Binding Of Chemotactic Peptide Research Articles

Alteration of cellular cytosolic calcium and chemotactic peptide binding by an inhibitor of neutrophil function.

The lung is frequently exposed to particulate material that can potentially stimulate release of factors that attract polymorphonuclear neutrophils (PMN). However, few PMN are noted in the airways of normal subjects, suggesting there is some mechanism to dampen influx of these cells. We have isolated from bronchial lavage a peptide that inhibits PMN chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP). In the present study we examined effects of this molecule on 1) chemotaxis to other agonists, 2) FMLP-stimulated PMN superoxide production, 3) PMN calcium fluxes, and 4) binding of FMLP. Our results show that purified inhibitor attenuates PMN chemotaxis to C5a and leukotriene B4. This molecule also inhibits PMN superoxide release in response to FMLP. Exposure to this inhibitor causes an abrupt rise in cytosolic calcium concentration due to a pertussis toxin-sensitive shift of intracellular calcium and attenuates subsequent influx of extracellular calcium in response to FMLP. Binding studies demonstrate the inhibitor induces increased FMLP binding at 37 degrees C but has no effects at 4 degrees C. Inhibition of chemotaxis and increased FMLP binding mediated by this molecule are attenuated by buffering PMN calcium transients. These studies suggest an inhibitor of neutrophil function present in the bronchial environment alters PMN through effects on calcium homeostasis.

Binding of chemotactic peptide to the outer surface and to whole human spermatozoa with different affinity states

Binding of N-formyl-methionyl-L-leucyl-[3H]phenylalanine (fML[3H]Ph) to human ejaculated spermatozoa and to its isolated plasma membrane was studied. Our data confirm the presence of specific receptors for f-MLPh in the human spermatozoa and suggest that whole spermatozoa receptors exist in two affinity states, one high-affinity, low-capacity specific receptor (Kd = 12.3 +/- 0.5 nM, n = 22,285 +/- 65,008 binding sites per sperm cell) and a second one (Kd = 700 +/- 47 nM) that is not saturable, indicating a low-affinity, high-capacity nonspecific site. In contrast, sperm membrane showed only one class of binding site (Kd = 6.4 +/- 0.12 nM), which was statistically different from that of the high-affinity binding site of intact spermatozoa. To explain this difference we discuss the possibility that first, the two binding affinities represent two interconvertible states of a single receptor population, which, depending on the metabolic activity of spermatozoa, may change its physicochemical properties; or second, they reflect two different processes, binding and/or transport into the spermatozoa.

Reduced Expression of Surface Receptors for Synthetic N-Formylated Chemotactic Oligopeptides by Stimulated Peripheral Blood Neutrophils in Psoriasis

Receptors for synthetic N-formylated chemotactic peptides on peripheral blood neutrophils were studied by the binding of fluorescein-labeled hexapeptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys) to the cells in vitro at the range of concentrations 0.01-100 nM. Mean fluorescence of neutrophils was quantitated by a flow cytometry using FACS III. Comparison was made between 27 patients with psoriasis vulgaris and 14 normal controls. Various receptor states related to cell activities were induced by different temperatures, by incubation of cells with cytochalasin B and by preincubation with nonlabeled N-formyl-Met-Leu-Phe. This allowed us to distinguish between the specific binding of fluoresceinated hexapeptide to plasma membrane receptor already present (0 degree C), modulation of receptors by peptide and cytochalasin B stimulated degranulation (25 degrees C), and net binding, including internalization of peptide and receptor recycling system (37 degrees C). At peptide concentrations of 1-10 nM, the labeling of neutrophils at 25 degrees C and 37 degrees C, but not at 0 degree C, was found to be about 10-35% lower in psoriatic than in healthy subjects (p less than 0.002). The amount of fluorescein-labeled peptide bound to the cells at 25 degrees C was markedly increased by cytochalasin B, but to a much lower extent in psoriatic patients than in normal controls. Although the number of plasma membrane receptor for chemotactic peptides in the nonstimulated neutrophils was not altered in psoriasis, the receptor up-regulation induced by preincubation of the cells with 1-10 nM of nonlabeled N-formyl-Met-Leu-Phe at 37 degrees C was reduced when measured by subsequent fluoresceinated hexapeptide uptake at 0 degree C. Receptor recycling, as measured by an increase with time (0-30 min) in the binding of chemotactic peptide by neutrophils in which receptors had been down-regulated, was found to be within normal range in patients with psoriasis. These data indicate that nonstimulated, circulating neutrophils have a normal number of chemotactic peptide receptors on the cell surface, but are less able to recruit intracellular receptors to the cell surface. This finding may be related to smaller internal pools or less efficient translocation of these receptors.

