Initial characterization of the vitamin D binding protein (Gc-globulin) binding site on the neutrophil plasma membrane: evidence for a chondroitin sulfate proteoglycan.

The vitamin D binding protein (DBP) is a multifunctional plasma protein that can modulate certain immune and inflammatory responses. The diverse cellular functions of DBP appear to require cell surface binding to mediate these processes. Numerous reports have detected DBP bound to the surface of several cell types and would support the concept of a cell surface binding site for DBP. However, direct evidence for such a molecule has been lacking and essentially nothing is known about its basic biochemical properties. In the present study, radioiodinated DBP was used as a probe to characterize biochemically the neutrophil DBP binding site. Radiolabeled DBP binds to and remains associated with the plasma membrane and is not degraded. Quantitation of DBP binding to either intact cells or purified plasma membranes showed nonsaturable (linear) binding with positive cooperativity, possibly suggesting DBP oligomer formation. Solubilization of cell bound 125I-DBP with various nonionic and zwitterionic detergents demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fraction. Moreover, this molecule does not associate with the cytoskeleton. Cross-linking of radiolabeled DBP bound to plasma membranes increased the amount of protein that partitioned to the insoluble fraction, and analysis of these complexes by SDS-PAGE revealed that they may be very large since they did not enter the gel. Finally, treatment of plasma membranes with either proteases or chondroitinase ABC completely abrogated membrane binding of DBP, suggesting that the protein binds to a chondroitin sulfate proteoglycan.     

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.     

CD44 and annexin A2 mediate the C5a chemotactic cofactor function of the vitamin D binding protein

The vitamin D binding protein (DBP) is a plasma protein that significantly enhances the chemotactic activity of C5a and C5a(desArg) (cochemotactic activity). The objective of this study was to investigate how DBP mediates this process using neutrophils and U937 cells transfected with the C5a receptor (U937-C5aR cells) and comparing chemotaxis to C-activated serum (DBP dependent) vs purified C5a (DBP independent). Binding to the cell surface is essential for this protein to function as a chemotactic cofactor, and DBP binds to a chondroitin sulfate proteoglycan (CSPG) on neutrophil plasma membrane preparations. To determine whether a CSPG also functions to mediate cochemotactic activity, U937-C5aR cells were grown in chlorate to inhibit CSPG sulfation or treated with chondroitinase AC. Either treatment significantly inhibited chemotaxis only to C-activated serum. CD44 is a major cell surface CSPG on leukocytes, and functions to facilitate chemotaxis. Treatment of cells with anti-CD44 blocks chemotaxis of neutrophils and U937-C5aR cells to C-activated serum but not purified C5a. DBP binds to CD44 on the cell surface as evidenced by coimmunoprecipitation, confocal microscopy, and cell binding studies. Annexin A2 associates with CD44 in lipid rafts; therefore, its potential role in mediating cochemotactic activity was investigated. Results demonstrate that anti-A2 inhibits neutrophil and U937-C5aR chemotaxis specifically to C-activated serum, blocks DBP binding to cells, and colocalizes with anti-DBP on the cell surface. These results provide clear evidence that CD44 and annexin A2 mediate the C5a chemotactic cofactor function of DBP.     

Inhibition of the activation of multiple serine proteases with a cathepsin C inhibitor requires sustained exposure to prevent pro-enzyme processing

Cathepsin C is a cysteine protease required for the activation of several pro-inflammatory serine proteases and, as such, is of interest as a therapeutic target. In cathepsin C-deficient mice and humans, the N-terminal processing and activation of neutrophil elastase, cathepsin G, and proteinase-3 is abolished and is accompanied by a reduction of protein levels. Pharmacologically, the consequence of cathepsin C inhibition on the activation of these serine proteases has not been described, due to the lack of stable and non-toxic inhibitors and the absence of appropriate experimental cell systems. Using novel reversible peptide nitrile inhibitors of cathepsin C, and cell-based assays with U937 and EcoM-G cells, we determined the effects of pharmacological inhibition of cathepsin C on serine protease activity. We show that indirect and complete inhibition of neutrophil elastase, cathepsin G, and proteinase-3 is achievable in intact cells with selective and non-cytotoxic cathepsin C inhibitors, at concentrations approximately 10-fold higher than those required to inhibit purified cathepsin C. The concentration of inhibitor needed to block processing of these three serine proteases was similar, regardless of the cell system used. Importantly, cathepsin C inhibition must be sustained to maintain serine protease inhibition, because removal of the reversible inhibitors resulted in the activation of pro-enzymes in intact cells. These findings demonstrate that near complete inhibition of multiple serine proteases can be achieved with cathepsin C inhibitors and that cathepsin C inhibition represents a viable but challenging approach for the treatment of neutrophil-based inflammatory diseases.     

