Permeabilized Neutrophils Research Articles

NEW SELECTIVE PEPTIDYL DI(CHLOROPHENYL)‐PHOSPHONATE ESTERS TO VISUALIZE AND BLOCK 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 developed selective phosphonate inhibitors with the structure: Ac‐peptidyl‐P(O‐C6H4‐4‐Cl)2. The peptidyl moiety was deduced from molecular modeling and kinetic studies comparing the substrate specificities of PR3 with a single residue PR3 mutant and HNE. The P4 and P2 positions were essential to discriminate between PR3 and HNE. We then synthesized N‐terminally biotinylated peptidyl phosphonates to identify 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).

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.

Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes

Neutrophils kill micro-organisms using microbicidal products that they release into the phagosome or into the extracellular space. The secretory machinery utilized by neutrophils is poorly characterized. We show that the small GTPase Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Cell fractionation analysis revealed that Rab27a and the Rab27a effector protein JFC1/Slp1 (synaptotagmin-like protein 1) are distributed principally in the low-density fraction containing a minor population of MPO-containing granules. By immunofluorescence microscopy, we detected Rab27a and JFC1/Slp1 in a minor subpopulation of MPO-containing granules. Interference with the JFC1/Slp1-Rab27a secretory machinery impaired secretion of MPO in permeabilized neutrophils. The expression of Rab27a was dramatically increased when promyelocytic HL-60 cells were differentiated into granulocytes but not when they were differentiated into monocytes. Down-regulation of Rab27a in HL-60 cells by RNA interference did not affect JFC1/Slp1 expression but significantly decreased the secretion of MPO. Neither Rab27a nor JFC1/Slp1 was integrated into the phagolysosome membrane during phagocytosis. Neutrophils from Rab27a-deficient mice efficiently phagocytose zymosan opsonized particles and deliver MPO to the phagosome. We conclude that Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes. Our results suggest that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

The Role of the Small GTPase Rab27a in the Regulated Secretion of Granulocytes.

Neutrophils kill microorganisms using microbicidal products that they release into the phagosome or the extracellular space. Mature neutrophils contain four types of exocytosable storage organelles: azurophilic granules that contain myeloperoxidase, specific granules, gelatinase granules and secretory vesicles. Since the uncontrolled release of the contents of these organelles is potentially harmful, granule exocytosis must be tightly regulated. However, the secretory machinery utilized by neutrophils is poorly characterized. Here, we evaluate the role of Rab27a and its effector JFC1 in the regulated secretion of granulocytes. First, we show that JFC1 and Rab27a are highly expressed in human neutrophils. Furthermore, in HL-60 promyelocytic cells, the expression of JFC1 and Rab27a dramatically increased when they are differentiated to neutrophil-like granulocytes by DMSO treatment, supporting a role for these proteins in the secretory machinery of granulocytes. We found that while Rab27a distribution is restricted to the membrane fraction after cell fractionation, JFC1 is distributed between the membrane fraction and the cytosol. The localization of Rab27a in neutrophils was confirmed by immuno-electron microscopy. Colocalization of endogenous JFC1 and Rab27a was observed by immunofluorescence analysis. After cell fractionation, JFC1 and Rab27a were mainly associated with the tertiary granules (γ fraction) which are enriched in MMP-9 and cytochrome b558. A small proportion of the myeloperoxidase-positive granules were also detected in this fraction. Both Rab27a and JFC1 colocalized with VAMP2, a marker of secretory vesicles. The introduction of the plasma membrane binding domain (C2A domain) of JFC1 into permeabilized neutrophils significantly decreased exocytosis, however, neither Rab27a nor JFC1 integrated to the phagolysosome when neutrophils were exposed to opsonized particles, suggesting that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

Functional epitope on human neutrophil flavocytochrome b558.

mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.

