Immunomodulatory effects of lactoferrin on antigen presenting cells

Lactoferrin (Lf) is an 80 kDa iron-binding protein of the transferrin family that is abundantly expressed in most biological fluids. It is now recognized that this glycoprotein is a key element in the mammalian immune system, playing an important role in host defence against infection and excessive inflammation. Although the mechanisms underlying Lf immunomodulatory properties have not been fully elucidated yet, evidence indicates that the capacity of this molecule to directly interact with antigen presenting cells (APCs), i.e. monocytes/macrophages and dendritic cells (DCs), may play a critical role. At the cellular level, Lf modulates important aspects of APC biology, including migration and cell activation, whereas at the molecular level it affects expression of soluble immune mediators, such as cytokines, chemokines and other effector molecules, thus contributing to the regulation of inflammation and immunity. While the iron-binding property was originally believed to be solely responsible for the plethora of host defence activities ascribed to Lf, it is now known that other mechanisms contribute to the broad spectrum of anti-infective and anti-inflammatory properties of this protein. Recent results suggest that at least some of the immunomodulatory effects of Lf rely on its capacity to form complexes with lipopolysaccharide (LPS). This review focuses on the effects of Lf on APC biology and function, highlighting known and putative mechanisms that underlie Lf immunomodulatory effects. The importance of LPS-binding capacity of Lf and LPS receptors, as well as of Lf-induced type 1 interferon (IFN) expression in some of these effects is also discussed.     

Role of lactoferrin in the tear film

The surface of the eye provides an inert barrier against infection. Through its unique combination of antimicrobial action and anti-inflammatory activities lactoferrin (Lf) in the tear film plays an important role in the maintenance of ocular health. In order to maintain clarity the eye must provide immunological defense without immunopathology. Along with physical barriers, soluble plasma factors and other proteins such as lysozyme, Lf produced by the acinar cells of the lacrimal gland serves a number of roles in defense for this purpose. Lf in tears provides antimicrobial efficacy by binding free iron thus reducing the availability of iron necessary for microbial growth and survival as well as pathogenesis. Lf has been shown to inhibit biofilm formation and thus may play a role in protecting contact lens surfaces from colonization. Virus particles' entry into epithelial cells is inhibited by Lf while an excess of Lf in tear film is thought to limit the opportunistic Lf-mediated bridging of adenovirus and host cell that occurs in other tissues. Lf dampens the classical complement activation pathway by binding to markers of inflammation and immune activation while pathogen-associated molecular patterns such as lipopolysaccharide (LPS) are targeted by Lf for removal through tears and hydrodynamic flushing. This review focuses on the role of Lf in human tear film and its contribution to ocular health during contact lens wear.     

Molecular Cloning and Expression of Yak (Bos grunniens) Lactoferrin cDNA in Pichia pastoris

cDNA encoding lactoferrin from yak was isolated by RT-PCR and then sequenced. The cloned cDNA (2127 bp) encodes a 709 amino acid precursor molecule of yak lactoferrin with a signal peptide of 19 amino acids. The yak lactoferrin cDNA was expressed in Pichia pastoris. The recombinant protein, purified by Ni-NTA affinity column, had a molecular weight of 76 kDa and reacted with an antibody raised against native bovine lactoferrin. The iron-binding behavior and antimicrobial activity of the purified protein indicated that it was correctly folded and functional.     

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity

Synthetic peptides derived from human and bovine lactoferricin, as well as tritrpticin sequences, were assayed for antimicrobial activity against wild-type Escherichia coli and LPS mutant strains. Antimicrobial activity was only obtained with peptides derived from the bovine lactoferricin sequence and peptides corresponding to chimeras of human and bovine sequences. None of the peptides corresponding to different regions of native human lactoferricin showed any antimicrobial activity. The results underline the importance of the content of tryptophan and arginine residues, and the relative location of these residues for antimicrobial activity. Results obtained for the same assays performed with LPS mutants suggest that lipid A is not the main binding site for lactoferricin which interacts first with the negative charges present in the inner core. Computer modelling of the most active peptides led to a model in which positively charged residues of the cationic peptide interact with negative charges carried by the LPS to disorganise the structure of the outer membrane and facilitate the approach of tryptophan residues to the lipid A in order to promote hydrophobic interactions.     

Variation in antimicrobial activity of lactoferricin-derived peptides explained by structure modelling

Antimicrobial peptides bovine lactoferricin (LfcinB) and human lactoferricin (LfcinH) are produced from the respective lactoferrin, but are more active than their precursors. Despite sequence homology, the bovine peptide and its derivatives are more active than their human homologs. Such differences between not only the peptides and their precursor but also between the bovine and the human peptides could relate to structural differences. Upon sequence alignment of both peptides with their parental proteins, the structural differences observed between the bovine lactoferrin (BLf) and LfcinB were also found between the human lactoferrin (HLf) and the LfcinH. The helical structures in HLf are replaced by beta-strands separated by a strong turn in LfcinH suggesting an antiparallel beta-sheet structure similar to LfcinB. MIC assays with HLP-2 and BLP-2, 11-residue peptides derived from the active core of both Lfcins, against Escherichia coli, showed that the bovine derivative, BLP-2, is more active than its human homolog HLP-2. Both 3D models for HLP-2 and BLP-2 showed that the beta-strand is centred between the aromatic residues giving both side chains the same orientations. The displacement towards the N-terminus observed for the beta-strand in HLP-2, compared with its central location in BLP-2, could be less favourable to membrane interaction and therefore responsible for the decrease in activity. Such a model suggests for LfcinH a mechanism similar to the one observed for LfcinB, where the absence of long-range interaction, present in lactoferrin, destabilises the first alpha helix, as observed in solution and, upon interaction with the membrane, could result in the formation of a beta-strand, as observed in the presence of LPS. The location of the beta-strand in relation to the positive charges, seems to define the efficiency of the activity of the peptide and may explain the difference in activity obtained between HLP-2 and BLP-2.     

