Wheat germ agglutinin specifically inhibits formyl peptide-induced polymorphonuclear leukocyte chemotaxis.

Evidence that surface membrane glycoproteins of polymorphonuclear leukocytes (PMN) are involved in stimulus-response coupling prompted us to examine effects on these cells of various plant lectins. We have found that wheat germ agglutinin (WGA) (1.0 microgram/ml) completely, specifically, and irreversibly inhibits directed migration (chemotaxis) of human PMN toward the synthetic peptide, N-formylmethionyl-leucyl-phenylalanine (FMLP) (0.1 to 100 nM). This effect of WGA was not shared by subagglutinating concentrations of either concanavalin A or Bandeirea simplicifolia lectin. In contrast to its effects on FMLP-induced chemotaxis, WGA did not influence other FMLP-induced PMN responses (i.e., selective discharge of lysosomal enzymes from cytochalasin B-treated cells, generation of superoxide anion radical(s). WGA also did not influence PMN chemotactic responses to either the complement-derived peptide, C5a, or the lipoxygenase product, leukotriene B4. Inhibition of FMLP-induced chemotaxis by WGA was not reversed by washing WGA-treated cells, but was reversed (and prevented) by N-acetyl-D-glucosamine (not by N-acetyl-D-galactosamine or mannosamine). WGA did not affect either orientation or stimulated random motility of PMN, and did not interfere with specific binding to PMN of (3H)-FMLP. A derivative of WGA with 10-fold less agglutinating activity for human erythrocytes was prepared by treating the native lectin with cyanogen bromide and formic acid. The derivative also inhibited FMLP-induced PMN chemotaxis specifically and selectively. These data suggest that WGA specifically inhibits FMLP-induced PMN chemotaxis by attaching to N-acetyl-D-glucosamine residues at a locus on the PMN plasma membrane that is distinct from the binding site of the FMLP receptor.

Chemokinetic activity of N-formyl-methionyl-leucyl-phenylalanine on human neutrophils, and its modulation by phenylbutazone.

Phenylbutazone (PBZ) is known to inhibit the oriented migration of human polymorphonuclear leukocytes (PMNs) induced by formyl-methionyl-leucyl-phenylalanine (FMLP), and to protect these cells against the deactivation caused by their prior incubation with FMLP. To gain insight into the mechanism of these effects, we measured the oriented PMN migration under agarose induced, in the presence and absence of PBZ, by FMLP, zymosan-activated serum and Klebsiella pneumoniae culture supernatant. The two components of this migration, i.e. the speed (chemokinesis), and direction of locomotion (chemotaxis), were also assessed. At concentrations ranging from 10(-8) to 10(-5) M, FMLP displayed similar chemotactic activity but the speed of PMN locomotion was maximal for 10(-7) M, and lower for concentrations above and below this level. Oriented migration was proportional to the mean cell locomotion speed during the experiments. PBZ inhibited both the oriented migration and locomotion speed induced by 10(-7) M FMLP, but did not affect its chemotactic activity. At concentrations of 10(-6) and 10(-5) M, PBZ increased oriented migration and locomotion speed, again without influencing FMLP chemotactic activity. Oriented migration induced by zymosan-activated serum was not affected by PBZ but the migration induced by Klebsiella pneumoniae culture supernatant diminished slightly. These results demonstrate that PBZ modulates the chemokinetic effect of FMLP on PMNs and thus alters oriented PMN migration.

Mechanism of dexamethasone inhibition of chemotactic factor induced granulocyte aggregation.

