Modulation of human lymphocyte transformation by bacterial products and leukocyte lysates.

Blast transformation of human peripheral blood lymphocytes by PHA is shown to be modulated by lipoteichoic acid (LTA) of Streptococcus mutans, by a cell-sensitizing factor of Actinomyces viscosus, as well as by a frozen and thawed extract of human leukocytes (LE). While small amounts of LE (5-50 micrograms/10(6) cells) significantly enhanced PHA-induced transformation, higher amounts showed a lesser effect on the blastogenic response. Both LTA and the A. viscosus extract did not cause any lymphocyte blastogenic effect when used alone. On the other hand LTA had an inhibitory effect and the A. viscosus extract had an enhancing effect when lymphocytes were pretreated by these agents and then exposed to PHA.

The interaction of leukocytes and their hydrolases with bacteria in vitro and in vivo: the modification of the bactericidal and bacteriolytic reactions by cationic and anionic macromolecular substances and by anti-inflammatory agents.

Acid hydrolases from extracts of human blood leucocytes lyse Staph.aureus, Staph.albus and Strep.faecalis in vitro. The leucocyte enzymes can be substituted by a lytic mixture which contains crude trypsin, lysolecithin, phospholipase C and lysozyme, which lyse other bacterial species, e.g. E.coli and Listeria which are resistant to leucocyte enzymes. Bacteriolysis by the lytic agents is strongly inhibited by the anionic polyelectrolytes, heparin, chondroitin sulphate, DNA, dextran sulphate and other sulphated mucopolysaccharides, by the cationic materials, histone, protamine sulphate, leucocyte cationic proteins and polylysine. Other strong inhibitors are trypsan blue and congo red, the phospholipids phosphatidyl serine and ethanolamine, gold thiomalate, extracts of coffee and tea and the anti-inflammatory agents, ultracorten-H, and ultracortenol. Bacteriolysis is also strongly inhibited by normal human serum and by synovial fluids from patients with a variety of joint diseases. The inhibitors in these body fluids are associated with the globulin fractions. Since mixtures of anionic and cationic polyelectrolytes, at equimolar concentrations, failed to inhibit bacteriolysis by leucocyte enzymes, it is postulated that a delicate balance between positively and negatively charged inhibitors control the degradation of cell wall components of bacteria in inflamed areas. Such bacterial components, induce 'storage type' granulomas. The possible role played by polyelectrolytes in the control of the inflammatory process induced by leucocyte hydrolases will be discussed.

The effect of leukocyte hydrolases on bacteria : VII. Bactericidal and bacteriolytic reactions mediated by leukocyte and tissue extracts and their modifications by polyelectrolytes.

Crude extracts of human blood leukocytes were employed as a source of bactericidal and bacteriolytic agents againstStaphylococcus aureus. While the bactericidal action of the extracts was a very rapid process, bacteriolysis is a very slow process. Both the killing and the lysis of staphylococci depended on the age of the culture, maximal effects being obtained only with young cells. The killing of staphylococci by the extracts was absolutely dependent on the density of bacteria employed. On the other hand, bacteriolysis was only very slightly affected when large numbers of bacteria were employed. Both the bactericidal and bacteriolytic reactions were optimal at pH 5.0. Under similar conditions, extracts of pus and the "cocktail" of enzymes were both bactericidal and bacteriolytic, but extracts of small intestine and of platelets were not significantly bactericidal. Experiments, designed to differentiate between the bactericidal and bacteriolytic properties of the extracts showed that both properties were preserved following heating in acid solutions but were completely destroyed following heating in alkaline solutions. The bactericidal factor in the lysates could be readily adsorbed on large numbers of viableStaph. aureus andE. coli, but the bacteriolytic properties of the extracts could not be removed by adsorption. The bactericidal effect of the extracts could not be inhibited by a variety of anionic polyelectrolytes, but all these agents strongly inhibited the bacteriolytic effect. Moreover, several of the anionic substances potentiated the bactericidal effects mediated by the extracts. Potentiation of these effects was also caused by protamine sulfate and by polylysine, which were highly bactericidal by themselves. The only substance that was found to abolish the bactericidal effects of the extracts is ultracorten H. Historie and polylysine (which are highly bactericidal) lost their effects when mixed with certain concentrations of heparin or polyglutamic acid, which by themselves are not bactericidal, indicating that an appropriate balance between cationic and anionic substances may determine the bactericidal effects of cationic substances. Since the bactericidal properties of the lysates could not be abolished by any of the anionic macromolecular substances employed; it is suggested that the bactericidal agents present in crude whole lysates of leukocytes comprise a complex mixture of agents, some of which are not identical with cationic substances. Thus, the data suggest that the employment of highly purified cationic proteins of leukocytes and tissues to study bactericidal models may not reflect the actual conditions that prevail in inflammatory exudates. The possible role played by cationic and anionic polyelectrolytes in the control of bacterial survival and lysis in inflammatory exudates is discussed.

