peptide isolated from amebocytes of Limulus polyphemus inhibits the biologic activities of endotoxin in vitro, including gelation of Limulus amebocyte lysate. A recombinant version of Limulus anti-lipopolysaccharide factor, termed endotoxin neutralizing protein, has now been expressed in yeast. Endotoxin-neutralizing protein was evaluated for its potential prophylactic and therapeutic effects in rabbits challenged with Escherichia coli endotoxin. Controlled animal trial. Animal research laboratory. A total of 112 New Zealand white rabbits were studied. Rabbits were challenged with an LD80 dose of E. coli endotoxin (100 micrograms/kg); control animals (n = 52) were treated with saline solution at the time of endotoxin challenge; experimental animals received endotoxin-neutralizing protein 2.5 mg/kg prechallenge (n = 20), 5.0 mg/kg prechallenge (n = 20), or 5.0 mg/kg 30 mins postchallenge (n = 20).