Plasma Membrane Of Human Neutrophils Research Articles

Organization and Mobility of CD11b/CD18 and Targeting of Superoxide on the Surface of Degranulated Human Neutrophils

A monoclonal IgM, specifically recognizingbothCD11b and CD18 of human neutrophils, was used to examine the organization and mobility of CD11b/CD18 in the plasma membrane of human neutrophils degranulated by dihydrocytochalasin B (dhCB) treatment and fMet-Leu-Phe (fMLF) stimulation. Subcellular fractionation analysis of untreated or dhCB-treated control neutrophils indicated that 20% of CD11b/CD18 cosedimented with plasma membrane and the remainder with specific granules. In contrast, fMLF stimulation of dhCB-treated cells caused a major reorganization of CD11b/CD18, in which 60–70% of CD11b/CD18 sedimented in dense plasma membrane fractions that were also enriched in superoxide-generating NADPH oxidase activity. Similarly pretreated neutrophils were fixed, immunogold labeled, and examined by scanning electron microscopy. Immunogold particles were distributed uniformly over the symmetrically ruffled surface of unstimulated neutrophils. On dhCB-treated cells, immunogold was mostly uniformly distributed on a smooth membrane with a small percentage of particles lining up into linear arrays. After fMLF + dhCB stimulation, CD11b/CD18 gold label was more abundant on the cell surface and formed large aggregates on polarized membrane protrusions. However, when cells were adhered to an albumin-coated quartz surface and stimulated with fMLF in the presence of dhCB, immunogold was excluded on the articulated and rounded cell body but concentrated on the periphery of adherent lamellae. Fluorescence photobleaching recovery indicated that in unstimulated cells 38 ± 3% of CD11b/CD18 was mobile (R) with a diffusion constantDof 3.1 ± 0.3 × 10−10cm2/s. Treatment with dhCB raisedRandD24 and 74%, respectively. Stimulation using 1 μM fMLF with dhCB loweredDandRto near control levels. Since NADPH oxidase and CD11b/CD18 cosediment in high-density plasma membrane domains after fMLF + dhCB stimulation, we speculate that a stimulus-induced reorganization of CD11b/CD18 and NADPH oxidase to common membrane domains may occur in fMLF + dhCB-degranulated neutrophils.

Increased saturated triacylglycerol levels in plasma membranes of human neutrophils stimulated by lipopolysaccharide

Neutrophils isolated from patients with bacterial infections or stimulated in vitro with lipopolysaccharide (LPS) produce a high resolution, lipid-dominated spectrum on 1H-NMR spectroscopy (May et al, 1993. J. Infect. Dis. 168: 386-392). We have investigated the origin of this lipid signal using NMR and chemical analyses of both whole neutrophils and purified plasma membranes. Plasma membranes from neutrophils that had been stimulated with 50 microg/ml LPS exhibited the high resolution 1H-NMR signal, and contained double the triacylglycerol (TAG) content of plasma membranes isolated from resting cells. Chemical analysis of the whole cells indicated that the TAG also increased at the cellular level (1.7-fold) after stimulation with LPS. Diradylglycerol increased 2- to 3-fold in both whole cells and plasma membranes after stimulation, but was only a minor component compared with TAG. The plasma membrane protein/phospholipid ratio increased 2.6-fold, whereas cholesterol (free and esterified) was unchanged. The membranes from LPS-stimulated neutrophils exhibited increased fluidity, as judged by increased merocyanine 540 binding, consistent with a 2-fold reduction in cholesterol/phospholipid ratio. LPS induced a shift in fatty acid content of whole cell polar lipids towards more oleic acid and less palmitic acid, whereas the neutral lipid fraction contained increased amounts of palmitic and stearic acids. The TAG fraction of plasma membrane lipids contained increased amounts of palmitic acid when prepared from cells stimulated with LPS. We conclude that the 1H-NMR signal in LPS-stimulated neutrophils arises from increased amounts of plasma membrane TAG with an elevated content of palmitic acid.

Fc receptor-triggered insertion of secretory granules into the plasma membrane of human neutrophils: selective retrieval during phagocytosis.