Polymorphonuclear leucocyte function and previous yersinia arthritis: enhanced chemokinetic migration and oxygen radical production correlate with the severity of the acute disease.

Polymorphonuclear leucocyte (PMN) functions (migration in vitro, chemiluminescence, O-2 production, binding of chemotactic peptide, and aggregation) were studied in HLA-B27 positive patients with previous yersinia arthritis (YA). PMNs of patients whose disease had been severe showed chemokinetic and chemiluminescence responses significantly higher than the PMNs of those with a mild disease. The results support the view that enhanced PMN function contributes to inflammatory symptoms in patients with YA.

Chemotactic peptide binding by rabbit polymorphonuclear leukocytes. Presence of two compartments having similar affinities but different kinetics.

N-Formylnorleucylleucylphenylalanine (f-Nle-LeuPhe) bound to rabbit peritoneal polymorphonuclear leukocytes at 4 degrees C exists in at least two compartments that can be differentiated by their off rates. The off rate of one compartment is similar to that of the receptor characterized previously, about 0.4 min-1 (Aswanikumar, S., Corcoran, B., Schiffmann, E., Day, A. R., Freer, R. J., Showell, H. J., Becker, E. L., and Pert, C. B. (1977) Biochem. Biophys. Res. Commun. 74, 810-817; Sullivan, S. J., and Zigmond, S. H. (1980) J. Cell Biol. 85, 703-711); the off rate of the second compartment is about 0.005 min-1. Lysis of the cells at 4 degrees C with 1% Triton does not affect the peptide release from either compartment. Accumulation of peptide at 4 degrees C into the fast off-rate compartment is rapid, reaching a plateau in about 5 min, while peptide in the slow off-rate compartment continues to increase for up to 4 h. The rate of accumulation in the slow off-rate compartment is approximately proportional to the amount of peptide bound to the fast off-rate compartment. Cells lysed at 4 degrees C before binding are still able to accumulate peptide into both compartments. Three possible models to explain the data are presented.

Leishmania donovani: Surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 × 10 6 cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O 2 consumption, O 2 − production, and H 2O 2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15–25% of control values. The acid phosphatase also affected concanavalin A-stimulated O 2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O 2 − production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH 4) 6[P 2Mo 18O 62]·9H 2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O 2 − production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.

Myeloperoxidase-Mediated Modulation of Chemotactic Peptide Binding to Human Neutrophils

Methionine-containing chemotactic peptides, such as formyl-methionyl-leucyl-phenylalanine (FMLP), are inactivated via a neutrophil-derived, myeloperoxidase-mediated oxidation of the methionine residue. We report that extracellular inactivation of FMLP by myeloperoxidase modulates the apparent binding of methionine-containing chemotactic peptides to their surface receptors. Inhibitors of myeloperoxidase enhanced FMLP binding. At subsaturating concentrations of 3H-FMLP (20 nM), 1 mM cyanide (KCN) increased the binding of 3H-FMLP to human neutrophils (PMN) by 51% +/- 12%. Similar increases occurred with 0.1 mM azide and 10 mM aminotriazole (ATZ). KCN had little effect on maximal 3H-FMLP binding to PMN at saturation (control-17,040 +/- 910 receptors/PMN; KCN-16,820 +/- 1,940 receptors/PMN), but decreased the concentration of 3H-FMLP required to half-saturate the PMN receptors (control-39 +/- 3 nM; KCN-17 +/- 1 nM). ATZ gave similar results. The binding to PMN of the non-methionine-containing chemotactic peptide 125I-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (125I-FNLPNTL) was unaltered by KCN. Also, the binding of 3H-FMLP to myeloperoxidase-deficient PMN was unaltered by KCN. Both KCN and ATZ decreased the oxidation of FMLP by PMN. Finally, ATZ (but not KCN) enhanced the chemotactic migration of PMN in response to submaximal concentrations of FMLP. These studies show that intact PMN inactivate methionine-containing chemotactic peptides by a pathway that is sensitive to myeloperoxidase inhibitors and is absent in myeloperoxidase-deficient PMN. This action results in an apparent decrease in the affinity of the chemotactic peptide receptor for methionine-containing chemotactic peptides, which may modulate chemotatic events in inflammatory loci.

