The role played by bacterial lipopolysaccharides (endotoxins) (LPS) in the initiation of tissue damage during bacterial infections, is well established. It is accepted that LPS is released from the invading bacteria following autolysis, and the interaction of the solubilized LPS with tissues and body fluids lead to the initiation of the physiological, pharmacological and pathological sequelae seen after infections with Gram negative bacterial. Previous studies from our laboratories have shown that the binding of LPS to membranes of RBC is markedly enhanced by heat-labile leukocyte factors2, and that leukocyte factors are also capable of activating ‘LPS’ for binding to cell surfaces. Since the ‘activation’ of LPS caused by this factor \s inhibited by protease inhibitors, it was postulated that proteases present Hn leukocytes and in inflammatory exudates may enhance tissue damage by increasing the passive sensitization of mammalian cells by LPS to subsequent lysis in the presence of antibodies and complement.

FURTHER STUDIES ON THE BACTERICIDAL AND BACTERIOLYTIC EFFECTS OF HUMAN LEUKOCYTE EXTRACTS: MODULATION BY ANTIBODIES CATIONIC PROTEINS, LYSOZYME, ANTIBIOTICS AND BY AUTOLYTIC SYSTEMS AND THE ROLE PLAYED BY SOME OF THESE FACTORS IN THE EXTRACTION OF LIPOTEICHOIC ACIDS FROM BACTERIA

A heat-stable factor present in extracts of human blood leukocytes is capable of lysing young Staphylococcusaureusat pH 5.0. Lysis is character- ized by breakdown of cell-wall components as judged by electron microscopic and biochemical analysis. The leukocyte extracts can be replaced by a variety of agents known to injure cell membranes, e.g., leukocyte cationic protein histone, polymyxin B, colimycin, phospholipase A, and lysolecithin. The mechanisms by which all these agents bring about the degradation of the sta- phylococcal walls was studied. By using ~4C-labeled cell walls devoid of cytoplasmic structures, it was demonstrated that none of the above-men- tioned agents had a direct lytic effect on purified cell walls. On the other hand, when any of these agents first interacted with intact staphylococci, a factor (presumably an autolysin) was generated that directly lysed the cell walls. Lysis of cell wails in the presence of intact staphylococci used as a source of autolysin was strongly inhibited by a variety of anionic poly- electrolytes such as heparine and liquoid. The possible role olayed by bacterial autolysins in the generation of microbial cell-wall comp~,nents capable of triggering chronic inflammation is discussed.

Human leukocyte extracts, egg white lysozyme, cationic proteins, polymyxin, colimycin, and phenol are capable of releasing lipoteichoic acids (LTA) from group A streptococci and Strep. mutans. While the extraction of LTA by phenol is optimal at pH 4.7, the release of LTA from streptococci by the other agents is optimal at pH 7.4. LTA released by all agents was found to have the same sensitizing abilities, as determined by passive hemagglutination, and to have a similar chemical composition, as shown by thin-layer chromatography and radioactive scanning. The LTA-releasing capacity of all the agents is strongly inhibited by normal human serum. The possible role played by LTA released by leukocyte factors in the pathogenesis of tissue damage during bacterial infections is discussed.

Although a mass of evidence exists which supports the etiologic relationship between group A streptococcal infections and the pathogenesis of human disease (6, 36, 49, 103, 112), our knowledge of the mechanisms involved in the initiation of the tissue lesions characteristic of the sequelae of streptococcal infection in man is far from complete. The hallmarks of the poststreptococcal sequelae in humans are the development of rheumatic fever, arthritis, glomerulonephritis, chorea and other less defined clinical manifestations. Lasegue’s dictum (68) that “acute rheumatism licks the joints but bites the heart” still characterizes the significance of cardiac and joint involvement as “major” manifestations, which are related to their importance as diagnostic criteria, but do not necessarily refer to their importance in the severity of the process, its activity, or prognosis.

