Comparative effects of azapropazone on cellular events at inflamed sites. Influence on joint pathology in arthritic rats, leucocyte superoxide and eicosanoid production, platelet aggregation, synthesis of cartilage proteoglycans, synovial production and actions of interleukin-1 in cartilage resorption correlated with drug uptake into cartilage in-vitro. Azapropazone (APZ) has been compared with standard NSAIDs in title systems to establish aspects of its mode of action on cellular events at inflamed sites. APZ (150 mg kg-1 day-1) given for 10-13 days exhibited a reduction in joint pathology in established adjuvant arthritis in rats comparable with that of indomethacin (2 mg kg-1 day-1) and clobuzarit (20 mg kg-1 day-1). APZ was shown to be a potent inhibitor of the production of leucocyte superoxide and synovial interleukin-1 (IL-1)-like activity and stimulated articular cartilage proteoglycan synthesis, but was ineffective as an inhibitor of platelet aggregation or IL-1 induced cartilage degradation in-vitro. These in-vitro effects may have relevance to the mode of action of this weak inhibitor of prostaglandin synthesis.

Previous studies have demonstrated that a number of membrane-active agents are capable of binding to the surface of polymorphonuclear leukocytes (PMN) resulting in an augmentation of superoxide anion and hydrogen peroxide (H2O2) production in response to soluble stimuli. It is now demonstrated that these same membrane-active agents can bind to the surface of endothelial cells and enhance their susceptibility to killing by H2O2. Membrane-active agents which are capable of synergizing with H2O2 include cationic proteins, cationic poly-amino acids, lysophosphatides and enzymes which are capable of degrading membrane phospholipids (e.g., phospholipase C, phospholipase A2 and streptolysin S). In each case, treatment of the target cells with the membrane-active agent and H2O2 produces greater damage than the sum of the damage produced by either agent separately. Since inflammatory lesions, particularly sites of bacterial infection, may contain a rich mixture of cationic substances, phospholipases and phospholipid breakdown products, these substances may contribute to the tissue damage observed at sites of inflammation by enhancing endothelial cell sensitivity to PMN-generated H2O2 as well as by augmenting the generation of H2O2 by PMNs.

Insoluble complexes of poly-L-histidine (polyhistidine) and catalase were prepared by mixing the two reactants together in solution at pH 5.5 and subsequently elevating the pH to approximately 7.0, at which point they precipitated. Complexes formed at optimal ratios of polyhistidine to catalase contained essentially all of the catalase present in the original solution. The catalase present in such complexes contained greater than 50% of the H2O2-inhibiting activity of the native catalase used to prepare the complexes. The insoluble complexes rapidly bound to viable endothelial cells and were resistant to removal by extensive washing. The presence of polyhistidine-catalase complexes on the cell surface protected the cells against injury mediated by H2O2 or activated polymorphonuclear leukocytes. These data show that polyhistidine-catalase complexes can be prepared that have a high affinity for cells and that retain catalase activity. These complexes may be useful in treating inflammatory conditions in which it is necessary to maintain a high local concentration of inhibitor.

Human neutrophils which are pretreated with subtoxic concentrations of a variety of lysophosphatides (lysophosphatidylcholine, lysophosphatidylcholine oleoyl, lysophosphatidylcholine myrioyl, lysophosphatidylcholine stearoyl, lysophosphatidylcholine gamma-O-hexadecyl, lysophosphatidylinositol, and lysophosphatidylglycerol) act synergistically with neutrophil agonists phorbol myristate acetate, immune complexes, poly-L-histidine, phytohemagglutinin, and N-formyl-methionyl-leucyl-phenyalanine to cause enhanced generation of superoxide (O2-). None of the lyso compounds by themselves caused generation of O2-. The lyso compounds strongly bound to the neutrophils and could not be washed away. All of the lyso compounds that collaborated with agonists to stimulate O2- generation were hemolytic for human red blood cells. On the other hand, lyso compounds that were nonhemolytic for red blood cells (lysophosphatidylcholine caproate, lysophosphatidylcholine decanoyl, lysophosphatidylethanolamine, lysophosphatidylserine) failed to collaborate with agonists to generate synergistic amounts of O2-. However, in the presence of cytochalasin B, both lysophosphatidylethanolamine and lysophosphatidylserine also markedly enhanced O2- generation induced by immune complexes. O2- generation was also very markedly enhanced when substimulatory amounts of arachidonic acid or eicosapentanoic acid were added to PMNs in the presence of a variety of agonists. On the other hand, neither phospholipase C, streptolysin S (highly hemolytic), phospholipase A2, phosphatidylcholine, nor phosphatidylcholine dipalmitoyl (all nonhemolytic) had the capacity to synergize with any of the agonists tested to generate enhanced amounts of O2-. The data suggest that in addition to long-chain fatty acids, only those lyso compounds that possess fatty acids with more than 10 carbons and that are also highly hemolytic can cause enhanced generation of O2- in stimulated PMNs.

