key: cord-0933065-ikzmghjk
authors: Legrand, D.; Elass, E.; Carpentier, M.; Mazurier, J.
title: Lactoferrin: Lactoferrin: a modulator of immune and inflammatory responses
date: 2005-11-02
journal: Cell Mol Life Sci
DOI: 10.1007/s00018-005-5370-2
sha: 2e343a37eb39d557f599901a6cc63135adcdc13c
doc_id: 933065
cord_uid: ikzmghjk

Lactoferrin is an iron-binding glycoprotein of the transferrin family. Abundant expression and secretion of lactoferrin, in particular in milk and fluids of the digestive tract, are related to its implication in the first line of host defense. Lactoferrin is also a prominent component of the secondary granules of neutrophils (PMNs) and is released in infected tissues and blood during the inflammatory process. In addition to its direct antimicrobial properties, the abilities of lactoferrin to regulate the immune response and to protect against infection and septic shock have been described in numerous in vitro and in vivo studies. Although the cellular and molecular mechanisms that account for the modulation of the inflammatory and immune responses by lactoferrin are not yet totally elucidated, many are now established. At the cellular level, lactoferrin modulates the migration, maturation and function of immune cells. At the molecular level and in addition to iron binding, interactions of lactoferrin with a plethora of compounds, either soluble or membrane molecules, account for its modulatory properties. This paper reviews our current understanding of the cellular and molecular mechanisms that explain the regulatory properties of lactoferrin in host defence.

When lactoferrin (Lf) was fi rst discovered in milk [1], it was named lactotransferrin, suggesting a functionally related variant of transferrin. Since Lf is homologous to serum transferrin (Tf), it was indeed tempting to consider the molecule solely as an iron-binding molecule. The possibility of Lf having functions other than just simple iron sequestration emerged as soon as it was reported that Lf binds to microbes, host cells and components of the immune system. Besides its direct effects in host defense on bacteria, fungus and parasites, possible roles in the modulation of the immune response were reported. These properties were illustrated with many in vitro and in vivo experiments carried out in humans and animals, sometimes controversial but often leading to similar conclusions. Controversial conclusions are not unexpected, since modulation means both positive and negative effects, sometimes neutral effects, depending on the physiological status of the organism. Furthermore, the regulation of the immune system calls for so many factors that determining the exact role of Lf is very diffi cult. At the moment, however, we are at a stage where more questions are asked than answers are given about the molecular mechanisms governing the modulating properties of Lf in the immune and infl ammatory responses. The present review will attempt to detail our current knowledge of these properties.

Host defense involves innate and acquired immune systems, the latter being divided into humoral and cellmediated immunities. Lf clearly belongs to the innate, non-specifi c immune system. However, several lines of evidence indicate that it may also contribute, at least indirectly, to acquired immunities. Additionally, host defense includes protective systems, of which Lf is an essential element, against deleterious effects of infl ammation. It is now accepted that Lf plays a direct anti-microbial role in secretions and at the surface of epithelia by limiting the proliferation and adhesion of microbes and/or by killing them. These properties are the subject of an ac-companying review, however, and will not be developed further here. Briefl y, these properties are mainly related to the ability of Lf either to sequester iron in biological fl uids or to destabilize the membranes of microbes [12] . The constitutive expression of Lf in large amounts in all secretion fl uids and the massive delivery of Lf from PMNs at infl ammation sites [13] ensure the effi ciency of such a protective effect. Besides its direct antimicrobial effects, Lf may be responsible for up-and downregulation of immune cells and cells involved in the infl ammatory process. These regulatory activities are due to the iron-binding properties of Lf and above all to its ability to interact with target molecules and cells. On the one hand, some in vitro experiments suggest that it may regulate the proliferation, differentiation and activation of immune cells, thus strengthening, either directly or indirectly, the immune response. On the other hand, Lf mediates anti-infl ammatory activities that can lower the harmfulness of the response. When tissues are infected, reactive oxygen species are abundantly produced, either generated by free iron released from necrosed tissues or overproduced by activated granulocytes. This oxidative burst, together with the excessive release of pro-infl ammatory cytokines, mainly interleukin-1 (IL-1) and tumor necrosis factor a (TNF-a) contributes to the pathogenesis of septic shock [14] . The protective anti-infl ammatory activity of Lf lies in its ability to bind not only free ferric ion but also exogenous pro-infl ammatory bacterial components such as lipopolysaccharides (LPSs) and their receptors. Whereas Lf iron binding has benefi cial detoxication effects in infected and pathological tissues, binding to pro-infl ammatory molecules has downregulating effects on both the activation and recruitment of immune cells in infl amed tissues. The molecular and cellular bases for these assertions are developed in the following sections.

