Human neutrophils contain an intracellular pool of putative receptors for the chemoattractant N-formyl-methionyl-leucyl-phenylalanine.

Purified human peripheral blood neutrophils were disrupted by nitrogen cavitation or sonication and fractionated on sucrose density gradients in order to separate the plasma membranes and granule fractions. Quantitatively, the fractions containing the specific granules by marker enzyme/protein enrichment contained the most tritiated N-formyl-methionyl-leucyl-phenylalanine (fmet-leu-[3H]phe)-binding activity. Competitive binding experiments using unlabeled formyl peptide analogues indicated that the intracellular binding sites display the same structure-function specificity as formyl peptide receptors on intact polymorphonuclear leukocytes (PMN) or isolated plasma membranes. Analysis of the fractions for membrane, primary, and secondary granule markers, as well as the distribution of 125I-labeled plasma membranes in sucrose density gradients, indicated that the specific fmet-leu-[3H]phe binding to granule-containing fractions was not due to contamination by plasma membranes. In addition, membranes isolated from PMN previously stimulated with phorbol myristate acetate (PMA) demonstrated increased binding sites, while isolated membranes exposed to PMA under the same conditions failed to show such increases. The data lend direct support to the concept that there is an intracellular pool of fmet-leu-phe receptors that serves as a source of new surface membrane constituents and receptor material that may allow PMN to maintain functional responsiveness during chemotaxis.

N-formylmethionyl-leucyl-[3H]phenylalanine binding, superoxide release, and chemotactic responses of human blood monocytes that repopulate the circulation during leukapheresis.

Human blood monocytes comprise two subpopulations: one migrates to the chemoattractant, N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), and has saturable binding sites for this peptide; the other does not migrate and exhibits little peptide binding. To determine if expression of binding sites was a function of monocyte maturation, we depleted human subjects of blood monocytes by leukapheresis so that the circulation was repopulated by monocytes released from the bone marrow. Pre- and postleukapheresis monocytes were then compared for fMet-Leu-[3H]Phe binding, superoxide generation, and chemotactic responses. No significant differences in peptide binding curves were found, suggesting that receptor expression was stable over the maturational span represented by these two groups of cells. This supports the hypothesis that there are two distinct lineages of monocytes with respect to expression of receptors for fMet-Leu-Phe. An additional finding of interest was that the number of chemotactically responsive cells immediately postleukapheresis was half the control. This was a transient state; monocyte responses were normal 3 hr after termination of leukapheresis, suggesting that they rapidly become functionally mature.

FMLP-induced enzyme release from neutrophils: a role for intracellular calcium.

The ability of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) to stimulate beta-glucuronidase release and 45Ca2+ release from rabbit neutrophils was studied. FMLP stimulated enzyme release from cytochalasin B-treated cells either in the presence or the absence of extracellular calcium. Depletion of cell calcium, by exposure to either ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid or the calcium ionophore A23187, blocked the ability of FMLP to stimulate enzyme release and 45Ca2+ release in the absence of extracellular calcium. The ability of A23187 to lower the 45Ca2+ content of neutrophils, to block FMLP-stimulated 45Ca2+ release, and to inhibit FMLP-stimulated enzyme release in the absence of calcium was dose dependent over the same concentration range (10(-8) to 10(-6) M A23187) for all three actions. In contrast, FMLP stimulated enzyme release from A23187-treated cells, provided that extracellular calcium was present. This secretory response was normal as judged by cell ultrastructure and FMLP dose-response relationships. It is concluded that A23187 depletes a pool of intracellular calcium usually released by FMLP and that release of calcium from this pool is necessary for initiation of enzyme secretion in the absence of extracellular calcium.

Release of leukotriene B4 from human neutrophils and its relationship to degranulation induced by N-formyl-methionyl-leucyl-phenylalanine, serum-treated zymosan and the ionophore A23187.