Monolayers of murine fibrosarcoma cells that had been treated either with histone-opsonized streptococci, histone-opsonized Candida globerata, or lipoteichoic acid-anti-lipoteichoic acid complexes underwent disruption when incubated with human polymorphonuclear leukocytes (PMNs). Although the architecture of the monolayers was destroyed, the target cells were not killed. The destruction of the monolayers was totally inhibited by proteinase inhibitors, suggesting that the detachment of the cells from the monolayers and aggregation in suspension were induced by proteinases releases from the activated PMNs. Monolayers of normal endothelial cells and fibroblasts were much resistant to the monolayer-disrupting effects of the PMNs than were the fibrosarcoma cells. Although the fibrosarcoma cells were resistant to killing by PMNs, killing was promoted by the addition of sodium azide (a catalase inhibitor). This suggests that the failure of the PMNs to kill the target cells was due to catalase inhibition of the hydrogen peroxide produced by the activated PMNs. Target cell killing that occurred in the presence of sodium azide was reduced by the addition of a "cocktail" containing methionine, histidine, and deferoxamine mesylate, suggesting that hydroxyl radicals but not myeloperoxidase-catalyzed products were responsible for cell killing. The relative ease with which the murine fibrosarcoma cells can be released from their substratum by the action of PMNs, coupled with their insensitivity to PMN-mediated killing, may explain why the presence of large numbers of PMNs at the site of tumors produced in experimental animals by the fibrosarcoma cells is associated with an unfavorable outcome.

Killing of rat pulmonary artery endothelial cells by activated polymorphonuclear leukocytes (PMNs), as measured at 4 hours, is catalase sensitive, iron dependent, and unaffected by addition of protease inhibitors. If the time course for exposure of endothelial cells to activated PMNs is extended to 18 hours, progressive injury occurs. Endothelial cell injury resulting at 18 hours is partially inhibited by catalase and partially inhibited by soybean trypsin inhibitor. Together, these two inhibitors function synergistically to protect the cells from injury. Exposure of endothelial cells to reagent H2O2 and purified proteolytic enzymes (trypsin, chymotrypsin, elastase, and cathepsin G) mimics the effects of activated PMNs: H2O2 alone is cytotoxic with maximal killing achieved by 4 hours; proteolytic enzymes produce cytotoxicity only at high concentrations and only after prolonged incubation (longer than 8 hours); and, in combination, H2O2 and proteolytic enzymes act synergistically. These data provide compelling evidence that PMN-mediated injury of endothelial cells involves interaction between oxygen products and proteases.

Bacteria and yeasts which are "opsonized" with cationic polyelectrolytes (poly-L-arginine, poly-L-histidine and arginine-rich histone) are avidly endocytosed by both "professional" and "non-professional" phagocytes. The cationized particles also strongly activate the respiratory burst in neutrophils and in macrophages leading to the generation of chemiluminescence, superoxide and hydrogen peroxide. On the other hand, lysine and ornithine-rich polymers are poor opsonic agents. Poly L-arginine is unique in its capacity to act synergistically with lectins, with chemotactic peptides and with cytochalasin B to generate large amounts of chemiluminescence and superoxide in human neutrophils. Unlike polyarginine, polyhistidine, in the absence of carrier particles, is one of the most potent stimulators of superoxide generations, known. Neutrophils treated with cetyltrimethylammonium bromide fail to generate superoxide, but generate strong luminol-dependent chemiluminescence which is totally inhibited by sodium azide and by thiourea. Neutrophils injured by cytolytic agents (saponin, digitonin, lysolecithin) lose their chemiluminescence and superoxide-generating capacities upon stimulation by a variety of ligands. These activities are however regained by the addition of NADPH. Lysolecithin can replace polyarginine in a "cocktail" also containing lectins and cytochalasin B, which strongly activate the respiratory burst. This suggests that polyarginine acts both as a cytolytic agent and as a ligand. Arginine and histidine-rich polyelectrolytes enhance the pathogenic effects of immune complexes in vivo (reversed Arthus phenomenon) presumably by "glueing" them to tissues. Polyhistidine complexed to catalase or to superoxide dismutase, markedly enhances their efficiency as antioxidants. On the other hand polyhistidine complexed to glucose oxidase markedly enhances injury to endothelial cells suggesting that the close association of the cationized enzyme with the plasma membrane facilitates the interaction of hydrogen peroxide with the targets. A variety of cationic agents (histone, polyarginine, polyhistidine, polymyxin B) and membrane-active agents (lysophosphatides, microbial hemolysins) act synergistically with glucose oxidase or with reagent hydrogen peroxide to kill target cells. The mechanisms by which arginine- and histidine-rich polyelectrolytes activate the respiratory burst in neutrophils might involve interaction with G-proteins, the activation of arachidonic acid metabolism and phospholipase A2, or the interaction with myeloperoxidase. Naturally-occurring cationic proteins might modulate several important functions of leukocytes and the course and outcome of the inflammatory process.

Treatment of Staphylococcus aureus in vitro with cationic agents results in the activation of their autolytic wall enzymes and in the degradation of their cell walls. Exposure of staphylococci either to hydrogen peroxide or the proteinases abolished the autolytic process. This effect was totally reversed by catalase and by proteinase inhibitors, respectively. It is suggested that the failure of neutrophils and macrophages to effectively degrade microbial cell wall components in inflammatory sites might be due to the inactivation of the autolytic wall enzymes of bacteria by hydrogen peroxide and by proteinases generated by the activated leukocytes. This might explain the prolonged chronic inflammatory sequelae seen following infections.

Cationic particles interact by electrostatic forces with membrane components of diverse cell types, including lymphocytes. Contact with cationized streptococci was shown to induce a murine T-cell hybridoma to transcribe lymphokine mRNA as well as secrete interleukin-2. This activation was accompanied by a rise in intracellular calcium. Cationized streptococci-induced activation of this T-cell hybridoma could be specifically inhibited by either chelating extracellular calcium or by treating with CD4 monoclonal antibody. These data indicate that the in vitro behaviour of T cells can be modulated by charged microbial particles; such interactions may have relevance for chronic inflammation associated with some bacterial infections.