The effect of leukocyte hydrolases on bacteria : IV. The role played by artificial enzyme "cocktails" and tissue enzymes in bacteriolysis.

Acid hydrolases of human blood leukocytes are highly lytic toStaph. albus, Staph. aureus, andStrep. faecalis. On the other hand, group A and viridans streptococci, encapsulated staphylococci, a variety of Gramnegative rods, andMyc. smegmatis are highly resistant to lysis by leukocyte extracts. The lytic effect of the leukocyte extracts can be mimicked by an artificial "cocktail" which contains crude trypsin, lysolecithin, phospholipase C, and lysozyme. This enzyme mixture is lytic to certain Gram-negative bacteria and encapsulated staphylococci which are resistant to lysis by leukocyte enzymes. Both the leukocyte lysates and the artificial cocktail are more lytic to bacteria harvested from the logarithmic phase of growth than to older cells.Staph. albus andStrep. faecalis, which are not lysed to any appreciable extent by extracts of rabbit intestines, lymphocytes, and platelets, undergo extensive lysis upon the addition of lysozyme, indicating that these cells contain preparatory prolytic agents which are activated by lysozyme. On the other hand, the lysis ofStaph. aureus by extracts of all these cells is less dependent upon lysozyme, indicating that other non-lysozyme-dependent lytic factors are involved in the lysis of this microorganism by certain tissue extracts. It is suggested that the resistance to lysis by leukocyte enzymes of bacterial cell-wall constituents may contribute to the pathogenesis of chronic sequellae, and that artificial enzyme cocktails be used for in vivo treatment of certain chronic inflammatory processes induced by bacteria.

The effect of leukocyte hydrolases on bacteria : V. Modification of bacteriolysis by antiinflammatory agents and by cationic and anionic polyelectrolytes.

Lysis of(14)C-labeledStaph. aureus by human blood leukocyte lysates, by extracts of rabbit small intestines and pancreas, and by the "cocktail" of enzymes (containing trypsin, lysolecithin, and lysozyme) is strongly inhibited by anionic polyelectrolytes (e.g., heparin, chondroitin sulfate, liquoid (polyanethole sulfonic acid), and DNA). Most of the lytic agents employed were inhibited by cationic polyelectrolytes (e.g., histone, protamin sulfate and polylysin), as well as by gold thiomalate, normal human serum, synovial fluids obtained from patients with knee-joint trauma, extracts of coffee, tea, and cocoa, Ultracorten- and Dexamethasone. On the other hand, some antiinflammatory agents tested (e.g., indomethacin, aspirin, hydrocortisone acetate and succinate, and prednisolone acetate and tributyl acetate) were not inhibitory. All the cationic polyelectrolytes employed and liquoid were also strong inhibitors of lysozyme. Since mixtures of cationic and anionic polyelectrolytes at equimolar concentrations failed to inhibit bacteriolysis, it is postulated that the balance between charged macromolecular substances, which are likely to accumulate in inflammatory foci, may determine the fate of cellular components of bacteria in inflamed tissues. The possible role played by lysosomal enzymes and by tissue inhibitors in tissue damage and in the survival of bacteria in chronic inflammatory lesions is discussed.