Elmer L. Becker

Hereditary angioneurotic edema: II. Deficiency of inhibitor for serum globulin permeability factor and/or plasma kallikrein☆

Abstract Studies of a 33-year-old Caucasian woman with hereditary angioneurotic edema and her two sons, of whom only one is definitely affected with the disease, have revealed that the inborn biochemical lesion is likely an inherited deficiency of serum inhibitor to plasma kallikrein and/or serum globulin permeability factor. The patient showed a significantly greater bluing response to intradermal injections of diluted autologous serum or to plasma kallikrein than did the normal controls and this response, unlike that of the control subjects, continued to increase for a period of hours. By use of an enzymatic determination utilizing p-toluenesulfonyl-l-arginine methyl ester as the substrate, it is possible to demonstrate a deficiency in both patients to an inhibitor of plasma kallikrein. A variety of other known permeability factors have been excluded as the potential agent involved.

Demonstration of a receptor on rabbit neutrophils for chemotactic peptides

Abstract [3H]formylNorleu-Leu-Phe, a potent leucocyte chemoattractant, binds specifically to a site on rabbit neutrophils with an affinity of 1.5 × 10−9 M. Other acylated peptide chemoattractants displace this binding at concentrations closely related to those levels required to elicit chemotaxis. The binding fulfills the major criteria for demonstration of specific receptor sites and indicates that a natural bacterial product and synthetic formyl-peptides produce chemotaxis by the same receptor mechanism.

Substance P binds to the formylpeptide chemotaxis receptor on the rabbit neutrophil

Abstract Substance P, a potent vasodilatory and smooth muscle contracting agent, binds specificially to the formyl peptide receptor on the rabbit neutrophil. Substance P stimulates chemotaxis and induces lysosomal enzyme secretion in concentrations which similarily inhibit f Met-Leu-(3H)Phe receptor binding. Competitive antagonists of the formyl peptide receptor also inhibit the activity of Substance P. The finding of a naturally occurring eukaryotic peptide interacting with the neutrophil formyl peptide receptor is of importance.

In vitro studies of immunologically induced secretion of mediators from cells and related phenomena.

Publisher Summary This chapter describes the noncytotoxic release of mediators from cells induced in vitro by immunological stimuli. It discusses the release of mediators from these same cells by nonimmunological means. It focuses on demonstrating the overall resemblance of the release of mediators by immunologically induced noncytotoxic reactions to secretory processes, in general. The chapter describes the chemotaxis and phagocytosis by polymorphonuclear leukocytes; release of lysosomal enzymes from the latter cells by staphylococcal toxin; leukocidin; aggregation of platelets; and the specifically and nonspecifically induced transformation of lymphocytes. In the context of this chapter, the term “mediators” is restricted to substances whose release is directly or indirectly initiated by an immunological reaction and are responsible for one or more of the manifestations of the subsequent allergic response.

Thrombin, collagen and A23187 stimulated endogenous platelet arachidonate metabolism: differential inhibition by PGE1, local anesthetics and a serine-protease inhibitor.

Abstract The formation of malondialdehyde (MDA) and rabbit aorta contracting substance (RCS) induced by treatment of platelets with thrombin and collagen, but not that produced from exogenous arachidonic acid, is inhibited by prostaglandin E 1 (10 −8 − 10 −7 M), the local anesthetics tetracaine, SKF 525-A and dibucaine (1 mM), and the serine-protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The burst in oxygen consumption which accompanies platelet stimulation by thrombin and collagen in the presence of antimycin A, known to be due to the oxidation of endogenous arachidonate, is also markedly suppressed by PGE 1 , tetracaine and PMSF. The inhibitory effect of PGE 1 is strongly potentiated by theophylline (1.0 mM). Addition of the Ca 2+ ionophore A23187 to platelet suspensions overcomes PGE 1 and PMSF inhibition of MDA and RCS formation, and induces a vigorous increase in O 2 consumption. Tetracaine and dibucaine, however, block the responses to A23187. Formation of MDA and RCS (a mixture of PG endoperoxides and TXA 2 ) due to stimulation by thrombin and collagen depends upon activation of Ca 2+ -dependent phospholipase A 2 (PLA 2 ) to supply free arachidonate from specific membrane phospholipids. These experiments therefore indicate that increased cellular cAMP, induced by PGE 1 , antagonizes the mobilization of the Ca 2+ which is normally required for PLA 2 activity. Thrombin-stimulated platelets exhibit enhanced 45 Ca uptake which probably reflects exchange of extracellular Ca 2+ with an increased available pool of exchangeable intracellular Ca 2+ . PGE 1 strongly suppresses this 45 Ca uptake, providing more direct evidence supporting the view that cAMP prevents the rise in free cytoplasmic Ca 2+ induced by thrombin. Under conditions which make sufficient free cytoplasmic Ca 2+ available (i.e., A23187), despite high cellular cAMP, formation of RCS and MDA, and O 2 uptake are nearly normal indicating that activation of PLA 2 can occur. Local anesthetics on the other hand since they abolish the response to A23187 as well, appear to directly antagonize the ability of Ca 2+ to activate PLA 2 . The effect of PMSF suggests that stimulus-specific proteases may be involved in the thrombin and collagen-induced activation of PLA 2 activity.

