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.

Cultures ofStaphylococcus aureus, which are harvested from the stationary phase of growth, are extremely resistant to lysis by extracts of human blood leukocytes. Such bacteria are, however, rendered susceptible to bacteriolysis when cultivated in the presence of subinhibitory concentrations of penicillin G, nafcillin, or cloxacillin (0.05μg/ml). The lytic effect of the leukocyte extracts on the penicillin-grown bacteria is further augmented by the addition of egg-white lysozyme. Staphylococci, which are harvested from the logarithmic phase of growth in ordinary media, are susceptible to lysis by leukocyte extracts, maximal lysis being achieved with about 100μg/ml of leukocyte extracts. On the other hand, penicillin-grown staphylococci are lysed by much smaller amounts of leukocyte extracts (20μg/ml), and much shorter periods of incubation are needed to achieve maximal lysis. Similar results are obtained when the leukocyte extracts are substituted by a cocktail of lytic agents which contain crude trypsin, lysolecithin, and lysozyme. Lysis of the staphylococci by the leukocyte extracts, fortified by lysozyme, is optimal at pH 5.0 and is accompanied by the solubilization of the bulk of glucosamine, known to be mostly concentrated in the peptidoglycan of the cell wall. Penicillin-grown staphylococci are also more susceptible than controls to lysis by a mixture of histone and lysozyme. The lysis, by leukocyte extracts and by the cocktail of both regular and penicillin-grown staphylococci, is strongly inhibited to the same extent by heparin, liquoid, histone, protamine sulfate, IgG, and human serum. On the other hand, no inhibition of lysis is achieved by chloramphenicol, streptomycin, erythromycin, KCN, HgCl2, or by neutral polyelectrolytes. Group A streptococci, which are extremely resistant to degradation by leukocyte extracts or by the cocktail, when harvested from any phase of growth, also become susceptible to lysis by leukocyte extracts or by the cocktail when grown in the presence of small amounts of penicillin (0.004-0.008μ/ml). Bacteriolysis became even more pronounced when the reaction mixtures were incubated at 41 °C, a temperature likely to develop in patients with streptococcal infections. Electron-microscope examination of the staphylococci following treatment with leukocyte enzymes and penicillin revealed that both cell wall and cytoplasmic structures were severely damaged by the lytic agents. The mechanisms by which penicillin exposes the bacterial cell walls to cleavage by leukocyte extracts is discussed, and the phenomenon of enhanced susceptibility to lysis by leukocyte enzymes is related to the role played by undegraded bacterial constituents in the initiation of chronic inflammatory lesions.

The effect of proteases and of extracts of human blood leukocytes and platelets on the sensitization of human red blood cells (RBC) by lipopolysaccharides (LPS) and by the cell-sensitizing factor (SF) of group A streptococci, as determined by passive hemagglutination, was studied. While treatment of RBC by trypsin, papain (10-1500Μg/ml), and plasmin markedly increased the binding of SF to RBC as determined by the passive hemagglutination test, small amounts of leukocyte and platelet extracts (25Μg protein) failed to enhance the sensitization of RBC. On the other hand, high concentrations of leukocyte extracts (>250Μg protein) destroyed, to a large extent, the capacity of SF to sensitize RBC. The inhibitory effect of the leukocyte extracts on the SF system was optimal at neutral pH and was inhibited by heat treatment, by phenylmethyl sulfonyl fluoride (PMSF), and by liquoid, indicating the participation of neutral proteases in this reaction. Treatment of LPS with small amounts of leukocyte extracts activated the LPS molecule; this treatment could replace the alkaline treatment needed to enhance the capacity of LPS to sensitize RBC. Very high hemagglutination titers were, however, obtained when both LPS and RBC were simultaneously treated with leukocyte extracts (25Μg protein). On the other hand, larger amounts of extracts destroyed receptors for LPS on RBC. Both the enhancing and destroying capacities of the leukocyte enzyme on the LPS system were abolished by PMSF. The simultaneous sensitization of RBC by SF and LPS showed that SF is a more dominant sensitizing agent. Histone blocked receptors in RBC for both SF and LPS. The effect of the histone was abolished by trypsin. Histone also strongly bound LPS and SF and abolished to a large extent their cell-sensitizing properties. The possible role played by leukocyte extracts in the initiation of tissue damage induced by cell-sensitizing products of bacteria is discussed.

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 Role of Leukocytes and their Hydrolases in the Persistence, Degradation, and Transport of Bacterial Constituents in Tissues: Relation to Chronic Inflammatory Processes in Staphylococcal, Streptococcal, and Mycobacterial Infections and in Chronic Periodontal Disease Read More: http://informahealthcare.com/doi/abs/10.3109/10408417609106944?journalCo...