The release of leukotriene B4 (LTB4) from human neutrophils and its relationship to degranulation induced by the divalent cation ionophore A23187, serum-treated zymosan (STZ), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and arachidonic acid (AA) have been studied. Greatest release of LTB4, measured by specific radioimmunoassay, occurred in response to A23187 (5-10 ng/10(6) cells); lower concentrations were obtained after incubation with STZ (0.2-0.8 ng/10(6) cells) and AA (0.3-2.6 ng/10(6) cells) and low (0.02 ng/10(6) cells) or not detectable amounts from cells incubated with FMLP. Release of LTB4 induced by STZ, FMLP and submaximal concentrations of A23187 was potentiated by simultaneous addition of AA. Lower amounts (0.06-0.3 ng/10(6) cells) of thromboxane B2 (TXB2) were also released by these stimuli, however this release of TXB2 was not potentiated by exogenous AA. The secretion of beta-glucuronidase induced by A23187, STZ and FMLP was not quantitatively related to release of LTB4 or TXB2 and was not potentiated by exogenous AA. Furthermore, FMLP induced degranulation was cytochalasin B (Cyt B)-dependent, whereas LTB4 release in response to this stimulus was only marginally increased by pretreatment of the cells with Cyt B. These data indicate that LTB4 does not mediate degranulation induced by these stimuli.

Guanine nucleotides modulate the binding affinity of the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes.

The oligopeptide chemoattractant receptor on human polymorphonuclear leukocyte (PMN) membranes exists in two affinity states. Since guanine nucleotides regulate the binding affinity and transductional activity of several other types of receptors, we examined the effect of nucleotides on the binding of N-formyl-methionyl peptides to their receptors on human PMN membranes. The addition of guanylylimidodiphosphate (0.1 mM), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMN membrane preparations reduced the fraction of high-affinity receptors detected in equilibrium binding studies from 21.3 +/- 0.13 to 11.8 +/- 0.05% (P less than 0.03), without altering the binding affinities. Since the total number of receptors remained unchanged, the effect of guanylylimidodiphosphate was to convert a portion of the receptors from the high-affinity state to the low-affinity state. At the maximal concentration of guanine nucleotide tested, approximately 50% of the high-affinity sites were converted to low-affinity sites. The findings obtained by equilibrium binding were supported by kinetic studies since the dissociation of the radiolabeled oligopeptide chemoattractant N-formyl-methionyl-leucyl-[3H]phenylalanine from PMN membranes was accelerated in the presence of guanine nucleotide. The effect of guanine nucleotides was reversed upon washing, indicating that affinity conversion is bidirectional. The guanine nucleotide effects were greatest with nonhydrolyzable derivatives of GTP followed by GTP then guanosine diphosphate. Neither guanosine monophosphate nor any adenine nucleotide tested had an effect on receptor binding. These data suggest a role for guanine nucleotides in the regulation of stimulus-receptor coupling of chemoattractant receptors on human PMN.

Polymorphonuclear leukocyte function in psoriasis: chemotaxis, chemokinesis, beta-adrenergic receptors, and proteolytic enzymes of polymorphonuclear leukocytes in the peripheral blood from psoriatic patients.

Psoriatic patients, particularly those with psoriatic arthritis, have neutrophilic and eosinophilic leukocytosis. Isolated polymorphonuclear leukocytes (PMNLs) from psoriatic patients have normal concentrations of proteolytic enzymes and they have beta-adrenergic receptors of normal density and affinity. PMNLs from psoriatic patients responded normally to the synthetic chemotactic peptide, f-Met-Leu-Phe (formyl-methionine-leucine-phenylalanine). The chemotactic activities of sera from psoriatic patients were similar to those of normal sera. Sera from psoriatic patients enhanced chemokinesis of PMNLs more than normal control sera at a final concentration of 1%; no difference in chemokinetic response between psoriatic and normal sera was found at serum concentrations greater than 2.5%. This study suggests that the peripheral PMNLs from psoriatic patients are normal, but the sera of psoriatic patients has more chemokinetic activity for PMNLs than does normal serum.

