Lactoferrin stimulates killing and clearance of bacteria but does not prevent mortality of diabetic mice.

We have previously shown that bovine lactoferrin (BLF) given intravenously (i.v.) protected mice against a lethal dose of Escherichia coli and strongly stimulated both the clearing and killing activities in liver, lungs, spleen and kidney. Since some studies indicated a reduction of the manifestation of experimental pancreatitis with lactoferrin (LF), we decided to examine the protective activity of BLF against lethal E. coli infection in animals with alloxan (Alx)-induced diabetes. It appeared that 48 h diabetes substantially lowered the killing activity in all four organs as well as the clearing rate of E. coli from the circulation. BLF given i.v. reduced this undesirable effect of diabetes. However, in 10- and 20-day diabetic animals, the diabetes alone stimulated the killing activity in the organs investigated, and upregulated the clearing rate of E. coli from the circulation. Lactoferrin significantly increased both the killing and the clearing activity in these long-term diabetic animals. In some cases the stimulating effect of BLF was very high, suggesting a concerted action of BLF and diabetes in that category of mice. Despite these beneficial effects of BLF and diabetes on the killing process in the investigated organs, the survival time of animals from all the diabetic groups (48 h, 10 and 20 days) was not prolonged by BLF. The protective properties of BLF did not depend on the blood glucose levels in the diabetic animals. BLF partly delayed the development of experimental Alx-induced diabetes, measured by the glucose level, but only if administered shortly after Alx injection. In conclusion, we demonstrated that the state of diabetes alone could increase killing of bacteria in the investigated organs and LF enhanced this process. However, LF had no protective effect against the mortality of diabetic mice infected with a lethal dose of E. coli.

Antibacterial system generated by lactoferrin in mice in vivo is primarily a killing system.

A single dose of bovine lactoferrin (BLF) was given intravenously (i.v.) to CFW mice 24 hours (h) prior to the i.v. injection of the E. coli lethal dose (LD100). BLF strongly accelerated the clearance rate of E. coli from the blood as well as its killing rate in the liver, lungs, spleen and kidney. The highest clearing and killing rate was found 5 h after E. coli LD100 injection. The most intensive killing in the organs examined was found in the lungs and kidney. Analysis of organs of i.v. BLF-stimulated mice which survived up to day 30 after the infection by E. coli showed that not all animals were definitely pathogen-free. It was concluded that the defense system generated by BLF in mice in vivo is primarily a bacteria-killing one. The participation and cooperation of reticulo-endothelial (RE)-macrophages and granulocytes in the phagocytosis and killing of E. coli may thus be related to the protective activity of LF.

Lactoferrin lowers serum interleukin 6 and tumor necrosis factor alpha levels in mice subjected to surgery.

Mice subjected to thymectomy or splenectomy in general anesthesia release interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) into circulation reaching high concentrations after 4 h following operation. In the case of thymectomy IL-6 can be detected only on the day of operation and TNF-alpha attains a maximal value on day 3 postoperation. Splenectomy, which is a more extensive surgical operation, results in a higher, and more prolonged existence of IL-6 in circulation accompanied by higher levels of TNF-alpha. Bovine lactoferrin (BLF; 10 mg/mouse), given intravenously (i.v.) 24 h before thymectomy, reduced, on average, the level of serum IL-6 by 70% as measured 4 h after operation. The inhibiting effect of BLF on TNF-alpha production was smaller with a mean 30% reduction. The effects of BLF (i.v.) administration on the cytokine levels following splenectomy were less inhibitory. BLF caused an approximate 35% fall in IL-6 levels and even weaker effects (20% inhibition) on TNF-alpha release. Application of much lower (1-0.2 mg) per os doses of BLF was even more effective in lowering IL-6 levels after thymectomy (up to 90%) after 5 BLF doses, and by 55% of TNF-alpha. The data suggest that lactoferrin may find therapeutical application for diminishing manifestations of shock caused by clinical insults.

Lactoferrin regulates the release of tumour necrosis factor alpha and interleukin 6 in vivo.

The effects of bovine lactoferrin on the serum cytokine levels, induced by lipopolysaccharide (LPS) in mice, are described. Bovine lactoferrin (BLF) introduced intravenously, 24 hours before i.v. injection of 50 micrograms of LPS, significantly lowered the serum concentration of TNF-alpha. Doses of BLF lower than 100 micrograms as well as pretreatment of mice with BLF on days 6-2 or 12-2 hours before LPS challenge, were not effective. Moreover, BLF induces by itself a relatively high level of IL-6, peaking at 1 hour following injection. Pretreatment of LPS-injected mice with BLF causes, in addition, a small but statistically significant drop in IL-6 level. Human albumin, used as a control protein, did not cause any changes in the cytokine levels. The data reported herein provide a satisfactory explanation with regard to preventive activity of LF in infection.

Protective effect of tissue ferritins in experimental Escherichia coli infection of mice in vivo.

The effect of ferritins from horse (FH) and bovine (FB) spleen and murine liver (FM) on the survival rate of CFW mice lethally infected with Escherichia coli (strain 8440-78 K 80/B) was evaluated. Ferritins given intravenously 24 h before intravenous inoculation of bacteria, protected mice most effectively from death due to infection. The effect was dose dependent. At 500 micrograms of ferritin per mouse, the maximum survival rates were 86% (FH), 81% (FM) and 79% (FB), while only 5% of the control mice survived up to the 30th day. The survival rates of animals injected with bovine serum albumin (BSA) and heat-inactivated FB were 8 and 25%, respectively. Intraperitoneal injection of FB was as effective as intravenous in enhancing the resistance of mice against bacteria. These data provide evidence for the beneficial role of tissue ferritins in nonspecific antibacterial resistance.

Lactoferrin can protect mice against a lethal dose of Escherichia coli in experimental infection in vivo.

Experiments were undertaken to demonstrate and partially explain the protective effect of bovine lactoferrin (LB) when administered intravenously to mice 24 h before a challenge with a lethal dose of Escherichia coli. About 70% of mice pretreated with LB survived challenge. The survival rates in control mice treated with E. coli alone and pretreated with bovine serum albumin (BSA), were 4 and 8%, respectively. Human lactoferrin (LH) had almost the same protective effect as LB. Sufficient amounts of ferric ions were given to mice, in single and multiple doses, for full serum transferrin saturation 30 min before or after E. coli administration. The multiple dose of ferric ions did not change considerably the survival rate of mice pretreated with LB. In contrast, a single dose of ferric ions gradually decreased the survival rate of the mice after the first week of experiment. From day 14 this decrease was statistically significant in all groups of mice treated with a single dose of ferric ions when compared with mice pretreated only with LB, and the difference ranged from 25 to 35% on day 30. The possible mechanism(s) of protective effect of LB and role of iron ions are discussed.