Vitamin B12-binding Protein Research Articles

Identification of Neutrophil Gelatinase-Associated Lipocalin as a Novel Matrix Protein of Specific Granules in Human Neutrophils

AbstractNeutrophil gelatinase-associated lipocalin (NGAL) is a novel 25-kD protein of human neutrophils, that is in part covalently complexed with neutrophil gelatinase. However, both NGAL and gelatinase exist mainly in forms not associated with each other. An explanation for this phenomenon might be that the unassociated proteins reside in different subcellular compartments. The aim of the present study was to determine the subcellular localization of NGAL. An enzyme-linked immunosorbent assay (ELISA) for NGAL was developed using specific anti-NGAL antibodies. The assay was applied on subcellular fractions of neutrophils obtained after centrifugation of a postnuclear supernatant on a two-layer Percoll gradient. The distribution profile of NGAL was found to colocalize strictly with the distribution profile of lactoferrin. This was confirmed by immunogold double-labeling of frozen thin sections of neutrophils that showed a high degree of colocalization of NGAL and lactoferrin, and by exocytosis experiments, which showed lactoferrin, vitamin B12-binding protein, and NGAL to be similarly released upon stimulation. Therefore, NGAL is a novel matrix protein of specific granules and thus partly segregated from gelatinase, the major part of which is located in a separate compartment, the gelatinase granules. An ELISA specific for the NGAL/gelatinase complex was developed and the subcellular distribution and release of this complex was determined. The distribution and mobilization of the complex allowed us to confirm the existence of differentially mobilized granule subpopulations among peroxidase negative granules.

Exocytosis of a subpopulation of specific granules coincides with H2O2 production in adherent human neutrophils.

Unlike polymorphonuclear leukocytes (PMN) that are activated in suspension, PMN plated onto extracellular matrix (ECM) proteins exhibited a prolonged delay in the onset of the respiratory burst in response to PMA, FMLP, A23187, or GM-CSF. The present study was focused on examining the events leading to H2O2 release in adherent PMN. The time course of stimulated H2O2 release from PMN plated on fibrinogen (FG) kinetically paralleled cell spreading and lactoferrin release. In contrast, the release of another specific granule component, vitamin B12-binding protein, preceded H2O2 generation by at least 20 min, suggesting the differential mobilization of subpopulations of specific granules. FMLP-stimulated PMN from a patient with leukocyte adhesion deficiency (LAD), lacking CD11/CD18 integrins and unable to adhere to FG, attached and spread on thrombospondin (TSP) and generated substantial amounts of H2O2. However, PMN from a second LAD patient, able to attach but not spread on TSP, failed to generate oxidant. These data indicated that PMN spreading might be a prerequisite for H2O2 generation and that CD11/CD18 integrins are likely not the only surface receptors involved in this response. Exposing PMN to the intracellular Ca2+ chelators MAPTAM or BAPTA significantly reduced H2O2 generation in response to the receptor-mediated agonists, FMLP or GM-CSF, but did not affect PMA-stimulated H2O2 generation. Under conditions that resulted in reduced H2O2 generation (i.e., MAPTAM or BAPTA plus FMLP or GM-CSF), lactoferrin, but not vitamin B12-binding protein, release was also reduced, suggesting a link between the secretion of lactoferrin-containing specific granules and H2O2 production. Since neither MAPTAM or BAPTA blocked PMN spreading, it appeared that cell spreading alone was not sufficient for H2O2 production. Thus, the major requirement for H2O2 generation appeared to be the exocytosis of a distinct population of lactoferrin-containing specific granules. In support of this observation, PMN-like HL-60 cells, which lack specific granules, attached and spread on FG but failed to release H2O2. Thus, we postulate that the delivery of cytochrome b from lactoferrin-containing specific granules to the plasma membrane during activation contributes to oxidant production in PMN adherent to ECM proteins.

Secretion of intrinsic factor from cultured rat gastric chief cells.