A Novel Assay System Implicates PtdIns(3,4)P 2, PtdIns(3)P, and PKCδ in Intracellular Production of Reactive Oxygen Species by the NADPH Oxidase

Activated neutrophils assemble an NADPH oxidase enzyme complex to produce superoxide for microbial killing. Much of the initial oxidase assembly occurs on intracellular granules, followed by movement of the oxidase to phagolysosomes and the plasma membrane. We have developed a novel assay system using Streptolysin-O permeabilized neutrophils that recapitulates the initial intracellular activation process while maintaining the ultrastructural features of this granulocytic cell type. Using this system, we biochemically dissect molecular events and signaling pathways involved in NADPH oxidase assembly and demonstrate specific roles for PKCδ, PI(3,4)P 2/PI(3,4,5)P 3, and PI(3)P in the PMA-dependent intracellular activation process. This system should be of great utility for the study of cell signaling events that regulate the intracellular production of reactive oxygen species by neutrophils.

Essential Requirement of Cytosolic Phospholipase A2for Stimulation of NADPH Oxidase-associated Diaphorase Activity in Granulocyte-like Cells

We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation. In this study we used this model to investigate the physiological role of cPLA(2) in regulation of NADPH oxidase-associated diaphorase activity. A novel diaphorase activity assay, using 4-iodonitrotetrazolium violet as an electron acceptor, was used in permeabilized neutrophils and PLB-985 cells differentiated toward the granulocytic or monocytic phenotypes. Phorbol 12-myristate 13-acetate, guanosine 5'-3-O- (thio)triphosphate (GTP gamma S), or FMLP stimulated a similar diphenylene iodonium-sensitive diaphorase activity pattern in neutrophils and in differentiated parent PLB-985 cells. This diaphorase activity was not detected in undifferentiated cells, but developed during differentiation. Furthermore, diaphorase activity could not be stimulated in permeabilized neutrophils from X-linked CGD patients and in differentiated gp91(phox)-targeted PLB-985 cells that lacked normal expression of gp91(phox), but was restored to these cells following transduction with retrovirus encoding gp91(phox). The differentiated PLB-D cells showed no diaphorase activity when stimulated by either GTP gamma S or FMLP, and only partial activation when stimulated with phorbol 12-myristate 13-acetate. Diaphorase activity in response to either agonists was fully restored by the addition of 10 microm free AA. The permeabilized cell 4-iodonitrotetrazolium violet reduction assay offers a unique tool for the evaluation of NADPH oxidase-associated diaphorase activity in stimulated whole cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in activation of electron transfer through the FAD reduction center of NADPH oxidase.

Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cβ activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport

Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [γ-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCβ (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 °C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact.

Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cβ activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport

Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [gamma-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCbeta (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 degrees C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact.

Co-operation of phosphatidylinositol transfer protein with phosphoinositide 3-kinase gamma in the formylmethionyl-leucylphenylalanine-dependent production of phosphatidylinositol 3,4,5-trisphosphate in human neutrophils.

Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3) play an essential role in the regulation of neutrophil functions by the chemoattractant formylmethionyl-leucylphenylalanine (FMLP). Here we show that permeabilization of human neutrophils leads to loss of cytosolic components, including PI3Kgamma, and causes the loss of FMLP-dependent production of PIP3. FMLP-sensitive synthesis of PIP3 could be restored by reconstitution of permeabilized neutrophils with recombinant PI3Kgamma. Admixture of recombinant phosphatidylinositol transfer protein (PITP) to the reconstitution cocktail produced a further increase of PIP3 synthesis, whereas pertussis toxin suppressed the FMLP-dependent production of PIP3. We conclude that FMLP-sensitive PIP3 formation in human neutrophils involves the FMLP receptor, heterotrimeric G-proteins of the Gi type, PI3Kgamma and PITP.

Augmentation by calmodulin of ADP-ribosylation factor-stimulated phospholipase D activity in permeabilized rabbit peritoneal neutrophils.