A full member of the transferrin family but with specifi c features Lf belongs to the transferrin family, of which Tf is the best known member. Human Lf (hLf) consists of a single 692-residue polypeptide chain [15] whose three-dimensional (3D) structure has been thoroughly defi ned [16] . Although both Lf and Tf have very similar 3D structures, they differ signifi cantly in their primary structures; hLf is about 60% identical to human Tf. The differences are found especially in surface-exposed sequences, which contribute specifi cally to physicochemical and biological properties. In contrast to Tf, which is a rather acidic molecule (pHi ~ 6.5), Lf is a strongly basic protein (pHi Cell. Mol. Life Sci. Vol. 62, 2005 Multi-author Review Article 2 551 8. [5] [6] [7] [8] [9] . Much of the cationic charge of Lf is located on domain N1, especially in the so-called lactoferricin (Lfc) domain (fi g. 1A) which is known to be important for many functions of the molecule [17] . Lfc, a peptide isolated following pepsin digestion of hLf (residues 1-19 and 20-37) and bovine Lf (bLf) (residues 19-36), contains basic amino acid residue repeats in a b-sheet-ahelix structure that was shown to be responsible for most of iron-independent activities of Lf, not only bactericidal but also immunoregulatory and infl ammatory activities [18] . Recently, an a-helix-containing basic peptide (residues 268-284 in bLf), close to the Lfc domain, called lactoferrampin, was also recently shown to be involved in bactericidal activity against several bacterial strains [19] . Interestingly, a common feature of all Lfs is to retain iron to pH values as low as 3, whereas Tf releases iron at a pH of about 5.5. This difference of stability towards pH can be explained in part by the presence of a hydrogen bond between a pair of lysine residues in Tf that could provide a trigger for iron release on protonation [20, 21] , but in particular by cooperative interactions between the two lobes of Lf [22] . The high affi nity and stability of iron binding by Lf make the protein not only a powerful bacteriostatic agent but also an antioxidant protective molecule whose properties will be discussed below.