Intrinsic factor (IF) is a vitamin B12 binding protein that is secreted from the gastric mucosa. We tested secretagogues which stimulate IF secretion in rat gastric perfusion and found that carbachol and cholecystokinin octapeptide (CCK-8) stimulated secretion, but histamine and tetragastrin did not. To confirm these results, we examined IF secretion from isolated rat chief cells. For this purpose, we established an enzyme immunoassay (EIA) using an avidin-biotin peroxidase complex to measure small amounts of IF. To prepare an anti-rat IF, IF was isolated from the stomach, and was injected into a rabbit for immunization. Rat gastric chief cells were isolated from the gastric mucosa with Dispase and a Percoll gradient centrifugation, and were cultured. We examined the effects of chemicals by adding them to culture dishes of chief cells in a CO2 incubator. Released IF in culture medium was determined by EIA. Carbachol, CCK-8 and secretin stimulated IF secretion from cultured chief cells, while histamine and tetragastrin did not; Forskolin and A23187 also stimulated the secretion. We concluded that carbachol and CCK-8 stimulated IF secretion via an increase of intracellular Ca2+ concentration and that secretin did so via a cAMP accumulation.

Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria.

Extracellular solute-binding proteins of bacteria serve as chemoreceptors, recognition constituents of transport systems, and initiators of signal transduction pathways. Over 50 sequenced periplasmic solute-binding proteins of gram-negative bacteria and homologous extracytoplasmic lipoproteins of gram-positive bacteria have been analyzed for sequence similarities, and their degrees of relatedness have been determined. Some of these proteins are homologous to cytoplasmic transcriptional regulatory proteins of bacteria; however, with the sole exception of the vitamin B12-binding protein of Escherichia coli, which is homologous to human glutathione peroxidase, they are not demonstrably homologous to any of the several thousand sequenced eukaryotic proteins. Most of these proteins fall into eight distinct clusters as follows. Cluster 1 solute-binding proteins are specific for malto-oligosaccharides, multiple oligosaccharides, glycerol 3-phosphate, and iron. Cluster 2 proteins are specific for galactose, ribose, arabinose, and multiple monosaccharides, and they are homologous to a number of transcriptional regulatory proteins including the lactose, galactose, and fructose repressors of E. coli. Cluster 3 proteins are specific for histidine, lysine-arginine-ornithine, glutamine, octopine, nopaline, and basic amino acids. Cluster 4 proteins are specific for leucine and leucine-isoleucine-valine, and they are homologous to the aliphatic amidase transcriptional repressor, AmiC, of Pseudomonas aeruginosa. Cluster 5 proteins are specific for dipeptides and oligopeptides as well as nickel. Cluster 6 proteins are specific for sulfate, thiosulfate, and possibly phosphate. Cluster 7 proteins are specific for dicarboxylates and tricarboxylates, but these two proteins exhibit insufficient sequence similarity to establish homology. Finally, cluster 8 proteins are specific for iron complexes and possibly vitamin B12. Members of each cluster of binding proteins exhibit greater sequence conservation in their N-terminal domains than in their C-terminal domains. Signature sequences for these eight protein families are presented. The results reveal that binding proteins specific for the same solute from different bacteria are generally more closely related to each other than are binding proteins specific for different solutes from the same organism, although exceptions exist. They also suggest that a requirement for high-affinity solute binding imposes severe structural constraints on a protein. The occurrence of two distinct classes of bacterial cytoplasmic repressor proteins which are homologous to two different clusters of periplasmic binding proteins suggests that the gene-splicing events which allowed functional conversion of these proteins with retention of domain structure have occurred repeatedly during evolutionary history.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity of peroxidase-positive granules in normal human an Chédiak-Higashi neutrophils.

Studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. This study evaluated the heterogeneity morphometrically, morphologically, cytochemically, and biochemically. Intact human peripheral blood neutrophils collected from normal volunteers and a patient with Chédiak-Higashi syndrome (CHS) and isolated normal neutrophil granules were processed for ultrastructural morphology and peroxidase staining. Intact cells, nuclei, and granule profiles were analyzed by computer-assisted planimetry. Peroxidase-positive granules (PPG) represented about 40% of normal neutrophil granules and covered the entire spectrum of granule size. PPG in the least-dense fractions of isolated granules were significantly smaller than in higher-density fractions. PPG in low- and intermediate-density fractions differed from high-density fraction by moderate to strong vicinal glycol staining with Thiéry's periodate-thiocarbohydrazide-silver proteinate method. Differing ratios of % beta-glucuronidase/% myeloperoxidase (MPO) across granule fractions indicated PPG heterogeneity. Morphometric analysis of neutrophils treated with 1 microM calcium ionophore A23187 did not show significant differences in PPG size or number. Biochemically analyzed MPO in these cells was preserved, although the number of peroxidase-negative granules (PNG) and levels of vitamin B12-binding protein were markedly decreased. In CHS, about 20% of granules were PPG. Analysis of CHS neutrophils revealed the persistence of microgranules similar to normals. PNG number and volume fractions of PPG and TG were not different from normals. Complex heterogeneity of normal PPG was quantitated using morphometry and appeared preserved in ionophore-treated cells but was uniquely modified in CHS.

Quartz selectively down-regulates CR1 on activated human granulocytes

We have investigated the interaction between quartz and granulocytes with respect to complement receptor expression. When N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated leukocytes were exposed to quartz at 37 degrees C, CR1 was down-regulated but CR3 was not affected. This was a direct effect on granulocytes because it occurred in a similar fashion when mixed leukocyte suspensions and isolated granulocyte populations were used as targets for quartz. The observed down-regulation by quartz was not affected by the microfilament-disrupting agent cytochalasin B and the total detectable pool of CR1 was reduced after quartz exposure. When protease inhibitors, such as aprotinin or phenylmethanesulfonyl fluoride, were present during quartz exposure, the down-regulation of CR1 was less pronounced, but this was not the case not when protease inhibitors such as EDTA-Na2 and pepstatin were present. Exposure to quartz was not accompanied by a pronounced release of beta-glucuronidase (marker for the primary granules) or vitamin B12 binding protein (marker for the secondary granules). In contrast to quartz, exposure to alumina did not affect the expression of CR1 and CR3. The spontaneous mobilization of CR1 at 37 degrees C was reduced when quartz was present but the CR3 mobilization was unaffected. Our results indicate that quartz induces a granule protease-dependent selective shedding of CR1 but not CR3 despite a low degree of degranulation.

Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria.

Extracellular solute-binding proteins of bacteria serve as chemoreceptors, recognition constituents of transport systems, and initiators of signal transduction pathways. Over 50 sequenced periplasmic solute-binding proteins of gram-negative bacteria and homologous extracytoplasmic lipoproteins of gram-positive bacteria have been analyzed for sequence similarities, and their degrees of relatedness have been determined. Some of these proteins are homologous to cytoplasmic transcriptional regulatory proteins of bacteria; however, with the sole exception of the vitamin B12-binding protein of Escherichia coli, which is homologous to human glutathione peroxidase, they are not demonstrably homologous to any of the several thousand sequenced eukaryotic proteins. Most of these proteins fall into eight distinct clusters as follows. Cluster 1 solute-binding proteins are specific for malto-oligosaccharides, multiple oligosaccharides, glycerol 3-phosphate, and iron. Cluster 2 proteins are specific for galactose, ribose, arabinose, and multiple monosaccharides, and they are homologous to a number of transcriptional regulatory proteins including the lactose, galactose, and fructose repressors of E. coli. Cluster 3 proteins are specific for histidine, lysine-arginine-ornithine, glutamine, octopine, nopaline, and basic amino acids. Cluster 4 proteins are specific for leucine and leucine-isoleucine-valine, and they are homologous to the aliphatic amidase transcriptional repressor, AmiC, of Pseudomonas aeruginosa. Cluster 5 proteins are specific for dipeptides and oligopeptides as well as nickel. Cluster 6 proteins are specific for sulfate, thiosulfate, and possibly phosphate. Cluster 7 proteins are specific for dicarboxylates and tricarboxylates, but these two proteins exhibit insufficient sequence similarity to establish homology. Finally, cluster 8 proteins are specific for iron complexes and possibly vitamin B12. Members of each cluster of binding proteins exhibit greater sequence conservation in their N-terminal domains than in their C-terminal domains. Signature sequences for these eight protein families are presented. The results reveal that binding proteins specific for the same solute from different bacteria are generally more closely related to each other than are binding proteins specific for different solutes from the same organism, although exceptions exist. They also suggest that a requirement for high-affinity solute binding imposes severe structural constraints on a protein. The occurrence of two distinct classes of bacterial cytoplasmic repressor proteins which are homologous to two different clusters of periplasmic binding proteins suggests that the gene-splicing events which allowed functional conversion of these proteins with retention of domain structure have occurred repeatedly during evolutionary history.(ABSTRACT TRUNCATED AT 400 WORDS)