We studied the kinetics of secretion in human neutrophils stimulated by IgG-opsonized zymosan. Secretion of azurophilic and specific granules was quantified measuring the appearance of the granule markers CD63 and CD66b, respectively, at the cell surface. The kinetics of secretion was compared with the course of phagocytosis, revealed by the trapping of the fluid phase marker, Lucifer Yellow, in vacuoles containing zymosan particles. We found that secretion of both azurophilic and specific granules precedes phagosome sealing. An initial rapid phase of secretion was followed by a decrease in the amount of CD63 and CD66b at the cell surface. This subsequent disappearance of surface CD63 and CD66b was inhibited by cytochalasin B and probably represents internalization of the granular markers into the forming phagosome. The decrease in the amount of CD63 and CD66b exposed at the cell surface was not accompanied by a commensurate reduction in cell surface area, measured with the amphiphilic fluorescent dye FM1-43. These findings imply that CD63 and CD66b are selectively retrieved from the plasma membrane following secretion. Evidence is also presented that calcium is not the sole mediator of the rapid secretion of azurophilic and specific granules triggered by IgG-opsonized particles and that cytochalasin does not impair signaling of the calcium transient elicited by Fc receptors. Instead, actin disassembly appears to reduce the efficiency of the interaction between opsonized particles and their receptors, an effect that can be overcome by increasing the concentration of the stimulating particles.

Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane.

The NADPH oxidase that produces superoxide in professional phagocytic cells is a flavocytochrome b electron transport chain in the membrane, a heterodimer of gp91phox and p22phox, that is activated by a number of cytosolic proteins, including p47phox, p67phox, and the small GTP-binding protein p21rac, which translocate to the membrane and attach to the flavocytochrome on activation. The components of this oxidase were localized on the cytoplasmic surface of the plasma membrane of adherent unroofed neutrophils by immunolabeling. Components of the NADPH oxidase and p21rac were found together in punctate clusters occupying 0.03-0.1 microm2 of the cytoplasmic surface of the plasma membrane where the density of labeling of the cytosolic components was increased after stimulation with phorbol myristate acetate.

Near membrane Ca 2+ changes resulting from store release in neutrophils: detection by FFP-18

FFP-18 was incorporated into the inner face of the plasma membrane of human neutrophils by incubation with its acetoxymethyl ester. Conversion to the Ca 2+ sensitive intracellular indicator was monitored by the change in excitation spectra. The fluorescence from extracellularly facing FFP-18 was quenched by the membrane impermeant ion Ni 2+. Ratio fluorescence measurement of FFP-18 under these conditions permitted the detection of near membrane Ca 2+ changes resulting from the release of Ca 2+ from intracellular stores. Near membrane and cytosolic Ca 2+ changes were measured under conditions in which store release and Ca 2+ influx were triggered by FMLP, thapsigargin or immune complexes. There were significant differences in the timing and magnitude of Ca 2+ changes near the plasma membrane and bulk cytosolic Ca 2+ concentration, which were consistent with a Ca 2+ storage site deep within the neutrophil released by thapsigargin and FMLP, but Ca 2+ release sites with immune complex stimulation being close to the membrane. The use of FFP-18 to monitor Ca 2+ near the inner face of the plasma membrane thus provides evidence for the existence of two distinct Ca 2+ storage locations in neutrophils.

Effect of cetirizine on plasma membrane of human eosinophils, neutrophils and platelets: a dose response study

The effect of different concentrations of cetirizine (0.1, 0.3, 0.5, 0.7, 0.9 ug/ml) on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils and platelets were investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results demonstrate that cetirizine induced a significant increase in the lipid order in the exterior part of the membrane and a decrease in membrane heterogeneity of eosinophils at concentrations of 0.7 ug/ml and higher and that at a concentration of 0.9 ug/ml it does likewise in neutrophils and platelets. Moreover, cetirizine abolished the PAF induced changes in membrane fluidity in these cells. These data suggest that cetirizine interacts with the plasma membrane of eosinophils, neutrophils and platelets inducing functional changes.

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons.

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10(-8) M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10(-8) M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.