Activation of the membrane-bound phospholipase D (PLD) requires cytosolic factor(s), and ADP-ribosylation factor (ARF) has been identified as a cytosolic PLD activator. In the present study, we demonstrate that calmodulin (CaM) and ARF are both involved in PLD activation in rabbit peritoneal neutrophils. The PLD activity of streptolysin O-permeabilized, cytosol-depleted rabbit neutrophils was significantly enhanced when the permeabilized cells were reconstituted with bovine brain cytosol in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas there was little activation of the enzyme in the absence of cytosol. The GTP gamma S-stimulated PLD activity in the presence of cytosol was augmented on increasing the concentration of free Ca2+. The PLD activity stimulated by GTP gamma S and Ca2+ in this system was inhibited by the calmodulin inhibitor W-7. These findings suggest that CaM plays a role as a cytosolic PLD activator. Moreover, highly purified CaM alone, as well as partially purified ARF alone, promoted a slight stimulation of the PLD activity in permeabilized neutrophils. Interestingly, ARF-stimulated PLD activity was augmented by CaM in the presence of GTP gamma S and Ca2+. This augmentation was again inhibited by W-7, as well as by the structurally unrelated CaM inhibitor trifluoperazine. These data imply that CaM stimulates the PLD activity of rabbit neutrophils in concert with ARF.

Fixation traps formyl peptide receptors in high and low affinity forms that can be regulated by GTP[S] in the absence of ligand.

The formyl peptide receptor on human neutrophils recognizes bacterial, N-formylated peptides and initiates a cascade of intracellular signals via a pertussis toxin sensitive Gi protein. We used fluorescence techniques to investigate the interactions of ligand (L), receptor (R), and G proteins (G), the ternary complex, in both live and fixed human neutrophils. By lightly fixing permeabilized neutrophils with a procedure that retained ligand binding, we were able to "capture' R and G in different configurations in the absence of ligand. Fixed receptors were trapped in a high affinity form (attributed to LRG) that could not be rapidly converted to low affinity by the addition of GTP[S]. Adding saturating nucleotide prior to fixation trapped receptors in a low affinity form (attributed to LR). The low affinity receptors retained the sensitivity of the native receptors to the presence of NA+. The distribution between high and low affinity receptors was modulated by GTP[S] in a dose dependent manner. The ability to redistribute low and high affinity receptor forms prior to fixation was unique to GTP[S], as compared to other non-activating nucleotides, suggesting that GTP[S] can regulate the distribution between R and RG. We suggest that precoupled receptors that give rise to high affinity ligand binding are likely to exist in native membranes in human neutrophils.

5-Lipoxygenase contains a functional Src homology 3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins.

A short, proline-rich region spanning residues 566-577 in human 5-lipoxygenase is a binding site for the Src homology 3 (SH3) domain of growth factor receptor-bound protein 2 (Grb2), an "adaptor" protein for tyrosine kinase-mediated cell signaling. Purified 5-lipoxygenase bound to glutathione S-transferase fusion products of Grb2 and a truncated version of Grb2 containing its SH3 domain. A peptide corresponding to the proline-rich, SH3-binding motif inhibited formation of the 5-lipoxygenase.Grb2 complex in vitro. The peptide also inhibited the redistribution of 5-lipoxygenase from the cytosol to the membrane in intact or permeabilized neutrophils activated by calcium ionophore A23187. 5-Lipoxygenase did not bind to the SH3 domains of other signaling proteins, such as GTPase-activating protein and phospholipase C gamma; however, it bound to certain cytoskeletal proteins including alpha-actinin and actin. 5-Lipoxygenase contains a consensus guanine nucleotide-binding site at residues 296-299, and guanine nucleotides inhibit 5-lipoxygenase activity in vitro. Our results suggest that 5-lipoxygenase may have a previously unrecognized role in tyrosine kinase signaling, distinct from its catalysis of lipid mediator formation. Our results also clarify the molecular basis for compartmentalization and translocation of 5-lipoxygenase in myeloid cells, implying that it binds to proteins other than its activating protein.