The cationic nature of Lf accounts for its propensity to 'stick' to many anionic molecules and makes identifi cation of specifi c ligands contributing to the immunoregulatory roles of Lf diffi cult. At the surface of cells, the sulfated chains of proteoglycans present the main Lf binding sites, responsible for about 80% of total binding [10, 23] . Although the low affi nity (Ka ~ 10 6 M) and ionic nature of the interactions may raise questions about their physiological relevance, it is now accepted that binding to proteoglycans is responsible for high-density binding of Lf at the surface of cells (generally several millions of binding sites on many cells) [23] . It is thought that Lf binding to glycosaminoglycans is an important event in the infl ammatory process. The binding determinants in hLf for cell surface glycosaminoglycans and soluble heparin have been identifi ed as the basic stretches 1 GRRRRS 6 and 28 RKVR 31 [9, 10, 23] . These two sections of polypeptide (fi g. 1B) seem to act as a cationic cradle to bind sulfated chains [9] . Besides proteoglycans, a few cell receptors have been reported that could account for the signaling, endocytosis and nuclear targeting of Lf in cells. A specifi c 105-kDa receptor was formerly identifi ed on activated lymphocytes, platelets and mammary gland cells that could permit signaling in cells as well as endocytosis of Lf [25] [26] [27] . Very recently, we showed binding of Lf to nucleolin expressed at the surface of dividing cells that participates, together with proteoglycans, in the endocytosis and nuclear targeting of Lf [28] . There are many indications that nucleolin could represent the previously reported Lf receptor, though the receptor binding sites on Lf look different. Another important Lf receptor at the surface of cells is the low-density lipoprotein receptor-related protein (LRP), which has been shown to be responsible for Lf endocytosis but also to function as a mitogenic Lf receptor in osteoblastic cells [29] . Since the LRP is also present at the surface of immune cells, its role in the immuno-modulating activity of Lf may be considered. Finally, a specifi c receptor visualized as a 34-kDa protein under reducing conditions, responsible for Lf endocytosis, has been found at the surface of intestinal cells [30, 31] . Apart from cell surface target molecules, soluble anionic molecules can be bound by Lf, most of them being important actors in the infl ammatory response. First of all, the high-affi nity interaction of Lf with the lipid A moiety of Escherichia coli LPS was reported [32, 33] . Using E. coli LPS, two binding sites with dissociation constants (K d ) of 3.6 +/-1 nM and 390 +/-20 nM were found on the N-and C-lobes of hLf [34] . It was shown that both sequences 1 GRRRR 5 and 28 RKVRGPP 34 , the same involved separately or together in the interactions with the 105-kDa Lf receptor, the LRP, glycosaminoglycans and DNA [9, 10, 24] are required for the high-affi nity binding of Lf to LPS [34, 35] . Binding of Lf to other bacterial pro-infl ammatory components such as unmethylated CpG-containing oligonucleotides was also reported [36] . More important, high-affi nity interactions (K d ~ 16 +/-7 nM) were reported between Lf and sCD14, a serum-soluble LPS receptor. It was shown that hLf interacts not only with free sCD14 but also, though with different binding properties, with sCD14 complexed to LPS or lipid A-2-keto-3-deoxyoctonic acid-heptose [37] . As for LPS, the cationic N-terminal peptides of Lf are essential for binding [37] . Interestingly, it may be noted that most interactions of Lf with cell receptors and infl ammatory molecules also involve the N-terminal Lfc domain of Lf. The Lfc peptide itself was also found to interact with LPS [18, 34, 38, 39] . Recent studies, however, suggest that lipid A is not the main binding site for Lfc but the negative charges present in the inner core [40] .

The binding of Lf to the surface of cells suggests that it might directly trigger cellular responses such as differentiation, activation and proliferation. Several fi ndings, reported below, are in accordance with this postulate. However, it is not clear how exactly Lf could trigger signals in cells. Interestingly, LRP, a Lf receptor at the surface of many cells, has recently been shown to function as a mitogenic Lf receptor in osteoblastic cells, via p42/44 MAP kinase signaling [29] . Such MAP kinase signaling was also observed in Jurkat lymphoblastic T cells owing to the 105-kDa Lf receptor [41] . It is also hypothesized that Lf may enter the cell and be targeted to the nucleus where it can act as a transcriptional activator [42] . Recently, nucleolin ubiquitously expressed on dividing cells was pointed out as a possible Lf carrier between cell surface and nucleus [28] . Interestingly, it has also been shown that Lf may downregulate LPS-induced cytokines in THP1 through a mechanism involving Lf internalization, nuclear localization and interference with nuclear factor-kB (NF-kB) [43] . The mechanisms of interference of Lf with NF-kB, a transcription factor playing a critical role in immune responses and infl ammation, are not perfectly clear. However, Oh et al. [44] showed that overexpressed Lf acts as a p53 gene transactivator through the stimulation of the inhibitor of NF-kB (IkB)-kinase activity and NF-kB binding. These authors previously demonstrated a matrix metalloproteinase 1 gene transactivating activity by Lf through stress-activated mitogen-activated protein-kinase (MAPK) signaling modules [42] .