Is cytosolic ionized calcium regulating neutrophil activation?

The concentration of cytosolic ionized calcium, [Ca2+]i, was measured in intact neutrophils by use of a fluorescent indicator trapped in the icytoplasm. A given rise of [Ca2+]i elicited by the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) was associated with a much greater degree of superoxide generation and myeloperoxidase secretion than was the same or larger [Ca2+]i produced by a specific calcium ionophore, ionomycin, which bypasses cell surface receptors. Thus, FMLP appears to generate some important excitatory signal in addition to a rise in [Ca2+]i and exocytosis and superoxide generation in neutrophils may not be simply dependent on [Ca2+]i as is widely supposed.

Decrease in apparent Km for oxygen after stimulation of respiration of rat polymorphonuclear leukocytes.

The respiratory burst of polymorphonuclear leukocytes, induced by the addition of chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine) and cytochalasin B was found to consist of two phases. The first phase of very rapid oxygen uptake lasted 1-3 min. and was followed by a second more prolonged phase of lower magnitude. The apparent Km for oxygen of unstimulated cells was 9.6 +/- 0.67 microM, while that of the second phase of stimulation was 3.7 +/- 1.6 microM oxygen. The possibility that lowered oxygen concentrations may regulate polymorphonuclear leukocyte activity in some pathological conditions is discussed.

Abnormal distribution of complex carbohydrates in neutrophils of a patient with lactoferrin deficiency.

Previous studies have identified patients with susceptibility to bacterial infection associated with lactoferrin deficiency in dysmorphic neutrophils containing abnormal or no secondary granules and abnormal nuclear segmentation. We have investigated the subcellular distribution of vicinal glycol-containing complex carbohydrates in marrow and blood myeloid cells of such a patient using the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining method and have examined the response of these neutrophils to the degranulating agents N-formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). As in normal specimens, immature primary granules were strongly PA-TCH-SP reactive; however, unlike normal specimens, masking of PA-TCH-SP reactivity did not occur in mature primary granules. Endoplasmic reticulum demonstrated moderately strong PA-TCH-SP staining, in contrast to absent staining of this organelle in normal promyelocytes and consistent with abnormal primary granule genesis. Small abnormal elongated granules (0.1-0.2 micron in diameter) were identified at the myelocyte state of development and were the predominant granule type in late neutrophils. These granules were identified as secondary granules on the basis of their PA-TCH-SP positivity and were differentiated from primary and tertiary granules on the basis of a lack of peroxidase, acid phosphatase, and sulfate staining. When the neutrophils were exposed to PMA, cell aggregation occurred, and the abnormal granules degranulated in a manner similar to the degranulation observed with normal secondary granules. Although PA-TCH-SP staining of the plasma membrane appeared normal, a decrease in FMLP receptors was demonstrated. Thus, a defect(s) is present in complex carbohydrate distribution and staining that involves primary and secondary granules and possibly the plasmalemma of neutrophils from this patient. This results in abnormal packaging of primary granules and synthesis of normal numbers of secondary granules that are qualitatively and morphologically abnormal, but can be recruited to degranulate with PMA.

Enhancement of human neutrophil adherence by synthetic leukotriene constituents of the slow-reacting substance of anaphylaxis.

The functionally predominant constituents of the slow-reacting substance of anaphylaxis (SRS-A), designated leukotrienes C4 and D4 (LTC4 and LTD4), as well as the leucocyte chemotactic factor leukotriene B4 (LTB4) enhance the adherence of human neutrophils to Sephadex G-25. Enhancement of neutrophil adherence was significant at leukotriene concentrations of 3 X 10(-9) M -3 X 10(-7) M, and reached a maximum level for each of the leukotrienes that was similar in magnitude to that evoked by the neutrophil chemotactic peptide N-formyl-methionyl-leucylphenylalanine (FMLP). The leukotrienes and FMLP elicited optimum increases in neutrophil adherence within 1-2 min at 37 degrees. Indomethacin inhibited the increase in neutrophil adherence evoked by LTC4 and LTD4 and the concurrent elevation in the concentration of endogenous thromboxane B2. The smooth muscle contractile and vasoactive factors LTC4 and LTD4, which lack chemotactic activity for leucocytes, are as active as LTB4 in stimulating human neutrophil adherence, and the effect may be mediated in part by neutrophil-derived thromboxane A2.

