Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+.

Lactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactoferrin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.

A radiolabel ratio method for measuring pulmonary clearance of intratracheal bacterial challenges.

Calculation of bacterial clearance is a fundamental step in any study of in situ lung antibacterial defenses. A method is described whereby about 85% of a radiolabeled bacterial inoculum was consistently introduced into the bronchopulmonary tree of a mouse by the intratracheal route. Mice were then killed 1 and 4 hours later; their lungs were removed aseptically and homogenized, and viable bacteria and radiolabel counts were determined. Radiolabel counts fell slowly, and more than 80% of the original radiolabel was still present in homogenized lung samples from animals sacrificed 4 hours after challenge. Bacteria/isotope ratios for the bacterial inoculum and homogenized lung samples from animals sacrificed immediately after challenge were very similar. Bacterial clearance values were the same whether computed from bacterial counts alone or according to a radiolabel ratio method whereby the change in the bacteria/isotope ratio in ground lung aliquots was divided by a similar ratio from bacteria used to inoculate animals. Some contamination resulted from oral streptococci being swept into the bronchopulmonary free during the aspiration process. This contamination was not a problem when penicillin was incorporated into the agar and penicillin-resistant strains were used for the bacterial challenges.

Release of lactoferrin by polymorphonuclear leukocytes after aerosol challenge with Escherichia coli.

Mice with cyclophosphamide-induced granulocytopenia were challenged with aerosolized Escherichia coli, their lungs were lavaged at 1 and 4 h, and total cell counts, differential counts, and levels of lactoferrin, transferrin, and albumin were measured in the lung lavage fluid. Lung lavage fluid from cyclophosphamide-treated mice had few neutrophils and no increase in lactoferrin levels, whereas control mice had significant increases in both. Transferrin levels did not change in either group. Neutrophils are the source of increased lactoferrin levels in lung lavage fluid after aerosol challenge.

Effect of aerosolized Escherichia coli and Staphylococcus aureus on iron and iron-binding proteins in lung lavage fluid.

Iron-binding proteins have antibacterial activity; they have been identified in lung secretions, but their role in pulmonary antibacterial defenses is unclear. Murine lactoferrin and murine transferrin were used to generate polyclonal antiserum to lactoferrin and to transferrin, and the specificity of both antisera was shown by western blot. Mice were exposed to either aerosolized Escherichia coli or Staphylococcus aureus; they were killed 1, 4, 24, or 48 hr later; and their lungs were lavaged. We measured the levels of transferrin, lactoferrin, and albumin and did a cell count for the lavage fluid. The predominant iron-binding protein in resting animals was transferrin. Aerosolized E. coli caused a brisk PMNL response in the lungs that was associated with a major increase in the levels of lactoferrin. Challenge with S. aureus was associated with a moderate increase in the number of macrophages and a moderate decrease in the levels of transferrin and iron but no change in the levels of lactoferrin. The levels of iron-binding protein can vary according to the type of inflammatory response.

Effect of zinc and phosphate on an antibacterial peptide isolated from lung lavage.

Incubation of Escherichia coli (10(4) organisms per ml) in cell-free rabbit lung lavage for 30 min at 37 degrees C resulted in a 70% reduction in colony counts on deoxycholate agar. A low-molecular-weight peptide (about 3,400 daltons), with zinc as a cofactor, was responsible for this activity. The peptide was isolated by Sephadex G-15 separation of lyophilized rabbit lung lavage which had been centrifuged to remove macrophages and suspended phospholipids and passed through a 10,000-dalton (pore size) membrane filter. Peptide activity against E. coli was inhibited by phosphate buffer but not by borate, Tris, or barbital buffer. Bacteria incubated in phosphate buffer and then washed in saline were resistant to peptide activity. Antibacterial activity was also inhibited when peptide-exposed bacteria were incubated in phosphate buffer before deoxycholate treatment. 32P-radiolabeled E. coli cells lost about 20% of their radiolabel after 15 min of incubation with peptide.

Sublethal damage of Escherichia coli by lung lavage.

Incubation of Escherichia coli (10(4)/ml) in cell-free rabbit lung lavage for 30 min at 37 degree C resulted in a 70% reduction in microbial counts on deoxycholate agar but no decrease on blood agar. This effect was not due to agglutination, and the order of exposure was important, i.e., activity was seen only if lavage incubation proceeded deoxycholate treatment. After high speed centrifugation of lung lavage (50,000 X g), activity remained in the supernatant and not in the surfactant pellet, Ultrafiltration of the supernatant (UM to filter) yielded an active ultrafiltrate and an inactive retent. Ultrafiltrate activity was unaffected by heating to 95 degrees C but could be removed by treatment with trypsin or bentonite. Sephadex G-15 fractionation of lyophilized ultrafiltrate yielded three active peptide peaks. Electron photomicrographs showed that incubation with the initial G-15 peak followed by deoxycholate resulted in the disappearance of intracellular material in about half the cells, a finding not seen with deoxycholate or peptide along, and EDTA reversed activity of the G-15 peptide and ultrafiltrate. Rabbit lung lavage contains a complex antimicrobial system that facilitates bile acid destruction of bacteria.

Heightened lung bactericidal activity in mice after aerosol immunization with Re 595 Salmonella minnesota: importance of cellular rather than humoral factors.

Intrapulmonary bactericidal activity was enhanced against aerosolized Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus two weeks after aerosol immunization with Re 595 Salmonella minnesota. To define better this nonspecific stimulation of antibacterial lung defenses, mice were simultaneously challenged with S. aureus and S. marcescens up to 30 days after aerosol immunization with Re. Enhanced bactericidal activity against both organisms was noted, although activity against Serratia was more pronounced during the first week after immunization. Repetitive aerosol immunization with Re also resulted in enhanced bactericidal activity. Macrophages harvested from mice after aerosol immunization were "activated" by ultrastructural criteria and had enhanced intracellular bactericidal activity against S. aureus. Aerosol immunization also caused an increase in polymorphonuclear leukocytes in lung lavage fluid, which may have been important in activity against Serratia. Mice systemically immunized with Re developed high antibody titers in serum and lung washings but had no stimulation of lung antibacterial activity.