To date there has been no direct in vivo evidence for a regulatory role of Lf in the immune system, although knockout animals for Lf have been produced [45] . Involvement of Lf in the regulation of the immune system was suggested in 1980 [46] , when a total absence of Lf in neutrophils, but normal Lf content in glandular secretion [47] , was observed for a patient suffering recurrent infections. More recently, hLf-transgenic mice have been shown to clear bacteria signifi cantly better than congenic littermates [48] . This effect is the consequence of direct inhibition of the growth of Staphylococcus aureus, and of the enhancement of the T helper (Th) type 1 response due to overexpression and constitutive presence of Lf in animal tissues. Furthermore, the susceptibility to tuberculosis of b-2-microglobulin knockout mice was abolished by Lf treatment [49] . Oral administration of Lf also revealed host-protecting effects against microbial infections [50] , during lethal bacteraemia in mice [51, 52] and against oral candidiasis [53] . Finally, orally administered Lf was shown to protect piglets against septic shock [54] . At the molecular level, altered expression of cytokines, mostly pro-infl ammatory interferon g (IFN-g), interleukin (IL)-1b, IL-6 and TNF-a, and granulocyte-macrophage colony-stimulating factor (GMCSF) have been detected in the presence of exogenous Lf [48, [55] [56] [57] [58] , with a decrease of IL-5 and IL-10 production. In contrast, upregulation of anti-infl ammatory IL-4 and IL-10 was found after oral Lf administration in rats with colitis [59] . At the cellular level, there seems to be an increased number of natural killer (NK) cells [60, 61] , increased phagocytosis-enhancing effect [18, 62] , an increased recruitment of neutrophils in blood [63] and modulation of myelopoiesis [56] .

It has previously been shown in vitro that Lf may induce cell proliferation and maturation by acting as an alternative iron donor for T cells [25, 64] , but recent in vivo studies establish that Lf mainly acts by scavenging iron [48] and correcting iron overload [49] . In fact, most mechanisms through which Lf upregulates the immune system involve direct Lf interactions with cells. It is assumed that more or less specifi c receptors bind Lf and are key effectors for cell signaling, casual endocytosis and/or nuclear targeting [65] . Unfortunately, data on these putative receptors and pathways are disparate and sometimes contradictory. Lf is likely to regulate lymphocyte maturation and activation. Lf differentiation effects were previously described Cell. Mol. Life Sci. Vol. 62, 2005 Multi-author Review Article 2 553 on isolated thymocytes and splenic B cells [66, 67] , and it was shown that Lf interactions with Jurkat T-cells upregulate the expression of CD4 antigen [41] . Furthermore, in cervical cancer patients, a recent fi nding indicates that Lf can regulate the expression of the x chain of the T-cell receptor [68] . Promotion of lytic cell activity seems to be another important aspect of Lf function. Lf is already expressed on resting PMNs where it could participate in the binding of micro-organisms [69] . It is then massively released from PMNs on stimulus by TNF-a and phorbols, and binds to PMN membranes [70, 71] . It was shown in vitro that both release and cell binding promote the activation and phagocytosis of PMNs and monocytes/macrophages. Lf was reported as a promoter of motility, superoxide production and release of pro-infl ammatory molecules such as NO, TNF-a and IL-8 [72] [73] [74] , and a recent study indeed demonstrates enhanced phagocytosis against S. aureus [75] . The molecular mechanisms underlying these activities are, however, highly controversial. Phagocytosis by PMNs is enhanced by the interaction of complement activation products, particularly complement factor C3. Nevertheless, it is unclear whether Lf activity is related to complement activation since Lf was shown either to inhibit [76] or to activate [75, 77] the classical and alternate pathways of complement. A recent report shows that the Lfc domain of either hLf or bLf inhibits the classical complement pathway but not the alternative complement pathway [78] . Direct Lf binding to PMNs and opsonin-like activity could also be involved [79] . The latest data supporting the immunotropic activity of Lf are recent reports showing its adjuvant effect in the generation of delayed-type hypersensitivity [80] and in the boost of Bacille Calmette-Guérin (BCG) vaccine effi cacy to generate T helper response in mice [81] . This adjuvant effect could be due to bovine Lf binding on the mannose receptor of immature antigen-presenting skin cells [80] . Such binding has been recently confi rmed in a study showing that bovine Lf binding to DC-SIGN on dendritic cells blocks its interaction with HIV gp120 and subsequent virus transmission [82] .