Inhibition of neutrophil function by aspirin-like drugs (NSAIDS): Requirement for assembly of heterotrimeric G proteins in bilayer phospholipid

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms that are independent of their effects on prostaglandin biosynthesis. We examined the effects of sodium salicylate and piroxicam on GTP/GDP exchange by a regulatory G protein (Gαi). Plasma membrane and cytosol of human neutrophils were prepared by nitrogen eavitation and discontinuous sucrose density centrifugation. Salicylate (3 mM) and piroxicam (50 μM) reduced [ 35S]GTPγS binding to purified plasma membranes [65 ± 3.7 and 75 ± 5.3% (P < 0.003) of control, respectively]. Membrane-associated Gα βγ was solubilized by treatment of plasma membranes with sodium cholate. NSAIDs did not inhibit binding of GTP to solubilized Gα βγ derived from detergent-treated plasma membranes. Lipid reconstitution was achieved by detergent dialysis followed by the addition of bilayer liposomes (phosphatidycholine). Salicylate and piroxicam inhibited GTPγS binding to Gα βγ derived from solubilized plasma membranes reconstituted in phosphatidylcholine vesicles (bilayer structures) but had no effect when phosphatidylethanolamine (hexagonal phase II structure) was used for reconstitution. Salicylate and piroxicam had no effect on GTP binding to cytosolic fractions in which soluble Gαi exists as a free subunit, suggesting that the effect required either assembly of Gαi βγ heterotrimer or the presence of a lipid bilayer. Although the addition of purified bovine βγ subunits to dialyzed cytosol increased both the total GIP binding capacity and the pertussis toxin-dependent ADP-ribosylation of Gαi, consistent with assembly of a G protein heterotrimer, NSAIDs had no effect on GTP binding. In contrast, NSAIDs inhibited GTP binding to heterotrimeric Gα cytosol βγ bovine when the complex was inserted into bilayer liposomes. The data indicate that salicylate and piroxicam disrupt neutrophil function via their capacity to interfere with GTP/GDP exchange at an α subunit of a regulatory G protein, an effect which requires assembly of the active heterotrimer Gαi βγ in a phospholipid bilayer.

Decrease in the phosphotyrosine phosphatase activity in the plasma membrane of human neutrophils on stimulation by phorbol 12-myristate 13-acetate

Phorbol 12-myristate 13-acetate (PMA) induced a decrease in the phosphotyrosine phosphatase (PTPase) activity in human neutrophils. The decrease in the activity induced by PMA was blocked by the treatment of the cells with staurosporine, indicating that protein kinase C is involved in the decrease. The PTPase activity was localized in the plasma membrane. The activity in the membrane with the optimum pH at 5.5 had a K m value for phosphotyrosine of 2.2 mM and V max of 2.0 μmol/min per mg of protein. No activity was observed against phosphoserine and phosphothreonine. Vanadate, molybdate, zinc and a sulfhydryl reagent, p- chloromercuribenzenesulphonic acid , inhibited the PTPase. The PMA-induced decrease in activity was almost completely recovered by treatment of the plasma membrane with Triton X-100 at low concentrations which did not solubilize the activity from the membrane. When the plasma membrane was treated with trypsin, the PTPase of the membrane from PMA-treated cells was mostly protected from the proteinase attack while that from the resting cells was not protected. Pretreatment of the plasma membrane with Triton X-100 enabled trypsin to gain access to all the PTPase in the membrane from both PMA-treated and resting cells. The PMA treatment affected neither subcellular localization of the PTPase nor the orientation of the plasma membrane vesicles. These findings suggest that conformational changes of the enzyme induced by PMA result in the decrease in PTPase activity.

Up-regulation of the amount of Giα2 associated with the plasma membrane in human neutrophils stimulated by granulocyte-macrophage colony-stimulating factor

Preincubation of human neutrophils with the human cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) results in an increase in the amount of alpha-subunit of Gi2 (Gi alpha 2) associated with the plasma membrane and a corresponding decrease in the amount associated with the granule fractions. Similar results are obtained with interleukin-8. GM-CSF has no effect on the distribution of Gi alpha 3. The effect of GM-CSF on Gi alpha 2 is time-dependent, and, although a significant effect can be observed after incubation for 5 min with GM-CSF, the enhancement increases with increasing time. Genistein, a protein tyrosine kinase inhibitor, and 1,2-bis-(O-aminophenoxyl)ethane-NNN'N'-tetra-acetic acid (BAPTA), an intracellular Ca2+ chelator, decrease the stimulatory effect of GM-CSF. On the other hand, the protein-synthesis inhibitor cycloheximide does not affect the action of GM-CSF. Also, although preincubation of human neutrophils with GM-CSF increases the levels of Gi alpha 2 in the plasma membrane it does not alter the total amount of cellular Gi alpha 2. In addition, the level of Gi alpha 2 mRNA, unlike that of the proto-oncogene c-fos, is not increased in cells treated with GM-CSF. This indicates that the observed increase in the amount of Gi alpha 2 associated with the plasma membrane is not due to the synthesis of new Gi alpha 2. These data provide insight into the mechanism by which GM-CSF may prime human neutrophils for increased responsiveness to subsequent stimulation by G-protein-dependent agonists.