The reaction of FMLP with granulocytes causes aggregation and degranulation and enhances adherence to endothelium. To evaluate whether prevention of granule extrusion could impair these granulocyte activities, granulocytes were treated with either dexamethasone or hydrocortisone prior to treatment with FMLP. Dexamethasone was added to suspensions of cytochalasin B-treated granulocytes; it markedly impaired the aggregation response of the granulocytes of FMLP. When cytochalasin-B was not used, granulocyte aggregation in response to FMLP or PMA was inhibited by dexamethasone. Although dexamethasone prevented aggregation of cells following stimulation with FMLP or PMA, it failed to prevent the aggregation of granulocytes induced by rabbit lactoferrin. Adherence of granulocytes to human endothelial monolayers was enhanced by FMLP; dexamethasone inhibited the enhancement. However, with the addition of human lactoferrin to the granulocytes exposed to dexamethasone, the cells were able to adhere as well to endothelium as the cells exposed to FMLP but free of dexamethasone. When cytochalasin-B-treated granulocytes were incubated with dexamethasone or hydrocortisone prior to the addition of FMLP, the subsequent release of lactoferrin was substantially blocked, whereas the release of the primary granule products, lysozyme and beta-glucuronidase, was attenuated but not completely blocked. Thus, corticosteroids might block chemotactic-factor-induced granulocyte aggregation by selectively preventing release of specific granule products that contribute to and sustain aggregation.

C3a-induced lysosomal enzyme secretion from human neutrophils: lack of inhibition by f met-leu-phe antagonists and inhibition by arachidonic acid antagonists.

C3a-induced lysosomal enzyme secretion from human peripheral neutrophils in a noncytolytic, dose-dependent (10-100 microgram/ml) process. Release of both primary and secondary granule constituents occurred when neutrophils were exposed to C3a plus cytochalasin B, however, C3 alone induced limited release of lysozyme. A competitive antagonist of the formyl-peptide receptor on neutrophils, t boc (phe-leu) 2-phe, did not block the release induced by C3a. Arachidonic acid antagonists, nordihydroguaiaretic acid and quercetin caused dose-dependent inhibition of release induced by C3a plus cytochalasin B, however, lysozyme release induced by C3a in the absence of cytochalasin B was minimally affected. Indomethacin at high concentration (greater than 10(-5) M) had similar inhibitory effects.

Inhibition of formylmethionyl-leucyl-phenylalanine-stimulated neutrophil chemiluminescence by human immunoglobulin A paraproteins.

Immunoglobulin A (IgA) paraproteins from patients with myeloma have been shown to inhibit human neutrophil chemotaxis to C5a, casein, and chemotactic factors produced by Escherichia coli. This study demonstrates that these paraproteins also inhibit neutrophil chemotaxis in response to the synthetic peptide formylmethionyl-leucyl-phenylalanine (f-MLP). Furthermore, the neutrophil chemiluminescence response stimulated by f-MLP was markedly suppressed by the presence of IgA paraprotein. Maximal inhibition of chemiluminescence was observed when the paraprotein was present during the chemiluminescence response. The inhibitory activity was substantially reduced by removal of the Fc region of IgA or by conversion of polymeric IgA to monomeric IgA by limited reduction and alkylation. Additional experiments showed that these IgA paraproteins inhibited C5a but not phorbol myristate acetate-stimulated chemiluminescence. These observations are constant with the hypothesis that polymeric forms of IgA bind to human neutrophils and interfere with the binding of chemotactic factor to its receptor or the consequent receptor-mediated oxidative burst or both.

Inhibition of the n-formylmethionyl-leucyl-phenylalanine induced respiratory burst in human neutrophils by adrenergic agonists and prostaglandins of the E series.

Exogenous prostaglandins E1 and E2 and L-isoproterenol potently inhibited the production of superoxide anions by human neutrophils activated in vitro by n-formylmethionyl-leucyl-phenylalanine (FMLP). An estimated ID50 of 50 nM was found for all three agents while L-epinephrine and prostaglandin F2 alpha were 10 and 100 fold, respectively, less active. Inhibition occurred whether these agents were added before, together with, or after the addition of the tripeptide to cell suspensions. Cells treated with dibutyryl adenosine 3',5'-monophosphate also expressed reduced rates of superoxide synthesis thus suggesting that the hormonal inhibitors acted indirectly by stimulating membrane bound adenylate cyclase.

Inhibition of human polymorphonuclear leukocyte function by 2-cyclohexene-1-one. A role for glutathione in cell activation.