Effect of Vibrio cholerae non-O1 protease on lysozyme, lactoferrin and secretory immunoglobulin A

Abstract The effect of Vibrio cholerae non-O1 protease on host defense proteins (lysozyme, secretory immunoglobullin A and lactoferrin) was studied in relation to its virulence mechanism. The proteins treated with the protease were analysed by SDS-PAGE. There was no influence of the protease on lysozyme. The protease cleaved lactoferrin into two fragments of 50 kDa and 34 kDa. N-terminal amino acid sequencing of these fragments revealed that the cleavage site was near the hinge region, between serine 420 and serine 421. This cleavage could affect the transition from open to closed configuration which is involved in iron binding and release. The anti-bacterial activity of lactoferrin was not affected by protease treatment. Secretory immunoglobulin A yielded a 42-kDa protein as the cleavage product. The susceptibility of secretory immunoglobulin A to V. cholerae non-O1 protease suggests a mechanism by which bacteria might evade the effect of this immunoglobulin.     

Activation of rainbow trout, Oncorhynchus mykiss, phagocytic cells by administration of bovine lactoferrin

The chemiluminescent response of kidney phagocytic cells was significantly increased by the oral administration of bovine lactoferrin to rainbow trout, Oncorhynchus mykiss. The phagocytic activity of kidney cells was also increased by administration of bovine lactoferrin to the fish. Activation of kidney cells was also observed in vitro with cells cultured in the presence of bovine lactoferrin.     

Subcutaneous injection of interleukin 12 induces systemic inflammatory responses in humans: implications for the use of IL-12 as vaccine adjuvant

Interleukin 12 (IL-12) is a cytokine with important regulatory functions bridging innate and adaptive immunity. It has been proposed as an immune adjuvant for vaccination therapy of infectious diseases and malignancies. The inflammatory properties of IL-12 play an important role in the adjuvant effect. We studied the effect of s.c. injections of recombinant human IL-12 (rHuIL-12) in 26 patients with renal cell cancer and demonstrated dose-dependent systemic activation of multiple inflammatory mediator systems in humans. rHuIL-12 at a dose of 0.5 g/kg induced degranulation of neutrophils with a significant increase in the plasma levels of elastase (p<0.05) and lactoferrin (p=0.01) at 24 h. Additionally, rHuIL-12 injection mediated the release of lipid mediators, as demonstrated by a sharp increase in the plasma secretory phospholipase A₂ (sPLA₂) level (p=0.003). rHuIL-12, when administered at a dose of 0.1 g/kg, showed minimal systemic effects. In conclusion, when IL-12 is used as an adjuvant, doses should not exceed 0.1 g/kg, in order to avoid severe systemic inflammatory responses.     

Arginine 25 and Arginine 28 of lactoferrin are critical for effective heparin neutralization in blood

The interaction of lactoferrin with endogenous heparin-like molecules modulates glycosaminoglycan-mediated biological processes. We performed site-specific mutagenesis and expressed recombinant lactoferrin and lactoferrin mutants by the baculovirus insect cell expression system. Five basic residues at the lactoferrin N terminus; Arg 5, Arg 25, Arg 28, Lys 29, and Arg 31, were individually replaced by alanines. Heparin chromatography on fast-performance liquid chromatography system showed that the NaCl concentrations corresponding to the peak of each eluted recombinant protein from the column were 665, 620, 540, 550, 630, or 650 mM for wild-type recombinant lactoferrin, Arg 5, Arg 25, Arg 28, Lys 29, or Arg 31 recombinant lactoferrin mutant, respectively. We compared the ability of each mutated lactoferrin derivative to neutralize glycosaminoglycans in the thrombin serpin inhibition assays. In comparison to wild-type recombinant lactoferrin, all the mutants showed decreased ability to neutralize glycosaminoglycan in a dose-dependent manner. The mutations of lactoferrin at Arg 25 and Arg 28 demonstrated the most striking decrease in lactoferrin's ability to neutralize various glycosaminoglycans in both enzymatic and plasma clotting-based experiments. Therefore, our results suggest that Arg 25 and Arg 28 are the critical basic residues at the lactoferrin N terminus responsible for heparin binding. The other basic residues on the N terminus, Arg 5, Lys 29, and Arg 31, also contribute to heparin binding by presenting an additional cationic motif.     

In vitro evaluation of the anticancer effect of lactoferrin and tea polyphenol combination on oral carcinoma cells

We investigated the anticancer effects of green and black tea polyphenols alone and in combination with bovine milk lactoferrin (bLF) on human tongue squamous carcinoma (CAL-27) and normal human gingival fibroblast (HGF) cells. Both green (Polyphenon-E;P-E) and black tea polyphenols (Polyphenon-B;P-B) preferentially inhibit the growth of CAL-27 cells in a dose-dependent manner. Based on the IC(50) values, P-E was found to be more effective than P-B and the combination of P-E and bLF (1:2 ratio) exhibited synergistic inhibition of CAL-27 cells. Analysis of the mechanism revealed nuclear fragmentation and condensation with appearance of the A(o) peak indicative of apoptosis. Furthermore, tea polyphenols transduced the apoptosis signal via generation of reactive oxygen species and decrease in the Bcl-2/Bax ratio thereby inducing mitochondrial permeability transition with consequent activation of caspase-3. Overall, the potency of cytotoxic and apoptosis inducing effects of dietary agents on CAL-27 cells was in the order P-E and bLF combination (1:2 ratio)>P-E>P-B. These results suggest that a "designer" approach may be useful for oral cancer prevention strategies.