The anti-infl ammatory properties of Lf have been extensively studied for the last decade. Lf modulates the infl ammatory process mainly by preventing the release of cytokines which induce recruitment and activation of immune cells at infl ammatory sites. Actually, hLf suppresses TNF-, IL-1 and IL-6 production in mononuclear cells in vitro and in vivo, in response to LPS activation [43, 58, [83] [84] [85] . Bovine Lf regulates cytokine production by splenocytes of obstructive jaundiced rats [86] . In addi-tion, Lf enhances the secretion of the anti-infl ammatory cytokines IL-10 and IL-4, and reduces colitis in rats [59] . The downregulation of pro-infl ammatory cytokines can be partly related to the LPS-binding properties of Lf, through its Lfc domain [32] [33] [34] . Interestingly, it has been shown that Lfc itself neutralizes LPS activity [85, 87] . Lf competes with serum LPS-binding protein (LBP) for LPS binding and therefore prevents the transfer of endotoxin to mCD14 presented at the surface of macrophages [35] . Lf also suppresses the production of hydrogen peroxide mediated by the binding of LPS to L-selectin of neutrophils [88] . Furthermore, the interaction between Lf and soluble CD14 (sCD14) [37] inhibits the secretion of IL-8, a chemokine induced by the complex sCD14-LPS, by endothelial cells [89] . Apart from LPS and CD14 binding, other mechanisms of inhibition of pro-infl ammatory cytokines production have been described. The downregulation of IL-6 secretion induced by TNF-a [85] could result from the inhibition of NF-kB binding to the TNF-a promoter [43] following internalization of Lf in monocytic cells. Furthermore, the inhibition of immunostimulatory effects on human B cells can be correlated to the property of Lf to interact with the bacterial unmethylated CpG-containing oligonucleotides [36] . In collagen-induced and septic arthritis mouse models, peri-articular injection of hLf reduced infl ammation [90] . In agreement with this study, oral administration of bLf inhibited TNF-a and increased IL-10 secretion in adjuvant-stimulated arthritis rats [91] . Recombinant hLf and milk bLf also had a preventive effect on LPS-induced preterm delivery in mice through inhibition of IL-6 production [92] . Equally, virus-induced infl ammatory responses can be controlled by Lf. Recently, Sano et al. [93] have reported that Lf decreased both the infectivity of respiratory syncytial virus (RSV) and the RSV-induced IL-8 secretion by Hep2 cells through a direct interaction of Lf with a surface protein of RSV.

Several studies have reported that Lf, alone, activates macrophages and induces IL-8, TNF-a and nitric oxide (NO) [74] . The Lf-LPS complex could be under some conditions an inducer of infl ammatory mediators in macrophages, through Toll-like receptor 4 [94] . Moreover, after pretreatment with the Lf-LPS complex, cells are rendered tolerant to LPS challenge [94] . Lf restored the humoral immune response and increased production of IL-6 by peritoneal and alveolar cells in cyclophosphamide (CP)-immunocompromised mice [95, 96] . In the same experimental model, it was demonstrated that Lf strongly elevated the pool of CD3+ T cells and CD4+ T cell content. Lf, given orally to CP-immunosuppressed mice, could reconstitute a T cell-mediated immune response by renewal of the T cell pool [97] . A recent study 2554 D. Legrand et al. Lactoferrin modulates immune and infl ammatory responses has demonstrated that oral Lf administration prevents body weight loss and increases cytokine responses during herpes simplex virus type 1 infection of mice [98] . Bovine Lf or its pepsin hydrolysate induces IL-18 in mouse small intestine epithelial cells, which infl uences expression of a number of genes including IFN-g and other proinfl ammatory cytokines [99] . According to these authors, this effect of bLf may be a major mechanism by which bLf inhibits carcinogenesis and metastasis. Bovine Lf may also act as an inhibitor of angiogenesis, probably by inducing IL-18 production in serum and blocking endothelial functions [100] . A long-term bLf administration to chronic hepatitis C patients can produce a Th1-cytokine dominant environment in peripheral blood that favors the eradication of chronic hepatitis C virus by IFN therapy [101] . Upregulation of IFN-g and TNF-a production by cervical lymph node cells stimulated by heat-killed Candida albicans was observed in Lf-treated mice compared with non-treated mice [53] .