Selective defect in human neutrophil superoxide anion generation elicited by the chemoattractant N-formylmethionylleucylphenylalanine in pregnancy.

Pregnancy has been associated with alterations of polymorphonuclear neutrophil (PMN) function. Superoxide anion production was studied in pregnant women paired with nonpregnant women of childbearing age. There was a significant decrease in the amount of cytochrome c reduced in response to 1 microM N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) but not to phorbol myristate acetate, 4 or 20 ng/ml. Chemotaxis was also depressed. Binding of tritiated fMet-Leu-Phe to PMNs from pregnant women was not defective. Incubation of normal cells in up to 10(-6) M estradiol or progesterone did not mimic the defect, but 10(-7) M progesterone caused a decrease in chemotaxis. Serum pooled from women with the defect had no effect on superoxide anion production by normal PMNs. PMN rosetting with IgG-sensitized human erythrocytes was normal. Defective production of superoxide anion may contribute to the amelioration of connective tissue disease and increased susceptibility to infection often seen during pregnancy.

Modulation of polymorphonuclear leukocyte function by cetiedil.

Cetiedil citrate monohydrate inhibits sickling of red cells and aggregation of platelets. We assessed its ability to attenuate polymorphonuclear leukocyte (PMN) function. PMN aggregation in response to 2 X 10(-7) M formyl-met-leu-phe (FMLP) was inhibited in a dose-dependent fashion by cetiedil concentrations ranging from 60 to 250 microM. Additionally, 125 microM cetiedil inhibited PMN aggregation in response to 2 X 10(-7) M FMLP, 20 ng/ml phorbol myristate acetate (PMA), and 1 X 10(-6) M A23187 by 69% +/- 18%, 72% +/- 20%, and 65% +/- 4%, respectively. Inhibition of FMLP-induced aggregation was provided by only 5 min of incubation of the drug with the cells and was partially reversible. Cell viability was unaffected by exposure of PMN to the drug. Correspondingly, 125 microM cetiedil prevented the translocation of calcium from the PMN membrane as assessed by chlorotetracycline fluorescence. Paralleling the effect of the drug on PMN aggregation, 125 microM cetiedil inhibited release of superoxide by 55% and decreased the number of available 3H-FMLP receptors. However, its effect on release of the primary granule constituent, myeloperoxidase, was minimal (4.5% inhibition), while the effect on release of the specific granule product, lactoferrin (27% inhibition), was modest. These studies indicate that cetiedil affects PMN aggregation and superoxide release to a much greater extent than PMN degranulation. Thus, cetiedil may have potential uses in modulating inflammatory response in vivo.

Activation of the respiratory burst enzyme in human polymorphonuclear leukocytes by chemoattractants and other soluble stimuli. Evidence that the same oxidase is activated by different transductional mechanisms.