Myeloperoxidase-mediated modulation of chemotactic peptide binding to human neutrophils

Methionine-containing chemotactic peptides, such as formyl-methionyl- leucyl-phenylalanine (FMLP), are inactivated via a neutrophil-derived, myeloperoxidase-mediated oxidation of the methionine residue. We report that extracellular inactivation of FMLP by myeloperoxidase modulates the apparent binding of methionine-containing chemotactic peptides to their surface receptors. Inhibitors of myeloperoxidase enhanced FMLP binding. At subsaturating concentrations of 3H-FMLP (20 nM), 1 mM cyanide (KCN) increased the binding of 3H-FMLP to human neutrophils (PMN) by 51% +/- 12%. Similar increases occurred with 0.1 mM azide and 10 mM aminotriazole (ATZ). KCN had little effect on maximal 3H-FMLP binding to PMN at saturation (control-17,040 +/- 910 receptors/PMN; KCN- 16,820 +/- 1,940 receptors/PMN), but decreased the concentration of 3H- FMLP required to half-saturate the PMN receptors (control-39 +/- 3 nM; KCN-17 +/- 1 nM). ATZ gave similar results. The binding to PMN of the non-methionine-containing chemotactic peptide 125I-formyl-norleucyl- leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (125I-FNLPNTL) was unaltered by KCN. Also, the binding of 3H-FMLP to myeloperoxidase- deficient PMN was unaltered by KCN. Both KCN and ATZ decreased the oxidation of FMLP by PMN. Finally, ATZ (but not KCN) enhanced the chemotactic migration of PMN in response to submaximal concentrations of FMLP. These studies show that intact PMN inactivate methionine- containing chemotactic peptides by a pathway that is sensitive to myeloperoxidase inhibitors and is absent in myeloperoxidase-deficient PMN. This action results in an apparent decrease in the affinity of the chemotactic peptide receptor for methionine-containing chemotactic peptides, which may modulate chemotatic events in inflammatory loci.

Paradoxical effects of adenosine on neutrophil chemotaxis.

Chemotaxis of rabbit neutrophils is most sensitive to inhibition by 3-deazaadenosine, followed by 3-deaza-(+/-)aristeromycin, 3-deaza-(+/-)aristeromycinylhomocysteine, 3-deazaadenosylhomocysteine, and adenosylhomocysteine, in that order. Although adenosine by itself had no effect on the chemotaxis of neutrophils, it essentially abolished the inhibitory effects of 3-deaza-adenosine on chemotaxis and the reduction of nitroblue tetrazolium. Paradoxically, adenosine enhanced the inhibition of chemotaxis by 3-deazaadenosylhomocysteine slightly and that of 3-deaza-(+/-)aristeromycin significantly. Adenosine alone unexpectedly inhibited phospholipid methylation to the same extent as 3-deazaadenosine, and reduced protein carboxymethylation to a lesser degree. The inhibition of these two methylation reactions by 3-deazaadenosine was, however, not substantially altered in the presence of adenosine. Drastic changes in the ratio of adenosylmethionine/nucleosidylhomocysteine were observed in the presence of adenosine, 3-deazaadenosine, 3-deaza-(+/-)aristeromycin, or of adenosine in combination with each of the latter compounds. There was no significant effect on the binding of chemotactic peptide to receptors, or on the ratio of ATP/ADP in cells treated by the analogs. These results suggest that the inhibition of methylation reactions per se is not enough to account for the inhibition of both chemotaxis and the reduction of nitroblue tetrazolium by neutrophils.