Activation of progelatinase B by membranes of human polymorphonuclear granulocytes

Isolated human granulocyte plasma membranes contain progelatinase B. The binding of progelatinase B to the membrane, however, is relatively weak, and a considerable part of progelatinase B can be removed by simply washing the membrane with buffer. This detachment does not depend on the ionic strength of the buffer, indicating that electrostatic forces do not play an important role in the binding of progelatinase B to the membrane. A complete removal of progelatinase B is achieved by chromatography of neutrophil membranes on gelatin-agarose. The plasma membrane of human granulocytes activates added progelatinase B. This activation is inhibited by soybean trypsin inhibitor and is thus performed by membrane bound serine proteinases. In contrast to other reports that claimed an important role of elastase in activating progelatinase B, we found that this activation is mostly inhibited by chymostatin and not by elastatinal and is thus primarily due to cathepsin G. Proteinase 3 was shown to activate progelatinase B as efficient as neutrophil elastase, i. e. much weaker than cathepsin G. Binding of cathepsin G and elastase to the neutrophil membrane does not change their ability to activate progelatinase B. However, cathepsin G, the most potent activator of the three neutrophil serine proteinases, is only a weak activator, when compared to stromelysin-1. This, as well as only a weak binding of progelatinase B, make it doubtful that activation of membrane-bound progelatinase B by membrane-bound serine proteinases is of significant physiological importance.     

Interactions of serine proteases with cultured fibroblasts

This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.     

Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.     

A novel locust (Schistocerca gregaria) serine protease inhibitor with a high affinity for neutrophil elastase

We have purified to homogeneity two forms of a new serine protease inhibitor specific for elastase/chymotrypsin from the ovary gland of the desert locust Schistocerca gregaria. This protein, greglin, has 83 amino acid residues and bears putative phosphorylation sites. Amino acid sequence alignments revealed no homology with pacifastin insect inhibitors and only a distant relationship with Kazal-type inhibitors. This was confirmed by computer-based structural studies. The most closely related homologue is a putative gene product from Ciona intestinalis with which it shares 38% sequence homology. Greglin is a fast-acting and tight binding inhibitor of human neutrophil elastase (k(ass)=1.2x10(7) M(-1) x s(-1), K(i)=3.6 nM) and subtilisin. It also binds neutrophil cathepsin G, pancreatic elastase and chymotrypsin with a lower affinity (26 nM< or =K(i)< or =153 nM), but does not inhibit neutrophil protease 3 or pancreatic trypsin. The capacity of greglin to inhibit neutrophil elastase was not significantly affected by exposure to acetonitrile, high temperature (90 degrees C), low or high pH (2.5-11.0), N-chlorosuccinimide-mediated oxidation or the proteolytic enzymes trypsin, papain and pseudolysin from Pseudomonas aeruginosa. Greglin efficiently inhibits the neutrophil elastase activity of sputum supernatants from cystic fibrosis patients. Its biological function in the locust ovary gland is currently unknown, but its physicochemical properties suggest that it can be used as a template to design a new generation of highly resistant elastase inhibitors for treating inflammatory diseases.     

Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases

Serine proteinases of human polymorphonuclear neutrophils play an important role in neutrophil-mediated proteolytic events; however, the non-oxidative mechanisms by which the cells can degrade extracellular matrix in the presence of proteinase inhibitors have not been elucidated. Herein, we provide the first report that human neutrophils express persistently active cell surface-bound human leukocyte elastase and cathepsin G on their cell surface. Unstimulated neutrophils have minimal cell surface expression of these enzymes; however, phorbol ester induces a 30-fold increase. While exposure of neutrophils to chemoattractants (fMLP and C5a) stimulates modest (two- to threefold) increases in cell surface expression of serine proteinases, priming with concentrations of lipopolysaccharide as low as 100 fg/ml leads to striking (up to 10-fold) increase in chemoattractant-induced cell surface expression, even in the presence of serum proteins. LPS-primed and fMLP-stimulated neutrophils have approximately 100 ng of cell surface human leukocyte elastase activity per 10(6) cells. Cell surface- bound human leukocyte elastase is catalytically active, yet is remarkably resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that binding of serine proteinases to the cell surface focuses and preserves their catalytic activity, even in the presence of proteinase inhibitors. Upregulated expression of persistently active cell surface-bound serine proteinases on activated neutrophils provides a novel mechanism to facilitate their egress from the vasculature, penetration of tissue barriers, and recruitment into sites of inflammation. Dysregulation of the cell surface expression of these enzymes has the potential to cause tissue destruction during inflammation.     

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.     

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using alpha-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.     

Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils

Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1β-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population.     