Induction of actin polymerization in permeabilized neutrophils. Role of ATP.

We have used streptolysin-O (SO)-permeabilized neutrophils to investigate the signal transduction pathway through which chemoattractants induce actin polymerization. Chemoattractants stimulate phosphorylation of various proteins and lipids but whether these phosphorylations are required for actin polymerization is not known. Addition of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to SO-permeabilized neutrophils induced a doubling of the F-actin. This induction of F-actin, assayed by TRITC-labeled phalloidin binding, did not require the addition of ATP. Neither addition of apyrase to deplete residual ATP nor addition of ADP or UDP to compete with residual endogenous ATP inhibited significantly the GTP gamma S-induced polymerization. Addition of ATP on its own caused no increase in F-actin and did not affect the time course or concentration dependence of GTP gamma S-induced F-actin. Addition of ATP did increase the maximal amount of F-actin induced by GTP gamma S by about 20%. N-Formylnorleucylleucylphenalanine (formyl-peptide) in the presence of GTP, but not in its absence, also stimulated an increase in F-actin in SO-permeabilized cells. The F-actin induced by formyl-peptide plus GTP was inhibited by pertussis toxin. The induction did not require addition of ATP and addition of ADP to compete with residual ATP only slightly decreased the level of actin. However, addition of UDP significantly reduced the response to formyl-peptide plus GTP. Addition of ATP enhanced the increase in F-actin induced by optimal concentrations of GTP with formyl-peptide. ATP also lowered the apparent Km for GTP, but not for N-formyl peptide. The non-hydrolyzable ATP analog, adenosine 5'-(beta, gamma-imino)triphosphate, did not enhance the actin polymerization. Rather its presence inhibited the response induced by formyl-peptide plus GTP. The data suggest that actin polymerization can be induced by GTP gamma S in an manner that is largely ATP-independent. A role for ATP cannot be ruled out in the induction of actin polymerization by formyl-peptide plus GTP.

Farnesyl-L-cysteine analogs can inhibit or initiate superoxide release by human neutrophils

A series of farnesylcysteine analogs was studied with respect to their abilities to interfere with fMet-Leu-Phe (fMLP)-stimulated superoxide (O2-.) release by human neutrophils. Simple acyl derivatives of farnesyl-L-cysteine, such as the N-acetyl (L-AFC) and N-isobutyryl derivatives (L-iBFC), which are substrates for the isoprenylated protein methyltransferase, can block O2-. release. The N-butyryl analog (L-BFC), which is an isomer of L-iBFC and also a substrate for the methyltransferase, does not inhibit O2-. release but actually stimulates it in the absence of fMLP. Other analogs, including the N-pivaloyl derivative, which has been found to be neither a substrate nor an inhibitor of methyltransferase, also stimulate very large quantities of O2-. production. The stimulatory effects of these derivatives are saturable and exquisitively sensitive to small structural changes in the analogs. The signal transduction pathway(s) utilized by pivaloyl derivatives for triggering O2-. generation is very similar to that employed by fMLP. These data make it clear that farnesyl-L-cysteine analogs do not produce their pharmacological effects in neutrophils via methyltransferase blockade. This could be further demonstrated by showing that sinefungin and S-adenosylhomocysteine, both powerful and general methyltransferase inhibitors which bind at the S-adenosylmethionine site, had no effect in preventing the increased oxygen consumption associated with O2-. production in permeabilized neutrophils. These studies reveal that farnesyl-L-cysteine analogs interact with a hitherto undefined target in neutrophils that may be exploited for inhibiting or stimulating the inflammatory or antimicrobial responses of these cells.

Inhibition of neutrophil NADPH oxidase assembly by a myristoylated pseudosubstrate of protein kinase C.