Separation of human neutrophil plasma membrane from intracellular vesicles containing alkaline phosphatase and NADPH oxidase activity by free flow electrophoresis.

A putative reservoir of functional plasma membrane proteins, the secretory vesicle identified by latent alkaline phosphatase and tetranectin, has previously been demonstrated based on indirect evidence (Borregaard, N., Miller, L. J., and Springer, T. A. (1987) Science 237, 1204-1206; Borregaard, N., Christensen, L., Bjerrum, O. W., Birgens, H. S., and Clemmesen, I. (1990) J. Clin. Invest. 85, 408-416). Difficulties in separating plasma membranes from this entity by density gradient centrifugation has prohibited discriminative dynamic and quantitative studies of secretory vesicles and plasma membranes. By combining density centrifugation with free flow electrophoresis we overcame this obstacle. Freshly prepared unperturbed human neutrophils were subjected to nitrogen cavitation followed by density centrifugation on Percoll gradients. Light membrane fractions containing plasma membranes and secretory vesicles were applied to high voltage free flow electrophoresis on an Elphor VaP 22. Plasma membrane vesicles, identified by HLA class I antigen mixed enzyme-linked immunosorbent assay (Bjerrum, O. W., and Borregaard, N. (1990) Scand. J. Immunol. 31, 305-313) and 125I applied to cells before cavitation, were clearly separated from secretory vesicles. Electron microscopy revealed a morphology typical of plasma membranes in the former fraction and a population of vesicles with markedly different appearance in the latter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles demonstrated distinct differences in protein patterns between the two fractions. Superoxide generating capacity induced by sodium dodecyl sulfate and cytosol, an entity traditionally ascribed to the plasma membrane, was largely confined to fractions containing secretory vesicles. Thus, the majority of membrane-bound NADPH oxidase components of light membranes of human neutrophils colocalize with secretory vesicles.

G-protein activation by interleukin 8 and related cytokines in human neutrophil plasma membranes.

Interleukin 8 (IL-8) is a member of the rapidly growing superfamily of those cytokines which are thought to be involved in the regulation of inflammatory processes and cell proliferation. In neutrophils, IL-8 triggers a variety of cellular responses by interacting with specific cell-surface receptors. To examine whether IL-8 receptors are coupled to activation of guanine-nucleotide-binding proteins (G-proteins), we have investigated the influence of IL-8 on GTP hydrolysis by and guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to purified human neutrophil plasma membranes. IL-8 stimulated high-affinity GTPase about 2-fold at 100 nM, and half-maximal stimulation was observed at 1 nM. The peptide-stimulated GTPase was confined to plasma membranes upon subcellular fractionation, and was due to an increase in Vmax. rather than a decrease in Km. High-affinity binding of GTP[35S] to neutrophil plasma membranes was stimulated half-maximally and maximally (up to 5-fold) by IL-8 at about 10 nM and 100 nM respectively. GTP[35S] binding to the membranes was also stimulated by two IL-8-related cytokines, neutrophil-activating peptide 2 (NAP-2) and melanoma growth-stimulatory activity (gro/MGSA). Taken together, these results demonstrate that receptors for IL-8 and related cytokines are coupled to and activate G-proteins in neutrophil plasma membranes, indicating that G-protein activation is an important intermediate step in the induction of neutrophil functions by IL-8 and its congeners.

Protein kinase C activates an H+ (equivalent) conductance in the plasma membrane of human neutrophils.