A blood fluke serine protease inhibitor regulates an endogenous larval elastase

The larvae of Schistosoma mansoni invade their mammalian host by utilizing a serine protease, cercarial elastase (SmCE), to degrade macromolecular proteins in host skin. The catalytic activity of serine and cysteine proteases can be regulated after activation by serpins. SmSrpQ, one of two S. mansoni serpins found in larval secretions, is only expressed during larval development and in the early stages of mammalian infection. In vitro, (35)S-SmSrpQ was able to form an SDS-stable complex with a component of the larval lysate, but no complex was detected when (35)S-SmSrpQ was incubated with several mammalian host proteases. Formation of a complex was sensitive to the protease active site inhibitors PMSF, Z-AAPF-CMK, and Z-AAPL-CMK. Western blot analysis of parasite lysates from different life stages detected a complex of comparable size to SmCE bound to SmSrpQ using anti-SmSrpQ or anti-SmCE antibodies. SmSrpQ and SmCE are located in adjacent but discrete compartments in the secretion glands of the parasite. Fluorescence immunohistochemical analysis of simulated infection showed co-localization of SmCE and SmSrpQ in host tissue suggesting a post release regulation of parasite protease activity during skin transversal. The results of this study suggest that cercarial elastase degradation of skin tissue is carefully regulated by SmSrpQ.     

Enzymatic and Molecular Biochemical Characterizations of Human Neutrophil Elastases and a Cathepsin G-like Enzyme

Human neutrophil elastase (HNE, EC 3. 4. 21. 37) is a causative factor of inflammatory diseases, including emphysema and rheumatoid arthritis. Enzymatic characterization is important for the development of new drugs involved in the regulation of this enzyme. In this study, we investigated the enzymatic and biochemical properties of five different elastolytic enzymes, with a molecular mass between 24 kDa and 72 kDa. Three elastases, molecular masses of 27, 29, 31 kDa, might be elastase isozymes that have the same NH₂-terminal amino acid sequences of Ile-Val-Gly-Gly-Arg-Arg-Ala. The 24-kDa enzyme, which showed the identical NH₂-terminal amino acid sequences to elastase, was a degraded fragment of native elastase. The elastolytic activity was conserved at the 6/7 domain of the NH₂-terminal region. The inhibitory characteristics of PMSF, DipF were the same as those of native elastases. The 72-kDa molecule, which showed elastolytic activity, might be a trimer formed between native elastases (31 kDa and 29 kDa) and a cathepsin G-like enzyme, which did not show elastolytic activity but enhanced the elastolytic activity of neutrophil elastase. Although this cathepsin G-like enzyme showed weak cathepsin G activity, it has distinguishable NH₂-terminal sequences of Ile-Val-Gly-Gly-Ser-Arg-Ala- from those of elastase or cathepsin G. The potentiation of elastolytic activity could be a result of the trimerization of native elastase with a cathepsin G-like enzyme, and was then weakly inhibited by serine protease inhibitors, such as PMSF, DipF. Therefore, we suggest the cathepsin G-like enzyme to be a novel enzyme, which has an important role in the development of inflammation.     

The effects of various serine protease inhibitors on estrogen receptor steroid binding

Two serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP), were utilized to investigate the possible involvement of serine hydroxyl groups on 17 beta-estradiol binding to the rat estrogen receptor (ER). Single point saturation analysis and Scatchard analysis demonstrated that both 5 mM PMSF and 5 mM DFP were able to inhibit steroid binding to the ER after incubation at 37 degrees C, but neither were able to inhibit steroid binding of the nonactivated ER (0-4 degrees C). The reducing agent dithiothreitol (DTT) was used to differentiate between the interaction of PMSF with serine groups or with sulfhydryl groups of the receptor. When incubated in the presence of 5 mM PMSF, various concentrations of DTT up to 25 mM were not able to overcome the inhibition of this agent, indicating that there was no interaction of PMSF with sulfhydryl groups. Thus, these findings indicate that serine hydroxyl groups are involved in steroid binding of the rat ER.     

New Selective Peptidyl Di(chlorophenyl) Phosphonate Esters for Visualizing and Blocking Neutrophil Proteinase 3 in Human Diseases

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl^P(O-C₆H₄-4-Cl)₂. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases.     

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.     

NETosis occurs independently of neutrophil serine proteases

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.     

Neutrophil activation by surface bound IgG is via a pertussis toxin insensitive G protein

Pre-treatment of neutrophils with either pertussis or cholera toxins does not inhibit neutrophil activation by surface bound IgG. In contrast, pretreatment with the phorbol ester, phorbol myristate acetate, results in a dose dependent inhibition of degranulation by surface bound IgG. This inhibition is similar to that seen with soluble ligands where it is thought to be due to interference with the interaction of an activated guanine nucleotide binding protein with phospholipase C (J. Biol. Chem.,262,6121,1987). More directly, GTP binding and GTPase activity are enhanced when human neutrophil membranes are incubated in wells containing surface bound IgG. Neither of these G protein functions were inhibited when membranes were prepared in the presence of pertussis toxin, suggesting that neutrophil activation by surface bound IgG proceeds by a mechanism that involves a pertussis toxin insensitive G protein.