The interaction of Lf with LPS and sCD14 interferes not only with the activation of immune cells but also with the expression of adhesion molecules on endothelial cells, necessary for the local recruitment of immune cells at infl ammatory sites. In particular Lf inhibits the (sCD14-LPS)-induced expression of E-selectin, intercellular adhesion molecule 1 (ICAM-1) and IL-8 by human umbilical endothelial cells [88, 89] . These studies also pointed to the ability of Lf to compete with chemokines such as IL-8 for their binding to proteoglycans and their further presentation to leukocytes. Interestingly, a recent in vivo study has shown that orally administred recombinant hLf is able to prevent injury by non-steroidal anti-infl ammatory drugs in the intestine of rats and mice and that this effect could be linked to attenuation of neutrophil migration to the intestine [102] . Another recent study, however, has reported a role of Lf in the increased recruitment of neutrophils in blood, thus protecting mice from bacteremia [103] . Takakura et al. [53] also showed that alleviation of oral candidiasis by Lf feeding to mice is correlated with the enhancement of the number of leukocytes and their cytokine responses in regional lymph nodes against candida infection.

Lf is described as a potent molecule in the treatment of common infl ammatory diseases. A major anti-infl ammatory activity of Lf is related to the scavenging of free iron, which accumulates in infl amed tissues and catalyses the production of tissue-toxic hydroxyl radicals. Apo-Lf is released from PMNs at infl amma-tory sites and, owing to its iron-binding stability at low pH, participates in iron homeostasis and detoxifi cation. Interestingly, in neurodegenerative diseases, where iron deposits contribute to oxidative stress and neuronal death, overexpression of Lf was reported in some specifi c areas of the brain [104] . This event, together with transcytosis of plasma Lf through the blood-brain barrier during infl ammation [105] , could help to limit oxidative stress in the brain.

In vivo studies showed Lf protection against skin and lung allergies [106, 107] . Lf is overexpressed in patients with allergies [106] , a process which involves the activation of mast cells and basophils, and IL-1b and TNF-a-triggered migration of antigen-presenting cells [108] . In skin allergies, a mechanism by which Lf binds to keratinocytes and inhibits the release of TNF-a from these cells has been proposed [109] . Another explanation has been found in the ability of Lf to destabilize tryptase, a potent pro-infl ammatory protease released from mast cells [110] . Lf apparently displaces tryptase from heparin, which is known to maintain enzymatic activity. It was recently shown that inhibition occurs following Lf uptake by mast cells and interaction not only with tryptase but also with chymase and cathepsin G [111] . Recently, these authors also showed an inhibition of anti-immunoglobulin (Ig) E induced histamine and tryptase release from human colon mast cells by Lf [112, 113] .

Since Lf levels in blood and biological fl uids may greatly increase in septicaemia [5, 6] , it is tempting to consider Lf as a marker of infl ammatory diseases. Increased levels of Lf were measured in synovial fl uid but not in serum from patients with rheumatoid arthritis. Lf was thus proposed as a reliable marker of neutrophil activation at sites of infl ammation in rheumatoid synovitis, but does not represent a marker of disease activity [114] . The response of severe acute respiratory syndrome (SARS)-affected patients seems to be mainly an innate infl ammatory response, rather than a specifi c immune response. The gene expression of Lf in peripheral blood mononuclear cells is the most strongly increased during SARS (148-fold increase) [115] . This result opens new possibilities for designing new diagnostics and treatments for this disease. Studies using fecal Lf as a non-invasive diagnostic tool to evaluate the severity of intestinal infl ammation in patients presenting abdominal pain and diarrhea [116, 117] are more documented and interesting. Fecal Lf levels quickly increase with the infl ux of leuko-Cell. Mol. Life Sci. Vol. 62, 2005 Multi-author Review Article 2 555 cytes into the intestinal lumen during infl ammation. This biomarker has been shown to be a sensitive and specifi c marker of disease activity in chronic infl ammatory bowel disease [118] . Increase of fecal Lf is also observed in chronic infl ammatory bowel disease proctitis. Patient compliance and stability of the marker make Lf assay a promising method for clinical research [119] . Finally, Buderus et al. [120] identifi ed fecal Lf as a marker of intestinal infl ammation and therapeutic response in patients with Crohn's disease. All these studies indicate a possible use of Lf as a clinical marker of infl ammatory diseases.