Inhibition of neutrophil chemotaxis by soluble bacterial products.

BACTERIAL-NEUTROPHIL INTERACTIONS may be critical determinants of virulence in periodontal diseases. This study was undertaken to examine the ability of major bacterial species from the human oral cavity to inhibit (1) peripheral blood neutrophil chemotaxis, (2) chemotactic formylmethionyl peptide binding, and (3) phagocytosis. Included were cultured supernatants and sonic extracts obtained from strains of Capnocytophaga, Bacteroides gingivalis, Fusobacterium nucleatum, Bacteroides asaccharolyticus, Capnocytophaga species, Actinobacillus actinomycetemcomitans, Neisseria, Actinomyces viscosus, Bacterionema matruchotii, Streptococcus mitis, Streptococcus mutans, and Streptococcus sanguis. Chemotaxis was measured using Boyden chambers; phagocytosis was determined using Staphylococcus aureus as the indicator organism and radioactive chemotactic peptide binding was assessed by a rapid filtration assay. None of the test organisms were cytotoxic to neutrophils or inhibited neutrophil phagocytosis. Capnocytophaga species., Bacteroides species., A. actinomycetemcomitans, and F. nucleatum produced factors which specifically inhibited neutrophil chemotaxis. Activity was lost after dialysis. Extracts of Bacteroides species, A. actinomycetemcomitans and F. nucleatum, which were not chemotactic by themselves, inhibited binding of chemotactic peptide suggesting that in vitro chemotaxis inhibition was mediated by nonchemotactic components that compete for the chemotactic factor receptor on the neutrophil. The exception was Capnocytophaga which appeared to inhibit chemotaxis by inhibition of a post-binding event. Such chemotactic inhibitors from periodontopathic organisms that inhibit neutrophil function may be important determinants of virulence.

Two neutrophil populations in human blood with different chemotactic activities: separation and chemoattractant binding

Normal human blood contains a population of neutrophils that migrates to various chemoattractants and a population that fails to migrate. The percentage of neutrophils migrating to optimal concentrations of chemoattractants was quantified: 20 to 40% migrated to N-formylmethionyl-leucyl-phenylalanine, 30 to 50% migrated to human C5a, 25 to 35% migrated to human leukocyte-derived chemotactic factors, 20 to 30% migrated to casein, 15 to 20% migrated to pepstatin, and 1 to 5% migrated to medium alone. Neutrophil migration to the most active chemoattractant was not increased when other chemoattractants were added, indicating that the population of neutrophils migrating to the most active attractant was the same population that was migrating to the other attractants. The percentage of neutrophils migrating to a chemoattractant was not altered by prolonging the assay incubation period or by replacing the attractant with new chemoattractant during the assay, and the percentage was independent of the neutrophil concentration added to the chemotaxis chamber. Nonmigrating neutrophils were isolated with a chemotaxis collection chamber, and they were examined for radiolabeled chemotactic peptide binding. The binding of radiolabeled N-formylmethionyl-leucyl-phenylalanine by nonmigrating and migrating neutrophils was identical.

Corticosteroids block binding of chemotactic peptide to its receptor on granulocytes and cause disaggregation of granulocyte aggregates in vitro.