Development and application of an alpha-face-specific radioimmunoassay for vitamin B12.

The first development of an alpha-face-specific radioimmunoassay for vitamin B12 is described. Sheep, fed a cobalt-deficient diet, and immunized with a conjugate between Co-beta carboxypropyl cobalamin and keyhole limpet hemocyanin, were used to produce antisera. The antisera crossreacted with Co-beta derivatives of vitamin B12, but did not crossreact with the alpha-face vitamin B12 analog cobinamide. The antisera were used to develop a sensitive and reproducible radioimmunoassay that was free from contamination with the nonspecific vitamin B12 binding protein, R-protein. Both the radioimmunoassay and measurements of plasma concentrations of methylmalonic acid were applied to the diagnosis of cobalt/vitamin B12 deficiency in sheep. The assay correlated well with a commercially available radioassay and did not falsely detect normal vitamin B12 concentration in plasma samples containing elevated concentrations of methylmalonic acid.

Inhibition by Nedocromil Sodium of Superoxide Anion Production in Human Neutrophils

In this preliminary study, the effects of nedocromil sodium (3 or 300μM) on the Superoxide anion production and secretion of β-glucuronidase and vitamin B12-binding protein were investigated using human neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10 or 100nM) or phorbol-myristate-acetate (PMA; 100 μg/L). O−2 production was significantly inhibited (≈80%) in the presence of nedocromil sodium 300μM, but only when a low dose of FMLP (10nM) was used. In contrast, nedocromil was without effect on O−2 production induced by high doses of FMLP (100μM) or by PMA (100 μg/L), and on secretion induced by either of these stimulants. Studies are in progress to ascertain whether this inhibitory effect on receptor mediated O−2 production can be related to the therapeutic effect of nedocromil sodium.

Assessment of neutrophil leukocyte secretory response to fMLP in whole blood in vitro.

A simple, precise method has been developed for assessing neutrophil secretory responses (release of vitamin B12 binding protein from specific granules) to challenge of aliquots of whole blood with the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Dose-response studies performed on blood from normal healthy volunteers showed higher maximal secretory responses in males than females (33.3 +/- SEM 2.2 vs. 27.4 +/- 2.5, P less than .005) a left shift in dose-response curves after feeding compared to fasting (P less than .005), spontaneous up-regulation of responses in blood incubated at 37 degrees C for 1 h, and marked upregulation in response to preincubation with endotoxin. This whole blood challenge method may be used to study neutrophil responses in groups of individuals or patients without the confounding effects of changes in cell responses resulting from cell isolation procedures. The method may also be used as a bioassay for neutrophil-activating factors.

Postreceptor events associated with human neutrophil activation by interleukin-8.