Chemoattractant-receptor coupling triggers several biologic responses in phagocytic cells including activation of the respiratory burst. Prior evidence in intact cells implied that stimulation of the respiratory burst by chemoattractants was by a mechanism different from other soluble agents suggesting the possibility that different oxidative enzymes were responsible. We now show that the chemoattractants N-formyl-methionyl-leucyl-phenylalanine and a split fragment of the fifth component of complement (C5a) stimulate an NADPH oxidase activity, measured in the 50,000-g particulate fraction from human polymorphonuclear leukocytes (PMN). Levels of oxidase activity stimulated by the chemoattractants were both time and dose dependent and required the presence of cytochalasin B during stimulation. In contrast, activation by two nonchemotactic stimuli, the ionophore A23187 and phorbol myristate acetate (PMA), did not require cytochalasin B. Temporal patterns of oxidase activation suggested that different stimuli follow different transductional pathways. Chemoattractant-mediated activation was immediate (no lag); peaked by 45 s and declined rapidly to approximately 50% of maximal by 2 min. In contrast, activation by A23187 or PMA had a 15-30-s lag and increased more slowly. Stimulation by A23187 peaked at 5 min, then declined. Stimulation by PMA plateaued at 20 min and did not decline by 90 min. Comparison of Km values for NADPH and NADH obtained by Lineweaver-Burk analysis of the oxidase activity stimulated by N-formyl-methionyl-leucyl-phenylalanine, A23187, and PMA suggested that the same enzyme was activated by all stimuli. Thus, chemoattractants and other soluble stimuli appear to activate the same respiratory burst enzyme in PMN but they utilize different transductional mechanisms and are regulated differently.

Altered polymorphonuclear leukocyte responses in psoriasis: chemotaxis and degranulation.

Chemotactic activities of circulating polymorphonuclear leukocytes (PMN) were determined in twenty patients with psoriasis and twenty healthy control persons. After serial dilution of the complement split product C5a and the formylated tripeptide f-met-leu-phe (FMLP), chemotaxis profiles showed that PMN migration toward both chemotaxins was significantly increased in psoriasis. In addition, PMN from psoriatic patients responded to chemotaxins at much lower concentrations compared with controls. The liberation of (lysosomal) beta-glucuronidase was also determined in cytochalasin B-treated cells confronted with increased concentrations of the chemotaxins. Secretion of this marker enzyme started at lower concentrations in PMN derived from psoriatic patients. Our observations demonstrate migratory and secretory hyper-responsiveness of PMN from psoriatic patients. This may play a role in perpetuating the psoriatic tissue reaction.

Enhancement of chemotactic migration by the local anesthetic tetracaine.

The local amine anesthetic tetracaine added to a suspension of guinea pig or human neutrophilic granulocytes inhibited their random migration in Boyden chambers, but increased their chemotactic migration towards the chemotactic tripeptide f-Met-Leu-Phe, complement-activated normal guinea pig serum, and the eosinophil chemotactic factor ECF. Tetracaine not only increased the distance migrated by the leading cells, it also caused more cells to leave the upper filter surface and to migrate into the filter. The effect required the presence of the drug; cells preincubated with tetracaine and washed did not differ from control cells. It is suggested that tetracaine specifically enhanced a mechanism operative in a cell's response to a concentration gradient of a chemotactic factor.

Role of cell surface contact in the kinetics of superoxide production by granulocytes.

The complement-derived anaphylatoxin C5a and a putative analogue of bacterial chemotactic factor (N-formyl-methionyl-leucyl-phenylalanyl [fMLP]), as well as bacterial lipid A, all stimulate human granulocyte (PMN) adhesiveness and superoxide (O-2) production in a concentration-dependent manner. Since attachment of particulate matter to the PMN membrane is an early event in the triggering of respiratory burst of these cells, we further examined how adherence might modulate the release of O-2 induced by soluble mediators of inflammation. We found that both the quantity and kinetics of O-2 production depend on prior attachment of the cells to a surface. In stirred suspensions of PMN, fMLP induces only a short burst (2.5 min) of O-2 release associated with reversible PMN aggregation. The magnitude, but not the time course, of both these responses depend on the fMLP concentration. Unlike the short respiratory response of cells in suspension, PMN allowed to settle onto stationary petri dishes, then overlaid with fMLP, rapidly spread and attach to the surface where they remain and release O-2 throughout the 60-min test period. Prolonged O-2 release also follows fMLP stimulation in suspensions of PMN pretreated with cytochalasin B, in which case aggregation becomes irreversible during the 20-min burst. If fMLP is slowly infused into a suspension of cells at 37 degrees C or if PMN are challenged at 0 degrees C, and then warmed to 37 degrees C, O-2 release greatly decreases or becomes undetectable. Suspended PMN do not respond to a second challenge by the same stimulus regardless of the rate or temperature at which the first stimulus was added, a phenomenon formerly described as desensitization. However, if PMN challenged with fMLP in suspension undergo the short respiratory response and then are later placed in petri dishes, they adhere and resume production of O-2 without further stimulation. Chemotactic factor-induced adherence and O-2 release of PMN on a surface is entirely independent of either the mode of activation or prior O-2 release during preincubation in suspension. Human C5a also promotes PMN adherence and prolonged O-2 release in petri dishes. Furthermore, lipid A increases O-2 release and adherence of settled PMN, but fails to elicit either response from suspended PMN. These results indicate that cell surface contact plays an essential role in triggering the respiratory burst of PMN activated by soluble stimuli. This long-lasting O-2 release by chemotactic factor-stimulated PMN may play a significant role in inflammatory reactions when PMN become adherent in vivo.