2-cyclohexene-1-one and diethyl maleate specifically decrease reduced glutathione (GSH) levels in human polymorphonuclear leukocytes (PMN) by direct conjugation, and by interaction with the glutathione-s-transferase system. Using these two nontoxic reagents we have examined the effect of decreased GSH levels on five parameters of PMN activation: superoxide generation, release of the lysosomal enzymes lysozyme and beta-glucuronidase, and increases in the influx of Na+ and Ca2+. When PMN pretreated with 2-cyclohexene-1-one or diethyl maleate were incubated with formyl-methionyl-leucyl-phenylalanine (FMLP) or the proteolytic fragment of the fifth component membrane of complement, C5a, agents that interact with surface membrane receptors, increases in all five parameters were inhibited in a dose-dependent manner. For O-2 generation and lysosomal enzyme release the ID50 for 2-CHX-1 was 40--90 micrometers corresponding with a 30--50% decrease in intracellular GHS. In contrast stimulation of treated PMN by the divalent cation ionophore A23187 or 5-hydroxyeicosatetraenoic acid was much less sensitive to depressed GSH; the ID50 for 2-cyclohexene-1-one was 1 mM or greater, corresponding with an 80--90% decrease in GSH. The effect of lowered GSH was not the result of decreased binding of FMLP to surface receptors because [3H]-FMLP binding studies demonstrated a two- to three-fold increase in the number of available binding sites. These data indicate that normal GSH levels are necessary for the transduction of the activation signal from the exterior to the interior of the PMN, but once initiated the activation sequence proceeds normally despite markedly lowered intracellular GSH.

Receptor-directed inhibition of chemotactic factor-induced neutrophil hyperactivity by pyrazolon derivatives. Definition of a chemotactic peptide antagonist.

The two pyrazolon derivatives, phenylbutazone and sulfinpyrazone, selectively inhibit chemotactic peptide-induced effects on neutrophils. As they antagonize the induction of acute neutropenia in vivo and of cellular hyperadhesiveness, lysosomal enzyme release, hexose monophosphate shunt activity, and superoxide production in vitro, these effects occur with a specificity not shared with other prostaglandin biosynthesis inhibition by these drugs resembles the competitive type of antagonism and occurs at concentrations attainable in vivo under clinical conditions. The locomotory machinery, the direction-finding mechanisms, and the basic metabolic machinery of the cell are unaffected. These drugs interfere with specific binding of the formylpeptide to its receptor on neutrophils.

The effect of hemin, adenosine 3',5'-monophosphate and phosphorylated sugars on Met-tRNAfMet deacylase activity in rabbit reticulocyte lysates.

The effect of hemin, phosphorylated sugars, adenosine 3',5'-monophosphate (cyclic AMP) and a number of purines on a specific initiator tRNA deacylase activity in rabbit reticulocytes has been investigated. In the concentration range established to be optimal for maximal stimulation of translation (5.5-30.0 microM), hemin produces a 20-82% inhibition of Met-tRNAfMet deacylation. In contrast, all phosphorylated sugars tested, with the exception of fructose 1,6-diphosphate, are without effect. High concentrations of cyclic AMP (2-4 mM) also significantly inhibit the deacylase activity. The role of hemin and Met-tRNAfMet deacylase in the control of peptide initiation are discussed.

Inhibitor studies of methionyl-tRNA transformylase of Euglena gracilis.

Inhibitor studies of the only known eukaryotic methionyl-tRNA transformylase (10-formyltetrahydrofolate:L-methionyl-tRNA N-transformylase, EC 2.1.2.9) were carried out. All the natural pteroylglutamic acid derivatives examined, with the exception of pteroylglutamic acid, are inhibitors. The most effective is 5-methyltetrahydrofolate (5-CH3-H4PteGlu) (KI = 3 . 10(-6) M), which is the only noncompetitive inhibitor of the enzyme. All the other derivatives tested are competitive, and H4PteGlu shows a cooperative inhibition. These and other data obtained with pteroylglutamic analogues show that, in contrast to the bacterial enzyme, Euglena transformylase is also inhibited by compounds without a fully reduced pyrazine ring and is very sensitive to compounds with a methyl group in position 5 or 10 of the pteridine ring.