Metabotropic Glutamate Receptors Stimulate Phospholipase D via Different Pathways in the Adult and Neonate Rat Hippocampus

In order to characterize the ontogenetic profile of metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) we examined the effects of selected mGlu agents on PLD activity in immature and adult rat hippocampus. The group I mGlu receptor agonist 3,5-dihydroxyphenylglycine stimulated PLD in immature tissue, but reduced the PLD response evoked by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] in adult hippocampus. (2R,1S,2R,3S)-2-(2-Carboxy-3-phenylcyclopropyl)glycine (PCCG-13), a recently characterized selective antagonist of PLD-coupled mGlu receptors, displayed a much greater activity in reducing the PLD response to (1S,3R)-ACPD in adult than in neonate hippocampus. Our results lend support to the hypothesis that glutamatergic activation of PLD in the rat hippocampus is developmentally regulated.     

Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates

The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. The whole procedure, including neutrophil purification and kinetic measurements, can be done in 4-5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.     

Oxidative phosphorylation by mitochondria extracted from dry sunflower seeds

The role of mitochondria in the phosphorylation of ADP to ATP in the early steps of seed germination has been studied. Mitochondria were extracted from dry sunflower (Helianthus annuus) seeds. Adenylate kinase-dependent ATP synthesis was inhibited by p¹,p⁵-di(adenosine-5′)pentaphosphate. Synthesis of ATP was observed with the different substrates: citrate, α-ketoglutarate, succinate, malate, pyruvate or NADH. This synthesis was activated by cytochrome c, and inhibited by cyanide, oligomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and carboxyatractyloside. The ATP/O values with succinate were 0.85 and 1.2 in the absence or presence, respectively, of cytochrome c. Electron micrographs showed that mitochondria of dry tissues have different structures when observed in situ or in vitro after aqueous extraction, suggesting that profound changes occurred after the contact with the aqueous medium. These results confirm previous data obtained in vivo showing that mitochondria present in dry seeds are able to synthesize ATP as soon as the seeds are rehydrated.     

Catalytic Activity and Inhibition of Wegener Antigen Proteinase 3 on the Cell Surface of Human Polymorphonuclear Neutrophils

Proteinase 3 (Pr3), the main target of anti-neutrophil cytoplasmic antibodies, is a neutrophil serine protease that may be constitutively expressed at the surface of quiescent circulating neutrophils. This raises the question of the simultaneous presence in the circulation of constitutive membrane-bound Pr3 (mPr3) and its plasma inhibitor α1-protease inhibitor (α1-Pi). We have looked at the fate of constitutive mPr3 at the surface of circulating blood neutrophils and of induced mPr3 on triggered neutrophils. We found significant Pr3 activity at the surface of activated neutrophils but not at the surface of quiescent neutrophils whatever the constitutive expression. This suggests that constitutive mPr3 is enzymatically inactive or its active site is not accessible to the substrate. Supporting this conclusion, we have not been able to demonstrate any interaction between constitutive mPr3 and α1-Pi, whereas induced mPr3 is cleared from the cell surface when activated cells are incubated with this inhibitor. But, unlike membrane-bound elastase that is also cleared from the surface of activated cells, mPr3 remained bound to the membrane when inhibited by elafin or by a low molecular weight chloromethyl ketone inhibitor, which shows that it binds more tightly to the neutrophil membrane. mPr3 may thus be present at the surface of circulating neutrophils in an environment replete with α1-Pi. The permanent presence of inactive Pr3 at the surface of quiescent neutrophils may explain why Pr3 is a major target of anti-neutrophil cytoplasmic antibodies, whose binding activates neutrophils and triggers inflammation, as in Wegener granulomatosis.Proteinase 3 (Pr3)³ is a neutral serine protease (NSP) that is stored in the granules of circulating neutrophils (1, 2) and has been more recently located within secretory vesicles (3). Pr3, like its homologues neutrophil elastase (HNE) and cathepsin G (CG), participates in the intracellular degradation of phagocytized pathogens at inflammatory sites in combination with microbicidal peptides and the membrane-associated NADPH oxidase system (4). All three NSPs are also released from activated neutrophils and help destroy extracellular matrix components and regulate innate immunity, inflammation, and infection (5). Although NSPs are structurally and functionally related and are synthesized similarly (6), Pr3 differs from the other two by its bimodal, genetically determined, expression on the cell surface of quiescent neutrophils (7, 8). Thus, each individual has two subsets of neutrophils, mPr3^high and mPr3^low, whereas HNE and CG are not present in significant amount at the surface of resting neutrophils. Pr3 also differs from the other two NSPs by its storage within secretory vesicles that readily fuse with the plasma membrane (3). But it is not clear that this explains why Pr3 is constitutively expressed at the surface of a subpopulation of quiescent neutrophils. Supporting this hypothesis, it has been recently demonstrated that CD177 (also called NB1), which is also stored in secretory vesicles and has a bimodal membrane expression, is present on the plasma membrane of the same subset of neutrophils as Pr3 (9, 10).The presence of Pr3 on the surface of quiescent neutrophils would favor neutrophil activation by anti-neutrophil cytoplasmic antibodies (ANCAs) during Wegener granulomatosis (WG) (11). This explains why this protease, unlike HNE and CG, is a risk factor for this autoimmune disease characterized by necrotizing inflammation particularly of the respiratory tract, kidneys, and by small vessel vasculitis (12). Binding of anti-Pr3 antibodies to tumor necrosis factor-α-primed neutrophils is impaired by α1-Pi (13), which suggests that mPr3 activity and the protease-antiprotease balance are involved in neutrophil activation during WG.Measuring the Pr3 activity on the cell surface of quiescent and activated neutrophils requires specific substrates of Pr3 that were not available until recently (14, 15). Because of the storage of Pr3 in both secretory vesicles and primary granules and the presence of constitutive Pr3 at the surface of resting neutrophils, we have determined whether both constitutive and induced Pr3 are enzymatically active when bound to the cell surface, and how they are regulated by protease inhibitors. Pr3 activity is controlled by a variety of natural inhibitors, the most important of which are α1-Pi, elafin/trappin-2, and monocyte neutrophil elastase inhibitor. But none is specific for this protease, so it cannot be specifically targeted in vivo or ex vivo. We have previously shown that mHNE is rapidly cleared from the surface of activated neutrophils by α1-Pi and by EPI-hNE4, a low molecular weight recombinant inhibitor, with which it forms soluble, inactive complexes (16, 17). This raises the question of how mPr3 can be targeted by autoantibodies in the presence of α1-Pi, which efficiently inhibits its soluble form, although more slowly than it does HNE (18). We answered this question by investigating the enzymatic properties of mPr3 and its sensitivity to inhibitors. The behavior of mPr3 clearly differs from that of mHNE, which explains why it may be a preferential target for autoantibodies and so contributes to the pathogenicity of Wegener disease.     

Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates

Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.     

Inhibition of neutrophil elastase by alpha1-protease inhibitor at the surface of human polymorphonuclear neutrophils

The uncontrolled proteolytic activity in lung secretions during lung inflammatory diseases might be due to the resistance of membrane-bound proteases to inhibition. We have used a new fluorogenic neutrophil elastase substrate to measure the activity of free and membrane-bound human neutrophil elastase (HNE) in the presence of alpha1-protease inhibitor (alpha1-Pi), the main physiological inhibitor of neutrophil serine proteases in lung secretions. Fixed and unfixed neutrophils bore the same amounts of active HNE at their surface. However, the HNE bound to the surface of unfixed neutrophils was fully inhibited by stoichiometric amounts of alpha1-Pi, unlike that of fixed neutrophils. The rate of inhibition of HNE bound to the surface of unfixed neutrophils was the same as that of free HNE. In the presence of alpha1-Pi, membrane-bound elastase is almost entirely removed from the unfixed neutrophil membrane to form soluble irreversible complexes. This was confirmed by flow cytometry using an anti-HNE mAb. HNE activity rapidly reappeared at the surface of HNE-depleted cells when they were triggered with the calcium ionophore A23187, and this activity was fully inhibited by stoichiometric amounts of alpha1-Pi. HNE was not released from the cell surface by oxidized, inactive alpha1-Pi, showing that active inhibitor is required to interact with active protease from the cell surface. We conclude that HNE activity at the surface of human neutrophils is fully controlled by alpha1-Pi when the cells are in suspension. Pericellular proteolysis could be limited to zones of contact between neutrophils and subjacent protease substrates where natural inhibitors cannot penetrate.     

Computed Tomography (CT) Scanning Facilitates Early Identification of Neonatal Cystic Fibrosis Piglets

Background

Cystic Fibrosis (CF) is the most prevalent autosomal recessive disease in the Caucasian population. A cystic fibrosis transmembrane conductance regulator knockout (CFTR-/-) pig that displays most of the features of the human CF disease has been recently developed. However, CFTR ^-/- pigs presents a 100% prevalence of meconium ileus that leads to death in the first hours after birth, requiring a rapid diagnosis and surgical intervention to relieve intestinal obstruction. Identification of CFTR ^-/- piglets is usually performed by PCR genotyping, a procedure that lasts between 4 to 6 h. Here, we aimed to develop a procedure for rapid identification of CFTR ^-/- piglets that will allow placing them under intensive care soon after birth and immediately proceeding with the surgical correction.

Methods and Principal Findings

Male and female CFTR ^+/- pigs were crossed and the progeny was examined by computed tomography (CT) scan to detect the presence of meconium ileus and facilitate a rapid post-natal surgical intervention. Genotype was confirmed by PCR. CT scan presented a 94.4% sensitivity to diagnose CFTR ^-/- piglets. Diagnosis by CT scan reduced the birth-to-surgery time from a minimum of 10 h down to a minimum of 2.5 h and increased the survival of CFTR ^-/- piglets to a maximum of 13 days post-surgery as opposed to just 66 h after later surgery.

Conclusion

CT scan imaging of meconium ileus is an accurate method for rapid identification of CFTR ^-/- piglets. Early CT detection of meconium ileus may help to extend the lifespan of CFTR ^-/- piglets and, thus, improve experimental research on CF, still an incurable disease.