Purification and properties of rabbit alveolar macrophage lysozyme

We measured the microbicidal peptides MCP-1 and MCP-2 in rabbit alveolar macrophages (AM), comparing rabbits pretreated with complete Freund adjuvant with untreated control animals. Levels of MCP-1 increased from 4.7 ± 0.6,ug/107 resident AM to 13.2 ± 0.1 jig/107 complete Freund adjuvant-elicited AM. MCP-2 levels rose from 1.8 ± 0.1 ,ug/107 resident AM to 7.3 ± 0.4 ,ug/107 complete Freund adjuvant-elicited AM. The activities of five lysosomal hydrolases (,B-D-glucuronidase, ,B-D-galactosidase, acid phosphatase, N-acetyl-,f-D-galactosaminidase, and N-acetyl-fB-D-glucosaminidase) were 44 to 96% higher in complete Freund adjuvant-elicited AM, and lysozyme activity was threeto fourfold higher. As MCP-1 and MCP-2 are major constituents of rabbit AM and exhibit potent antibacterial and antifungal properties, they may contribute to the expression of microbicidal activity in both resident and activated states. Alveolar macrophages (AM) from rabbits pretreated with complete Freund adjuvant (CFA) kill ingested Candida albicans more rapidly or effectively than do resident alveolar or peritoneal macrophages (13). The candidacidal mechanisms of rabbit AM differ from those described in other mammalian phagocytes by being independent of peroxidase-mediated metabolic processes (12, 13). Recently, we described the presence of two microbicidal components, MCP-1 and MCP-2, in rabbit AM (16, 17). These highly cationic

Section: Distribution Of Proteinsupporting

confidence: 93%

Increased content of microbicidal cationic peptides in rabbit alveolar macrophages elicited by complete Freund adjuvant

Lehrer¹,

Szklarek²,

Selsted³

et al. 1981

“…The incubation mixture for the bactericidal assay consisted of blood fractions and inhibitors, where appropriate, and glucose-phosphate buffer containing 0.1% bovine serum albumin, to a volume of 0.9 ml. The addition of bovine serum albumin to reaction mixtures was found to enhance the stability of bactericidal serum fractions, as found previously for dilute lysozyme solutions (6), without affecting cell viability or resultant killing kinetics. After a 2-mmi preincubation at 370C, the experiment was initiated by adding 0.1 ml of the stock cell suspension (zero time; final concentration, 2 x 107 cells per ml).…”

Section: Materlals and Methodssupporting

confidence: 73%

“…Purification of rabbit lysozyme. Rabbit lysozyme was isolated from elicited alveolar macrophages and purified to homogeneity as previously described (6). Purified human leukemic lysozyme was a generous gift from Michael E. Selsted.…”

Section: Materlals and Methodsmentioning

confidence: 99%

“…Acrylamide gel electrophoresis and determination ofprotein. Analytical sodium dodecyl sulfatepolyacrylamide gradient gel electrophoresis was performed as previously described (6). Aqueous stock lysozyme solutions were prepared, using an El"Cm (280 nm) of 22.5 for rabbit lysozyme (6) and an El"Cm (280 nm) of 25.5 for both human (5) and hen egg white (33) lysozymes.…”

Section: Materlals and Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Role of Rabbit Lysozyme in In Vitro Serum and Plasma Serum Bactericidal Reactions Against Bacillus subtilis

Carroll

Martı́nez

1979

The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of "purified /3-lysin," fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified ,B-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components.

“…The individual fractions were frozen and thawed six times, and samples were removed to assay the following enzymes. (i) Lysozyme was measured by the method of Carroll and Martinez (8). (ii) ,-Galactosidase was measured by using ortho-nitrophenyl-fl-D-galactopyranoside (Sigma) as substrate in reaction mixtures containing in a total volume of 1 ml: 0.5 mg of ortho-nitrophenyl-,B-D-galactopyranoside, 0.1% Triton X-100, 0.01 M acetate buffer, pH 5.0, and 0.1 ml of sample was added to start the reaction.…”

mentioning

confidence: 99%

Microbicidal cationic proteins in rabbit alveolar macrophages: a potential host defense mechanism

Patterson-Delafield¹,

Martínez²,

Lehrer³

1980

Self Cite

143

Rabbit alveolar macrophages contain two highly cationic microbicidal proteins. These were shown to be distinct from histones and not to arise from granulocyte contamination. The macrophage proteins were especially active against Candida albicans and Candida parapsilosis. Gram-positive bacteria (Bacillus subtilis, Listeria monocytogenes, and Streptococcus faecalis) were also susceptible, whereas Escherichia coli and Salmonella typhimurium appeared more resistant. The proteins may be present in lysosomes, based on their solubilization by dilute acids and their distribution with lysosomal markers on sucrose density gradients. Such microbicidal proteins have not previously been demonstrated in any mammalian macrophage. They may play a significant role in the host-defense functions of the rabbit lung. The function of alveolar macrophages in host defense is well documented, and numerous reports describe their microbicidal proficiency (recently reviewed by Hocking and Golde [21]). Relatively little is known concerning the microbicidal mechanisms used by alveolar macrophages (4, 23). Recent studies in our laboratory (26) demonstrated that the mechanism(s) whereby rabbit alveolar macrophages killed Candida albicans differed substantially from the myeloperoxidase-H202-mediated antifungal systems predominantly operative in human monocytes (24) and neutrophils (25). Rabbit granulocytes have been shown to contain microbicidal lysosomal cationic proteins (41-43), certain of which kill C. albicans (27). As macrophages and granulocytes are thought to arise from a common stem cell (21), and as these cells contain certain constituents in common (e.g., lysozyme), we sought the presence of microbicidal cationic proteins with candidacidal activity in rabbit alveolar macrophages. This communication describes our findings. MATERIALS AND METHODS Cell preparations and fractionation. (i) Isolation of pulmonary macrophages. Macrophages were obtained by ravaging the airways of 2-to 3-kg New Zealand white rabbits according to the procedure of Myrvik (34) with the following modifications: (i) the lungs were ravaged in situ after the animal had been sacrificed by intravenous injection of 180 mg of sodium