Recombinant human monocyte-derived interleukin-8 (IL-8M), recombinant human endothelium-derived IL-8 (IL-8E), and a recombinant human truncated form of IL-8 (IL-8T) stimulated a time-dependent (t 1/2 approximately 2-3 s) and concentration-dependent (0.1-100 nM) release of azurophil (myeloperoxidase) and specific (vitamin B12 binding protein, gelatinase) granule constituents from cytochalasin B-treated human neutrophils (HNs) wherein IL-8T = IL-8M greater than IL-8E. An increase in the cytosolic free calcium concentration ([Ca2+]i) was greater in IL-8T- than in IL-8M- or IL-8E-activated HNs, and IL-8T was more potent than either IL-8M or IL-8E in sequentially desensitizing the HNs to the effects of the other IL-8 forms. IL-8 induced a time- and concentration-dependent (0.1-100 nM) increase in the production of inositol 1,4,5-trisphosphate (IP3) in HNs. U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione), a potent inhibitor of phospholipase C-catalyzed events in HNs, suppressed IL-8-triggered IP3 production, increased [Ca2+]i and granule exocytosis in HNs. The membrane-associated activity of the alpha and beta subtypes of protein kinase C was significantly enhanced in IL-8-activated cells.

Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules.

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.

Genomic structure and mapping of the chromosomal gene for transcobalamin I (TCN1): Comparison to human intrinsic factor

Transcobalamin I (TCI) is a vitamin B12 binding protein that is found in the secondary granules of mature neutrophils. The expression of the gene for TCI (TCN1) within neutrophils has been shown to be restricted to the later stages of myeloid development and can therefore be used as a marker for granulocyte differentiation. To study transcriptional control regions important in late stage myeloid gene regulation the genomic sequence for TCN1 has been cloned. Clones were isolated from a genomic library constructed in Charon 4A using homologous full-length cDNA probes. Southern blot analysis showed the gene to reside on five EcoRI fragments totaling 14 kb in length. Two overlapping phage clones, containing the entire 14 kb, were isolated and the introns and exons were mapped using Southern blotting and dideoxy sequencing of subclones. The cDNA is represented by nine exons contained within 12 kb of genomic DNA. Comparison of the genomic structure to gastric intrinsic factor (GIF), another vitamin B12 binding protein, revealed a strikingly similar intron/exon structure, with several positionally conserved splice sites. The gene was localized to chromosome 11 using in situ hybridization.

Inhibition of neutrophil respiratory burst and degranulation responses to platelet-activating factor by antagonists WEB 2086, CV 6209 and CV 3988.

The effect of three platelet-activating factor (PAF) antagonists, WEB 2086, CV 6209 and CV 3988, on neutrophil respiratory burst activity and degranulation in response to PAF was investigated. Both WEB 2086 and CV 6209 significantly inhibited the respiratory burst and degranulation in response to 400 nM PAF in a dose-dependent manner (10(-8)-10(-5) M). Higher concentrations of CV 3988 were required to inhibit these functions (10(-5) M and above). The three antagonists inhibited both the release of beta-glucuronidase (from azurophilic granules) and vitamin B12 binding protein (from specific granules) in response to PAF. Only a small nonsignificant inhibition of neutrophil function occurred in the absence of PAF. There was no loss of viability after incubation with the three antagonists at the concentrations tested. These antagonists will be useful tools to study the involvement of PAF in neutrophil-mediated tissue damage and inflammation.

Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells. Role of intracellular calcium changes and fusion of specific granules with the plasma membrane.

Chemoattractants, used at concentrations to invoke optimal neutrophil chemotaxis, induce rapid changes in neutrophils such as a transient increase in intracellular Ca2+ ([Ca2+]i). We have previously observed that neutrophils adhering to cytokine-activated endothelial cells (EC) also respond with a rapid rise in [Ca2+]i caused by an endothelial membrane-bound form of platelet-activating factor. After preloading with the intracellular Ca(2+)-chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), neutrophils were no longer able to respond with a rapid rise in [Ca2+]i toward the chemoattractant FMLP or to rIL-1 beta-pretreated EC. These neutrophils were still able to adhere and migrate under the conditions tested. The only difference was that the BAPTA/AM-treated neutrophils migrated a little slower than untreated control neutrophils. This discrepancy was not observed at later time points. The BAPTA/AM-preloaded neutrophils did not differ from unloaded neutrophils in actin polymerization responses. Whereas untreated neutrophils demonstrated an up-regulation of the specific granule markers CD11b, CD45, and CD67 during migration (without any release from the azurophil granules), the BAPTA/AM pretreatment completely prevented this process. The BAPTA/AM-preloaded neutrophils did not release vitamin B12-binding protein from the specific granules upon treatment with FMLP. The down-modulation of the selectin member LAM-1, as seen upon neutrophil activation, was not affected by BAPTA/AM pretreatment of the neutrophils. Thus, neither the rapid rise in [Ca2+]i nor specific granule fusion with the plasma membrane constitute a prerequisite for neutrophil migration across resting or cytokine-activated EC.