To further define the role played by protein kinase C (PKC) in the activation of the neutrophil NADPH oxidase, we have utilized a pseudosubstrate of PKC which was myristoylated at the N terminus. In electropermeabilized neutrophils, the myristoylated pseudosubstrate Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (myr-psi PKC) inhibited PMA-induced protein phosphorylations and activation of the NADPH oxidase, induced either by PMA or by the receptor agonist formyl-methionyl-leucyl-phenylalanine. Both the pseudosubstrate lacking the N-terminal myristate (psi PKC) and a myristoylated control peptide (Phe-Ala-Glu-Asp-Gly-Ala-Leu-Glu-Gln, myr-CP) were without effect on these responses. The myristoylated pseudosubstrate was also tested in a cell-free system, in which NADPH oxidase activation can be achieved by addition of SDS and guanosine 5'-3-O-(thio)triphosphate in a staurosporine-insensitive manner. Myr-psi PKC, but not psi PKC or myr-CP, proved to be a potent inhibitor of NADPH oxidase activity in the cell-free system, indicating that the inhibition observed in permeabilized neutrophils may have been caused by an effect other than PKC inhibition. In the presence of myr-psi PKC, translocation in the cell-free system of the cytosolic oxidase components p47-phox and p67-phox to the plasma membrane was inhibited. From these results we conclude that myristoylation profoundly increases the ability of pseudosubstrates of PKC to inhibit not only PKC-mediated phosphorylations, but also NADPH oxidase activation. The latter effect, however, is most probably not related to PKC inhibition but may indicate a critical role of the membrane surface charge in the translocation of the cytosolic oxidase components p47-phox and p67-phox.

Synthesis of phosphatidylinositol 3,4,5-trisphosphate in permeabilized neutrophils regulated by receptors and G-proteins

Formylated Met-Leu-Phe (fMLP), platelet-activating factor (PAF), ATP, and various nonhydrolyzable guanine nucleotides stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in intact human neutrophils. A protocol was devised to selectively inhibit the capacity of the nucleotide-sensitive receptors to elicit accumulation of PtdIns (3,4,5)P3. This enabled study of the regulation of phosphoinositide 3OH-kinase (PI3K) activities in permeabilized neutrophils free from interference due to activation of cell-surface nucleotide receptors. FMLP, PAF, and nonhydrolyzable GTP analogues stimulated an increase in the concentration, and rate of synthesis, of PtdIns(3,4,5)P3 in permeabilized neutrophils by increasing the rate of a PtdIns(4,5)P2-directed PI3K-catalyzed reaction. A number of characteristics of these responses, including their relative sensitivities to inhibition by pertussis toxin and guanosine 5'-beta-(thio)diphosphate, suggested that fMLP and PAF increased this PI3K activity via the actions of heterotrimeric G-proteins. Basal and guanosine 5'-gamma-(thio)triphosphate/fMLP-stimulated increases in PI3K activity were resistant to changes in free calcium concentrations, staurosporine, acute treatment with phorbol esters, and evidently to permeabilization. This, in conjunction with other work, indicates that the PAF and fMLP-induced increase in PtdIns(4,5)P2-directed PI3K activity is not being produced via activation of a currently defined G-protein regulated effector enzyme, or a protein tyrosine kinase coordinated mechanism of a type already known to regulate PI3K activities.