Lf shows effects on non-specifi c immune responses in fi sh [121] . The humoral immune response was not infl uenced by Lf feeding. Lf seems to affect innate immune cellular activity, mainly respiratory burst and natural cytotoxic activity. Lf was proposed as a possible immunostimulant for farmed gilthead seabream [121] . Liposomal Lf was shown to increase IFN-a production and NK activity in healthy volunters [122] .

Orally administered Lf lowers the concentration level of endotoxin in the gut of mice. Neither bifi dobacteria nor Lf stimulated an increase in B or T cells, or in cytokine production (IL-6, TNF-a, IFN-g), in Peyer's patches [123] . Interestingly, persorption of bLf from the intestinal lumen into the systemic circulation via the portal vein and the mesenteric lymphatics was demonstrated in growing pigs [124] . The mode of oral bLf administration, however, infl uences mucosal and systemic immune responses in mice [125] . The addition of Lf to the drinking water had no visible effect on the immune status. Gastric intubation, single buccal doses and continuous doses of Lf in the diet stimulated transient systemic and intestinal antibody responses against Lf. All of these oral modes of Lf exposure biased mucosal and systemic T cell responses toward Th2 types and elevated IgA production by mucosal cells. However, the less natural gastric intubation also promoted Th1-type responses, as evidenced by serum IgG(2a) antibodies and the secretion of Th1 cytokine by mucosal and systemic T cells in vitro. Thus, one should carefully consider the oral mode of administration for understanding regulation of immune responses by food proteins such as Lf.

It has to be recognized that in the last decade important breakthroughs have been made in the fi eld of 'lactobiology', in particular the evidence for neutralizing effects of exogeneous pro-infl ammatory molecules. Lf is a glycoprotein able to bind and sequester iron and LPS and thus to interfere in the signaling pathways and biological response induced by them. Additionally, Lf is able to interact with cell surface receptors: proteoglycans, apolipoprotein E/low-density lipoprotein (ApoE/LDL) receptor, nucleolin, lymphocyte and enterocyte receptors. These interactions result in cell capture and the induction of biological responses which, however, until now have been controversial. The biological properties of Lf depend on the regulation of its synthesis, secretion and presence at a well-defi ned time period and place. In normal physiological conditions, Lf is only secreted by glandular cells and covers the surface of mucosa, where it works as a powerful bacteriostatic and bactericidal molecule. During infl ammation and neurodegenerative diseases that lead to PMN degranulation at the site of infection and to activation of microglial cells and dopaminergic neurons [104] , respectively, the Lf secretion dramatically increases. Consequently, Lf binds to cell surface proteoglycans and receptors which will induce its endocytosis by various cell types. This increased Lf secretion can be monitored by the variation of its plasmatic concentration (fi g. 2). Lf will mainly act as a sequestrator of iron, LPS and CD14, and an activator/modulator of signaling pathways leading to negative feedback of the infl ammatory response, as shown by a decrease in production of reactive oxygen species and various pro-infl ammatory cytokines. In addition, orally administrated Lf induces interesting and benefi cial physiological responses that justify its use in the so-called foods for special health uses (FOSHU). In these applications, special care has to be given to the origin of Lf, whether milk or recombinant Lf from human or bovine species. Indeed, in addition to the biological properties played by the protein itself, one cannot exclude some 'non-specifi c activity' due to the glycan moiety. For example, the glycan moieties of bLf are of the high-mannose type and thus could allow them to be recognized by the DC-SIGN (dendritic cell-specifi c intercellular adhesion molecule3-grabbing nonintegrin receptor) and mannose receptor of monocytic cells and to interfere in the immune response.

tract of mouse, rat and hamster

Lactoferrin, an iron-binding protein in neutrophilic leukocytes

Breast milk lactoferrin levels in relation to maternal nutritional status

Lactoferrin content of peripheral blood cells

Development and evaluation of luminescence based sandwich assay for plasma lactoferrin as a marker for sepsis and bacterial infections in pediatric medicine