Effect of the cobalt‐N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin

The binding of several corrinoids to the binding site of human intrinsic factor, transcobalamin or haptocorrin was investigated, p-Cresolyl cobamide and 2-amino-vitamin B12 are complete corrinoids, whose nucleotide at the lower face of the corrin ring is not coordinated to the cobalt. These corrinoids were greater than or equal to 10(3) times less efficiently recognized by intrinsic factor or transcobalamin than vitamin B12, which contains a Co-coordinated nucleotide. Pseudovitamin B12, with a weak Co-N coordination bond, revealed only moderate affinity to intrinsic factor. From these findings it is concluded that the cobamide binding to intrinsic factor and transcobalamin is strongly affected by the Co-N coordination bonds of their lower cobalt nucleotide ligands. We suggest that the Co-N coordination bond positions the nucleotide at a critical distance to the corrin ring, which is recognized by the binding proteins. Human haptocorrin, however, disclosed to distinctive selectivity regarding the different corrinoid structures. The protein bound all corrinoids with similar efficiency, independent of the strength of their Co-N coordinations, or the structures of their lower Co alpha ligands. Hence, the corrin ring, rather than a structural feature induced by the Co-N coordination, has to be considered responsible for the corrinoid binding to haptocorrin.

Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony‐stimulating factor in patients with lymphoma

Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit the chemotaxis and enhance the oxidative burst response of human neutrophils in vitro. The present study describes the effect of recombinant GM-CSF on the neutrophil and monocyte function in patients with lymphoma undergoing GM-CSF treatment. Patients with either Hodgkin's or non-Hodgkin's lymphoma were treated with various dosages (2-16 micrograms kg-1 body weight per day for 5 days) of rhGM-CSF by intravenous or subcutaneous route. Prior to and on day 5 of rhGM-CSF treatment, neutrophil and monocyte chemotaxis and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Met-Leu-Phe and zymosan was enhanced by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence system may be enhanced by GM-CSF treatment and that this cytokine may be a useful therapeutic adjunct in compromised patients.

Oxidative Metabolism of Polymorphonuclear Leukocytes: Modulation by Adhesive Stimuli

Different agents such as phorbol myristate acetate (PMA), N-formyl-methionyl-leucylphenylalanine (fMet-Leu-Phe), or opsonized zymosan induced an oxidative burst in rat peritoneal polymorphonuclear leukocytes (PMNs) elicited by casein. Plastic adhesion of PMNs down-regulated superoxide (O2) release stimulated by PMA or fMet-Leu-Phe but had no effect on zymosan-induced O2 generation, indicating that the O2 forming enzyme, the NADPH oxidase, was not affected by modulation and that a common step of the transductional events induced by PMA or fMet-Leu-Phe might be involved in this regulation. We demonstrated that a differential translocation of protein kinase C (PKC) was not responsible for that modulation. PMA-induced secretion of granule content (vitamin B12-binding protein) was not susceptible to modulation, suggesting that the transductional pathways leading to O2 generation and granule secretion are partly separated. The adhesion of PMNs to different substrates (glass, plastic, albumin-, laminin-, fibronectin-, poly-lysine-, or concanavalin A-coated plastic) down-regulated to different extent superoxide release. Whether the nature of the biochemical signal induced by the diverse adhesive stimuli or a physical parameter such as binding strength was involved in this differential behavior remains to be elucidated. Since adhesiveness was dependent on the state of the cytoskeleton and O2 inducers were reported to stimulate actin polymerization, we studied the F-actin content and distribution of PMNs by using the specific fluorescent probe NBD-phallacidin and an original methodology allowing a quantitative analysis of fluorescence on both adherent and suspended cells. PMA induced a polarization of F-actin on suspended PMNs but had no effect on the intracellular distribution of F-actin in adherent PMNs. Thus, we suggest that the adhesion of PMNs induced an immobilization of F-actin, possibly correlated to the down-regulation of one of the transductional pathways involved in the NADPH oxidase activation.

