Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity

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Abstract

Lactoferrin (LF) is a key modulator of inflammatory response. Since bone and immune systems are genetically and functionally linked, we were interested to know if LF could influence bone remodeling. Bovine LF (bLF) inhibited in vitro bone resorbing activity (IC50, 200 μg/ml) in a rabbit mixed bone cell culture, consisting of authentic osteoclasts in an environment of osteoblast and stromal cells. Using human CD14 selected cells committed toward osteoclasts, bLF (10 μg/ml) stimulated cell proliferation, however, led to an inhibition of calcitonin receptor mRNA expression, a main marker of osteoclast phenotype, and decreased the global resorbing activity. No modulation of RANK mRNA expression was observed and mRNA for RANKL and OPG were not detected in this culture system, suggesting that bLF inhibits osteoclastogenesis and reduces bone resorption through a mechanism independent of OPG/RANKL/RANK. In conclusion, bLF appears to modulate bone remodeling. Its mechanism of action remains to be elucidated.

Section snippets

Materials and methods

Reagents. Bovine lactoferrin (bLF) was generously provided by DMV International, Veghel, The Netherlands. Soluble recombinant human RANKL was obtained from Alexis (Alexis, Switzerland). rh-M-CSF, PBS, α-MEM, and heat-inactivated fetal calf serum (FCS) were obtained from Sigma (Fluka, Buchs, Switzerland). Plastic dishes were obtained from Corning (Milan SA, Switzerland).

Rabbit pit assay. Unfractionated bone cells were prepared from long bones from 10-day-old New Zealand rabbits (Chavornay,

Effect of bLF on in vitro bone resorption in rabbit pit assays

We have tested the effect of bLF in a rabbit pit assay system. Rabbit bone cells were cultured for 72 h in the presence of several bLF concentrations (10 μg/ml to 1000 μg/ml). Osteoclastic bone resorbing activity was significantly and dose-dependently reduced. A 50% inhibition of bone resorption was obtained with 200 μg/ml (Fig. 1).

Generation of osteoclast-like cells from human CD14 selected cells

When human CD14 selected cells were cultured on bone slices in the presence of RANKL (25 ng/ml) and M-CSF (30 ng/ml) for 8, 14, and 21 days, multinucleated calcitonin

Discussion

To our knowledge, we have shown for the first time that bovine lactoferrin decreases (i) osteoclast activity in rabbit mixed bone cell cultures (pit assay system) and (ii) osteoclast differentiation of human CD14-selected cells towards osteoclast commitment.

Among the models available to study osteoclast biology, the rabbit pit assay is useful to assess osteoclast activity but not the osteoclast differentiation process. The mixed bone cell population consists of authentic osteoclasts in an

Acknowledgements

We are grateful to Dr. H. Green, Dr. P. Guesry (Nestle Research Center) for critical reading of the manuscript, and Dr. B.O. Oyajobi (University of Texas Health Science Center at San Antonio) for helpful discussions.

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