Although the aetiological agents responsible for the initiation of rheumatoid arthritis in the human are not known, the possibility that the disease is of bacterial origin has been considered. The bacterial factors involved may be small fragments of undegraded wall components which persist for long periods within macrophages and trigger the active release of lysosomal enzymes which cause tissue destruction. The failure to identify such wall components in diseases tissues may be due to the lack of adequate sensitive techniques to detect minute amounts of these wall components, shown to trigger chronic destructive arthritis in laboratory animals. Two models of arthritis caused by mycobacterial and streptococcal wall components are described and the possible role played by immune responses, to the persisting bacterial factors, in the pathogenesis of human arthritis is discussed.

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...

THE DETERMINATION of antistrep- tolysin 0 (ASO) in patients’ sera is most commonly performed as an aid in the diag- nosis of streptococcal infection and their sequelae.’ However, because of the differences in immune responses to a variety of streptococcal exoproducts in rheumatic fever and glomerulonephritis, it is advantageous to measure the level of more than one antis trep tococcal antibody, particularly in patients with low or borderline ASO levels. 2.3 The recently developed streptozyme test (A-STZ)l is a two-minute slide hemagglutina- tion procedure which quantitatively meas- ures multiple antibodies to streptococcal exracellular products. The reagents are sheep red blood cells sensitized simultaneously with streptolysin 0 (SLO), deoxyribonuclease B (DNase B), hyaluronidase (H), streptokinase (SK), and nicotinamide adenine dinucleotide glycohydrolase (NADG). A good correlation betweenA-STZandASOinseraofrheumatic fever patients has been demonstrated. In a comparative study’ of the STZ test with titers obtained with three of the antibody tests, ASO, ADNase B, and AH, the useful- ness of the streptozyme test for laboratories which perform only the ASO test has been demonstrated. Similar conclusions were drawn in a comparative study in our laboratory. his report deals with three main topics: 1) the streptozyme studies done in our laboratories on four of human categories sera: control group, acute pyodermal ne- tients and acute rheumatic fever patients; 2) the reproducibility and specificity of the streptozyme test; and 3) the development of ASTZ in rabbits immunized with nonviable and viable streptococci.

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.

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.

The lysis of 14C-labeled bacteria by hydrolases of human and rabbit leukocytes was studied in vitro. While Staphylococcus albus, Streptococcus faecalis, and Streptococcus mutans were highly susceptible to lysis, Staphylococcus auresus was intermediate in its susecptibility to lysis by the leukocyte enzymes. Group A Streptococcus, Listeria monocytogenes, Shigella flexneri, Escherichia coli, and Mycobacterium smegmatis were very resistant to degradation by these enzymes. The lytic activity of leukocyte lysates from human and rabbit blood was probably due to acid hydrolases of polymorphonuclear leukocytes. Extracts of human blood monocytes and of rabbit peritoneal and lung macrophages were less lytic for the bacteria tested. Lymphocytes and platelet extracts were not bacteriolytic. The lytic effect of the leukocyte lysates was not inhibited by KCN or sodium azide, but was abolished to a large extent by cationic polyelectrolytes such as protamine sulfate, histone and leukocyte cationic proteins, and poly-lysine, as well as by the anionic polyelectrolytes such as heparin, chondroitin sulfate, DNA, carrageenin, alginate sulfate, dextran sulfate, and ploy-L-glutamic acid. Other potent inhibitors of bacteriolysis were trypan blue, congo red, phosphatidic acid, normal immunoglobulins, and components of streptococcal cell wall.

The lysis of group A streptococci by muramidases of Streptomyces albus is strongly inhibited by human, rabbit, and calf serum as well as by human synovial fluids and pus. Rabbit antisera to heat-killed streptococci were no more inhibitory to the lysis of the streptococci by the lytic enzyme than normal rabbit serum. The results indicate that muramidases of S. albus will not be useful for the in vivo treatment of chronic granulomatous lesions which had been induced by insoluble cell wall components of streptococci.