Inhibition of complement-mediated granulocyte aggregation has recently been proposed as a mechanism of action of high-dose corticosteroids in shock states. Postulating that such inhibition might be effected through alteration of receptors function, we examined the effect of methylprednisolone (MP), hydrocortisone (HC), and dexamethasone (DEX) on the extent and kinetics of binding of the synthetic chemotaxin f-methionine-leucine-phenylalanine (FMLP) to its specific receptor on the granulocyte surface. Dose-dependent inhibition of binding was observed at corticosteroid concentrations paralleling plasma levels achieved with 30 mg/kg intravenous bolus therapy; the order of potency was MP greater than HC greater than DEX. Receptor number was unaffected by steroid exposure, but the steroids effected a decrease in association rate constant for the FMLP-receptor interaction (35% of N for 0.2 mg/ml MP), leading to decreased receptor-ligand affinity. Dissociation kinetics, as examined by cold-chase experiments, were unaltered by the corticosteroids. Furthermore, in addition to the inhibition of aggregation previously reported, aggregated granulocytes were found to disaggregate upon addition of corticosteroids; the order of potency was again MP greater than HC greater than DEX, with an MP concentration of approximately 2-3 mg/ml required to effect complete disaggregation. We conclude that corticosteroids can displace FMLP from the granulocyte surface by slowing association while allowing dissociation to proceed; altered kinetics of receptor-FMLP interaction may explain both the inhibition of granulocyte aggregation and granulocyte disaggregation. If these observations also hold for physiologic stimuli (such as C5adesarginine, which behaves similarly with respect to aggregation, inhibition, and disaggregation), such kinetic changes may be important in the clinical effects of very high-dose corticosteroids such as are administered in shock.

Reduced chemotactic peptide binding in juvenile periodontitis: A model for neutrophil function

Chemotactic peptide binding sites from peripheral blood neutrophils of Localized Juvenile Periodontitis patients and normal controls were quantitated using tritiated N-formylmethionylleucyl-phenylalanine and a rapid filtration assay. It was found that there is a significant reduction in the number of binding sites per cell on neutrophils from the patient group whereas binding affinity remained the same as control values. A direct correlation between in vitro neutrophil chemotactic response and density of binding sites was found. Since these patients present little clinical illness, localized juvenile periodontitis can be a valuable model for the study of human neutrophil function.

Surface membrane enzyme, chemotactic peptide binding activities, and chemotactic responsiveness of rabbit peripheral and peritoneal neutrophils.

The chymotrypsin-like esterase activity of plasma membrane and the ecto-5'nucleotidase and ecto-p-nitrophenyl phosphatase activities in peritoneal neutrophils are 2- and 3-fold higher than peripheral blood neutrophils. The binding of the chemotactic peptide, formyl norleucyl-leucyl-phenyl-alanine to peritoneal neutrophils is 6- to 7-fold higher than to peripheral blood neutrophils. The chemotactic responsiveness of peritoneal neutrophils to the chemotactic peptide is 5-fold greater than peripheral blood neutrophils. However, there was no significant difference in the chemotactic responsiveness to complement component C5a between the blood and peritoneal cells.

Cryptic receptors for chemotactic peptides in rabbit neutrophils

Specific binding of chemotactic peptides to receptors in rabbit neutrophils was enhanced in the presence of aliphatic alcohols — especially n-propanol and n-butanol. The effect was concentration dependent with maximum increases in specific binding of 2.6 and 3.0 fold in the presence of 2.5% n-propanol and n-butanol respectively. The results indicate that up to 65% of the receptors are cryptic and unavailable for ligand interactions. It is suggested that minute to minute modulation of chemotactic responsiveness could occur by controlling at the membrane level the number of receptors exposed to chemotactic stimuli.

The Evidence for the existence of chymotrypsin-like esterase activity in the plasma membranes of rabbit neutrophils and the specific chemotactic peptide binding activity of the subcellular fractions

1. 1. Light- and heavy-plasma membrane fractions have been isolated from rabbit neutrophils and a chymotrypsin-like esterase has been shown to be present in these fractions. 2. 2. The molecular weight of the chymotrypsin-like esterase of rabbit neutrophil plasma membrane was estimated to be about 200 000. 3. 3. About 93% of the chymotrypsin-like esterase of the plasma membranes is esterase 1 and the susceptibility to potential inhibitors was similar in light- and heavy-plasma membrane. 4. 4. Chemotactic peptide, [ 3H]formyl-norleucyl-leucyl-phenylalanine ([ 3H]-formyl-Nle-Leu-Phe) binding by subcellular fractions shows that the highest specific binding was observed in the light-plasma membrane fraction. The specific chemotactic peptide binding activity of light-plasma membrane was about 2-fold higher than the heavy-plasma membrane, about 37-fold higher than the nuclear fraction, about 3-fold higher than lysosomal fraction and about 10-fold higher than the microsomal fraction.