Inhibition by prostaglandins of leukotriene B4 release from activated neutrophils.

Chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) in the presence of cytochalasin B stimulates the release of leukotriene B4 (LTB4), superoxide (O2-), and N-acetylglucosaminidase from elicited rat peritoneal and human peripheral neutrophils [PMN (polymorphonuclear leukocytes)]. Prostaglandins E1 and E2 (PGE1 and PGE2) inhibit LTB4 release from PMN in a dose-related manner with an IC50 of 1 X 10(-8) M. This action is associated with increased levels of cyclic AMP. The inhibitory activity of a variety of PGs on LTB4 production by rat peritoneal PMN parallels their affinity for PGE receptors in other tissues. O2- release is also suppressed by low levels of PGE1 and PGE2 in a dose-related manner and this inhibition is enhanced by theophylline. In contrast, lysosomal enzyme release is only minimally affected by physiological levels of PGs. These data are consistent with an action of PGs at the level of the PG receptor on LTB4 and O2- release from the fMet-Leu-Phe-stimulated rat peritoneal PMN. In addition, the fMet-Leu-Phe-induced adherence of PMN to endothelial cells and inhibition of this phenomenon by PGs may now be explained by PG-mediated inhibition of LTB4 formation.

Relationship between the binding of N-formylmethionylleucylphenylalanine and the respiratory response in human neutrophils.

The results presented in this paper demonstrate that the chemotactic peptide N-formylmethionylleucylphenylalanine (f-Met-Leu-Phe) is rapidly inactivated by the products of the respiration of human neutrophils stimulated by the peptide itself. The process of inactivation is impeded by the addition of inhibitors of myeloperoxidase (KCN, NaN3), of catalase, of methionine but not by the addition of superoxide dismutase, indicating that the mechanism of inactivation is the oxidation of methionine residue by myeloperoxidase-H2O2-halide system. The oxidation of the peptide causes the rapid cessation of the respiratory burst, since the sulfoxide derivative loses its ability to bind the specific receptors of neutrophil surface and, hence, its biological activity. The comparison between the time course of the binding of f-Met-Leu-[3H]Phe to the specific receptors and the rate of the respiratory response of neutrophils in the presence and in the absence of the process of peptide oxidation was used to investigate the mechanism of the activation of the respiratory burst by the peptide-receptor complexes. In conditions where the inactivation of the stimulatory agent takes place the stimulated respiration slows down and resumes the resting state shortly after the cessation of the binding, although a substantial amount of the peptide remains bound to the specific receptors. In conditions where the degradation of the peptide does not occur the binding of the peptide and the respiratory burst continue for a longer period of time, but the rate of the respiration, calculated in terms of the instantaneous velocity (Vist), is not correlated to the amount of the ligand bound to the membrane receptors measured at various times, indicating that a summation of the effects of the ligand-receptor complexes does not occur as they form. These findings demonstrate, as far as the respiratory response is concerned, that the biological activity of the peptide-receptor complexes is short-lived and that continuous de-novo receptor occupancy is necessary for the maintenance of the activated respiration.