Cyclic AMP Inhibits Secretion From Electroporated Human Neutrophils

It has long been known that intracellular cAMP inhibits and cGMP enhances intact neutrophil function. However, these effects are modest and require relatively high concentrations of the cyclic nucleotides. We decided to re-examine the effects of cyclic nucleotides on Ca2(+)-induced secretion by electroporated cells. This system allowed us to bypass normal cell surface receptor-ligand interactions as well as to directly expose the intracellular space to native cyclic nucleotides. We found that concentrations of cAMP as low as 3 microM inhibited Ca2(+)-induced secretion; 30-300 microM cAMP was maximally inhibitory. cAMP was actually slightly more potent than dibutyryl cAMP, a membrane-permeant derivative. In contrast, cGMP was only slightly stimulatory at 3 microM and modestly inhibitory at 300 microM; dibutyryl cGMP was ineffective. A more detailed investigation of the effects of cAMP showed that inhibition was only obtained in the presence of Mg2+. Half-maximal inhibition by cAMP occurred at 10-30 microM. Inhibition by cAMP was achieved by shifting the Ca2+ dose-response curve for secretion to the right; this was observed for the release of both specific granules (vitamin B12 binding protein) and azurophil granules (B-glucuronidase). We previously showed that ATP could enhance Ca2(+)-induced secretion in the presence of Mg2+, apparently by interacting with a cell surface purine receptor. However, increasing concentrations of ATP could not overcome inhibition by cAMP; this suggested that cAMP acted at some site other than the purine receptor. Inhibition by cAMP was also less apparent in the presence of the protein kinase C agonist phorbol myristate acetate (PMA), suggesting that the cyclic nucleotide did not produce systemic desensitization of the neutrophils. In summary, these results demonstrate that low, physiologically relevant concentrations of cAMP can modulate neutrophil responsiveness.

Kinetics of N-formyl peptide receptor up-regulation during stimulation in human neutrophils.

The kinetics of receptor up-regulation was examined in isolated neutrophils and in whole blood by flow cytometry during cell activation. Stimulation of neutrophils prepared without exposure to LPS with chemoattractants induced fast up-regulation of N-formyl peptide receptors and C receptor type 3 (CR3). Biphasic N-formyl peptide binding curves were detected for saturating concentrations of N-formyl peptide at 37 degrees C. The bulk of the rapid binding during the first 30 to 60 s is attributed to already expressed binding sites whereas the slow binding over the next 3 to 4 min represents a time course of receptor up-regulation. Support for this interpretation comes from conditions under which the number of binding sites and the progress of the binding curves were affected. Cells treated with LPS, which caused expression of internal N-formyl peptide receptors, exhibited rapid, monophasic binding curves with increased total binding. In LPS-untreated, calcium-depleted cells, N-formyl peptide receptor up-regulation was inhibited and rapid, monophasic binding to a smaller total number of expressed sites was observed. Cytochalasin B enhanced the total number of available N-formyl peptide receptors in LPS-untreated but not LPS-treated cells. In both cases binding was rapid and monophasic suggesting that receptors were either fully or rapidly up-regulated. Although not studied in real-time, C receptor type 3 up-regulation was similar to N-formyl peptide receptor up-regulation in response to LPS, or stimulation by N-formyl peptide, C product C5a, leukotriene B4, and platelet-activating factor in isolated cells and in whole blood. After stimulation with formyl peptide, LPS, or C product 5a, the release of vitamin B12-binding protein paralleled up-regulation of receptors. These data indicate that untreated cells up-regulate N-formyl peptide receptors during cell response at a rate of approximately 10,000/min in a calcium-dependent manner whereas LPS-treated cells already express the bulk of their receptors. In cytochalasin B-treated, degranulating cells 30,000 to 50,000 receptors were up-regulated within a minute.