The rate of metabolic acid generation by neutrophils increases greatly when they are activated. Intracellular acidification is prevented in part by Na+/H+ exchange, but a sizable component of H+ extrusion persists in the nominal absence of Na+ and HCO3-. In this report we determined the contribution to H+ extrusion of a putative H+ conductive pathway and its mode of activation. In unstimulated cells, H+ conductance was found to be low and unaffected by depolarization. An experimental system was designed to minimize the metabolic acid generation and membrane potential changes associated with neutrophil activation. By using this system, beta-phorbol esters were shown to increase the H+ (equivalent) permeability of the plasma membrane. The direction of the phorbol ester-induced fluxes was dictated by the electrochemical H+ gradient. Moreover, the parallel migration of a counterion through a rheogenic pathway was necessary for the displacement of measurable amounts of H+ equivalents across the membrane. These findings suggest that the H+ flux is conductive. The effect of beta-phorbol esters was mimicked by diacylglycerol and mezerein and was blocked by staurosporine, whereas alpha-phorbol esters were ineffective. Together, these findings indicate that stimulation of protein kinase C induces the activation of an H+ conductance in the plasma membrane of human neutrophils. Preliminary evidence for activation of a separate, bafilomycin A1-sensitive H+ extrusion mechanism, likely a vacuolar type H(+)-ATPase, is also presented.

Down‐regulation by tumor necrosis factor‐α of neutrophil cell surface expression of the sialophorin CD43 and the hyaluronate receptor CD44 through a proteolytic mechanism

Adhesion of human neutrophils to endothelial cells is a crucial step during migration to the extravascular sites of inflammation. A large number of molecules, including the CD44 and LAM-1 antigens, have been described to participate in this process. We have investigated the regulation by human recombinant tumor necrosis factor-alpha (TNF-alpha) of human neutrophil plasma membrane expression of both CD44 and LAM-1 adhesion molecules, as well as that of CD43 sialophorin, which has been involved in adhesion and activation of leukocytes. The expression of these three antigens was down-regulated in neutrophils upon TNF-alpha treatment, as determined by immunofluorescence and immunoprecipitation experiments. However, the expression of other cell surface molecules, such as CD45 or CD11b, was up-regulated. Similar regulatory effects were also observed upon neutrophil treatment with other activating agents such as the chemoattractant peptide formyl-Met-Leu-Phe, the calcium ionophore A23187, or the phorbol ester phorbol 12-myristate 13-acetate. Protease inhibitors virtually abrogated the TNF-alpha-induced down-regulation of CD43 and CD44 expression, but not that of LAM-1, suggesting the involvement of a protease activity in this process. These results underline the role of TNF-alpha on the differential regulation of cell surface expression of neutrophil adhesion molecules, thus implying modifications in the neutrophil adhesive properties.

The chloride channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) uncouples mitochondria and increases the proton permeability of the plasma membrane in phagocytic cells

We present evidence that the potent chloride channel blocker NPPB has protonophoric activity in the mitochondria and across the plasma membrane of phagocytic cells. The resting O2 consumption of murine peritoneal macrophages was stimulated up to 2.5-fold in the presence of NPPB, with a K0.5 of 15 μM. The stimulatory effect of NPPB also O2 consumption, like that of the classical protonophore CCCP, was prevented by the mitochondrial respiratory chain inhibitors antimycin A, rotenone or cyanide. NPPB also mediated rheogenic proton transport across the plasma membrane of human neutrophils and macrophages in the direction dictated by the electrochemical proton gradient. As a consequence of its protonophoric activity, NPPB uncoupled mitochondrial ATP synthesis, resulting in partial depletion of cellular ATP. These observations indicate that, at the concentrations frequently used for blockade of anion channels, NPPB acts as an effective protonophore, potentially disturbing cytosolic pH and mitochondrial ATP synthesis.

Annexin-mediated membrane fusion of human neutrophil plasma membranes and phospholipid vesicles

Membrane fusion was studied using human neutrophil plasma membrane preparations and phospholipid vesicles approximately 0.15 μm in diameter and composed of phosphatidylserine and phosphatidylethanolamine in a ratio of 1 to 3. Liposomes were labeled with N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) and lissamine rhodamine B derivatives of phospholipids. Apparent fusion was detected as an increase in fluorescence of the resonance energy transfer donor, NBD, after dilution of the probes into unlabeled membranes. 0.5 mM Ca 2+ alone was sufficient to cause substantial fusion of liposomes with a plasma membrane preparation but not with other liposomes. Both annexin I and des(1–9)annexin I caused a substantial increase in the rate of fusion under these conditions while annexin V inhibited fusion. Fusion mediated by des(1–9)annexin I was observed at Ca 2+ concentrations as low as aproximately 5 μM, suggesting that the truncated form of this protein may be active at physiologically low Ca 2+ concentrations. Trypsin treated plasma membranes were incapable of fusion with liposomes, suggesting that plasma membrane proteins may mediate fusion. Liposomes did not fuse with whole cells at any Ca 2+ concentration, indicating that the cytoplasmic side of the membrane is involved. These results suggest that annexin I and unidentified plasma membrane proteins may play a role in Ca 2+-dependent degranulation of human neutrophils.