Arachidonic acid induced degranulation of rabbit peritoneal neutrophils.

Abstract We have found that arachidonic acid rapidly and selectively induces the release of lysosomal enzymes from cytochalasin B treated rabbit peritoneal neutrophils. 5, 8, 11, 14-eicosatetraynoic acid inhibits the arachidonate induced release with an apparent K D of 1.5 × 10 −6 M. 5,8,11,14-eicosatetraynoic acid (2.5 × 10 −5 M also inhibits the chemotactic factors and the A23187 induced release in the presence of cytochalasin B but does not affect the degranulation induced by A23187 alone. These observations strongly suggest a role for arachidonate metabolites in rabbit neutrophil physiology.

Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells

Abstract The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.

Calmodulin inhibitors block neutrophil degranulation at a step distal from the mobilization of calcium

Summary Two calmodulin inhibitors, trifluoroperazine and N-(6-aminohexyl-5-chloro-1-naphtalene sulfonamide, were found to be potent inhibitors of the chemotactic factor plus cytochalasin B induced lysosomal enzyme release from rabbit peritoneal neutrophils. On the other hand, these inhibitors did not affect the chemotactic factor induced intracellular calcium redistribution and membrane permeability increase. These results strongly suggest that calmodulin is involved in the expression of the various neutrophil functions. In addition, these compounds provide a unique experimental tool for the dissection of the excitation-secretion coupling sequence in neutrophils and other secretory cells.

Pertussis but not cholera toxin inhibits the stimulated increase in actin association with the cytoskeleton in rabbit neutrophils: Role of the “G proteins” in stimulus-response coupling

Treatment of rabbit neutrophils with pertussis toxin, but not cholera toxin, inhibits the increases produced by formylmethionyl-leucyl-phenylalanine, leukotriene B4 and the calcium ionophore A23187 in the amounts of actin associated with the cytoskeletons. The increase in the cytoskeletal actin produced by phorbol 12-myristate, 13-acetate on the other hand is not affected by pertussis toxin. Incubation of the neutrophils with cholera toxin, unlike pertussis toxin, did not inhibit the fMet-Leu-Phe induced rise in the intracellular concentration of free calcium, and caused only a shift to the right of the dose-response curve of N-acetyl-beta-glucosaminidase release. This shift was more marked in the presence of 1-methyl-3-isobutylxanthine. In addition, the stimulated breakdown of phosphatidylinositol 4,5 bis-phosphate was inhibited by pertussis toxin. These results suggest that pertussis toxin acts at an early step in the signal transduction and does not affect the sequence of reactions initiated by the activation of the protein kinase C. Furthermore, the guanine nucleotide regulatory protein Gi, but not Gs, is closely involved in signal transduction in these cells.

Unique inhibitory profile of platelet activating factor induced calcium mobilization, polyphosphoinositide turnover and granule enzyme secretion in rabbit neutrophils towards pertussis toxin and phorbol ester

Platelet activating factor has been found to increase the intracellular level of free calcium (as monitored by the fluorescent calcium indicator quin-2) and to stimulate the turnover of the polyphosphoinositides in rabbit neutrophils. Calcium mobilization induced by platelet activating factor, in contrast to previous reports with chemotactic factors, is unaffected by pertussis toxin; on the other hand, stimulated polyphosphoinositol hydrolysis and granule enzyme secretion are potently antagonized under the same conditions. The calcium, as well as the secretory responses to the lipid mediator are largely dependent on the presence of extracellular calcium. Internal contributions to the quin-2 signal are only detectable at relatively high concentrations of platelet activating factor. Calcium mobilization and secretion stimulated by platelet activating factor are inhibited following a short incubation with phorbol 12-myristate 13-acetate. These results are discussed in terms of the possibility that platelet activating factor activates neutrophils via dual pathways, the first involving direct interaction with phorbol ester inhibitable calcium channels and the other the stimulation in a manner dependent on a guanine nucleotide binding protein of the phospholipase C specific for polyphosphoinositides.