Regulation of tyrosine phosphorylation in neutrophils by the NADPH oxidase. Role of reactive oxygen intermediates

Neutrophils possess a multicomponent NADPH-oxidase that produces large quantities of superoxide, which can in turn generate other reactive oxygen intermediates. Superoxide and its dismutation product, hydrogen peroxide, are powerful oxidants. Because the activity of certain tyrosine kinases and phosphatases can be affected by their redox state, we considered the possibility that endogenously generated reactive oxygen intermediates (ROI) may alter phosphotyrosine formation and thereby function as intra- or intercellular messengers in neutrophils. Exposure of human neutrophils to exogenous oxidants such as diamide induced marked tyrosine phosphorylation of several cellular proteins. More importantly, activation of the NADPH oxidase in permeabilized neutrophils, by direct stimulation of GTP-binding proteins, also resulted in enhanced tyrosine phosphorylation. The latter was NADPH-dependent, paralleled by production of superoxide, and was inhibited by diphenylene iodonium, an inhibitor of the flavoprotein component of the oxidase. Neutrophils, from a patient with chronic granulomatous disease, which are deficient in the production of ROI, demonstrated no such phosphotyrosine accumulation. We conclude that ROI produced by the NADPH oxidase can regulate tyrosine phosphorylation in granulocytes, possibly by effects of oxidation-sensitive tyrosine kinases and/or phosphatases.

Strontium and barium induce exocytosis in electropermeabilized neutrophils

Calcium, strontium and barium induced an exocytotic response in electropermeabilized rabbit neutrophils while magnesium was without any effect. The extent of enzyme release was found to depend upon the concentration of these cations. For all cations, an optimum concentration was found with the same maximum enzyme release. At concentrations higher than optimum a decrease in lysozyme release was observed. Efficiency to induce enzyme release was in the order: Ca 2+>Sr 2+>Ba 2+. Enzyme release was significantly enhanced by guanosine-5′-[γ-thio]triphosphate (GTPγS) resulting in a shift to the left of the dose/response curve. The enhancement by GTPγS was strongest with Ca 2+, was less with Sr 2+, and was very little with Ba 2+. The time course of lysozyme release was the same for Ca 2+, Sr 2+, and Ba 2+ in the presence and absence of GTPγS when suboptimal cation concentrations were used. A decrease in responsiveness to the effectors after electropermeabilization was observed with Ca 2+, Sr 2+ and Ba 2+ in the presence and absence of GTPγS. The lysozyme release induced by the different cations was not inhibited by the protein kinase C inhibitor staurosporine and was slightly affected by pertussis toxin. Ca 2+ and Sr 2+, but not Ba 2+, potentiated formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) induced enzyme release in intact neutrophils. The divalent cation ionophore A23187 induced enzyme release in the presence of Ca 2+ and Sr 2+ but not in the presence of Ba 2+. The results obtained with electropermeabilized neutrophils indicate that Sr 2+ and Ba 2+ can act as substitutes for Ca 2+ in activating exocytosis, and that permeabilized neutrophils provide the best tool to investigate the effects of alkaline earth ions in exocytosis.

Differential up-regulation of specific and azurophilic granule membrane markers in electropermeabilized neutrophils

We have developed an alternative method to study the degranulation in electropermeabilized human neutrophils by measuring the up-regulation of the specific membrane markers CD63 (residing in the azurophilic granules of resting neutrophils) and CD67 (present in specific granules). The expression of these marker proteins was measured by the binding of specific antibodies to paraformaldehyde-fixed cells and subsequent flow cytometry. We first investigated whether the changes in CD63 and CD67 expression after stimulation of intact cells were comparable with earlier measurements of neutrophil degranulation, in which the release of soluble marker proteins was measured. These experiments indicated that this new method compare favourably with earlier studies, both with respect to kinetics and stimulus dependency. Subsequently, we applied this method (which does not include centrifugation of the cells)_to study degranulation in electropermeabilized neutrophils. In permeabilized neutrophils, a clear up-regulation of the specific granule marker CD67 was observed upon incubation with a free Ca 2+ concentration of 1 μM, a value of the cytosolic free Ca 2+ concentration occurring in formymethionyl-leucyl-phenylalanine (FMLP)-activated neutrophils. The azurophilic granule marker CD63 required GTP-γ-S besides 1 μM Ca 2+ for a significant up-regulation. Hence, our study indicates a different requirement for intracellular signals of the two main types of granules in human neutrophils.