Release of azurophilic granule contents in fMLP-stimulated neutrophils requires two activation signals, one of which is a rise in cytosolic free Ca 2+

We have used a continuous spectrofluorimetric method to analyse the role of cytosolic free Ca 2+ ([Ca 2+] i) in the lysosomal enzyme release from the azurophilic granules in human neutrophils stimulated with f-Met-Leu-Phe (fMLP) in the presence of cytochalasin B. Measurements were performed with the β-glucuronidase substrate 4-methylumbelliferyl-β- d-glucuronide. We found that the transient rise in [Ca 2+] i induced by fMLP is a necessary signal to obtain to obtain maximal degranulation. When this Ca 2+ transient is prevented by the Ca 2+ chelator BAPTA, degranulation can still be induced by a stimulated Ca 2+ influx, albeit to a lower extent. We also studied the degranulation process in the neutrophils of a patient with a generalized chemotactic defect. Release of β-glucuronidase from the patient's neutrophils could not be induced despite the occurrence of a normal Ca 2+ response and normal degranulation of specific granules. We conclude that, besides an increase in [Ca 2+] i], an additional signal is required for the fusion of azurophilic granules with the plasma membrane in human neutrophils.

Effect of Botulinum D Toxin on Human Neutrophilic Leukocytes and Localization of Its Substrates

Botulinum D toxin has been shown to ADP-ribosylate 22-kD proteins in neutrophilic leukocytes, but the function of these GTP-binding proteins remains unknown. In analogy to small GTP-binding proteins like SEC4 to YPT1, it has been suggested that botulinum D toxin substrates might be involved in secretory process of myeloid cells. Three main findings lead to the opposite conclusion. First of all, in human neutrophils, botulinum D toxin does not modify the release of azurophilic and specific granules induced by a chemoattractant (a formylpeptide) or a phorbol ester. Second, botulinum D toxin ADP-ribosylates 24 to 26-kD proteins that are only present in plasma membranes of human neutrophils. The membrane location of these substrates differs largely from that of the GTP-binding proteins involved in exocytosis and located in granules. Finally, since the same quantity of the toxin substrates is present in neutrophils as in their precursors, HL60 cells (which are devoid of specific granules and characterized by immature azurophilic granules and NADPH oxidase), it is unlikely that endogenous botulinum D toxin substrates are directly involved in the secretory responses of neutrophils.

Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils.

Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2-(p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N-formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL-SASD[125I]) revealed that the majority of receptors (greater than 60-90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G-protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.

The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils

Studies were performed to examine the lateral organization of the NADPH oxidase system in the plasma membrane of human neutrophils. Analysis of the subcellular fractionation of human neutrophils by isopycnic sedimentation of cavitated cell lysates suggested that there may be more than one population of plasma membrane vesicles formed upon cell disruption. One population (30-32% sucrose) contained surface accessible wheat germ agglutinin binding sites, alkaline phosphatase activity, and cytochrome b. Another population (34-36% sucrose) contained membrane-bound flavin and, when the cells were prestimulated with phorbol myristate acetate (PMA), NADPH-dependent superoxide generating activity. Approximately 25% of the neutrophil cytochrome b cosedimented with the heavy population, confirming our previous hypothesis (Parkos et al. (1985) J. Biol. Chem. 260, 6541-6547) that only a fraction of the total cellular cytochrome b is involved in superoxide production. The heavy plasma membrane fraction was also enriched in membrane associated actin and fodrin as detected by Western blot analysis. After extraction of the plasma membrane vesicles with detergent cocktails, the majority of superoxide generating activity remained associated with the detergent insoluble pellet. Western blot analysis demonstrated that the pellets were also enriched in actin. Further analysis of these pellets using rate-zonal detergent-containing sucrose density gradients indicated that the superoxide generating complex had an approximate sedimentation coefficient of 80 S, suggesting that the neutrophil superoxide generating system may form a complex on the plasma membrane which is associated with or somehow organized by the membrane skeletal matrix. This organization may be of functional relevance not only to the actual production of superoxide, but also to the targeting of microbicidal oxidants.