Interaction of human lactoferrin with the rat liver

Recognition of lactoferrin and aminopeptidase M-modifi ed lactoferrin by the liver: involvement of proteoglycans and the remnant receptor

Delineation of the glycosaminoglycan-binding site in the human infl ammatory response protein lactoferrin

The N-terminal Arg2, Arg3 and Arg4 of human lactoferrin interact with sulphated molecules but not with the receptor present on Jurkat human lymphoblastic T-cells

Immobilized lactoferrin is a stimulus for eosinophil activation

Lactoferrin: role in iron homeostasis and host defense against microbial infection

Lactoferrin, an iron-binding protein in neutrophilic leukocytes

Septic shock

Complete nucleotide sequence of human mammary gland lactoferrin

Structure of human lactoferrin at 3.2-resolution

Lactoferrin feeding augments peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans

Lactoferricin derived from milk protein lactoferrin

Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin

Dealing with iron: common structural principles in proteins that transport iron and heme

Comparative study of the ironbinding properties of human transferrins. I. Complete and sequential iron saturation and desaturation of the lactotransferrin

Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins

Role of heparan sulphate proteoglycans in the regulation of human lactoferrin binding and activity in the MDA-MB-231 breast cancer cell line

N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA

Expression of human lactotransferrin receptors in phytohemagglutinin-stimulated human peripheral blood lymphocytes

Lactotransferrin binding to its platelet receptor inhibits platelet aggregation

Effects of human lactoferrin on NK cell cytotoxicity against haematopoietic and epithelial tumour cells

Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells

The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells

Cellular internalization of lactoferrin in intestinal epithelial cells

Characterization of mammalian receptors for lactoferrin

Lactoferrin is a lipid A-binding protein

Biophysical characterization of lipopolysaccharide and lipid A inactivation by lactoferrin

Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affi nity binding to E. coli 055B5 lipopolysaccharide

Lactoferrin inhibits the endotoxin interaction with CD14 by competition with the lipopolysaccharide-binding protein

Lactoferrin binds CpG-containing oligonucleotides and inhibits their immunostimulatory effects on human B cells

Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by the CD14-lipopolysaccharide complex

Structural origin of endotoxin neutralization and antimicrobial activity of a lactoferrin-based peptide

Structure and association of human lactoferrin peptides with Escherichia coli lipopolysaccharide

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity

Lactoferrin upregulates the expression of CD4 antigen through the stimulation of the mitogen-activated protein kinase in the human lymphoblastic T Jurkat cell line

Human neutrophil lactoferrin trans-activates the matrix metalloproteinase 1 gene through stress-activated MAPK signaling modules

Lactoferrin down-regulates the LPSinduced cytokine production in monocytic cells via NF-kappaB

Neutrophil lactoferrin upregulates the human p53 gene through induction of NF-kappaB activation cascade

Lactoferrin: role in iron homeostasis and host defense against microbial infection

Lactoferrin defi ciency as a consequence of a lack of specifi c granules in neutrophils from a patient with recurrent infections

Glandular secretion of lactoferrin in a patient with neutrophil lactoferrin defi ciency

Enhanced Th1 response to Staphylococcus aureus infection in human lactoferrin-transgenic mice

Correction of the iron overload defect in beta-2-microglobulin knockout mice by lactoferrin abolishes their increased susceptibility to tuberculosis

In vivo antimicrobial and antiviral activity of components in bovine milk and colostrum involved in non-specifi c defence

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

Lactoferrin impairs type III secretory system function in enteropathogenic Escherichia coli

Effect of orally administered bovine lactoferrin on the immune response in the oral candidiasis murine model

The protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets

Lactoferrin stimulates colony stimulating factor production in vitro and in vivo

The opposing actions in vivo on murine myelopoiesis of purifi ed preparations of lactoferrin and the colony stimulating factors

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

Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced infl ammatory responses in mice

Oral administration of lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance

Lactoferrin-mediated protection of the host from murine cytomegalovirus infection by a T-cell-dependent augmentation of natural killer cell activity

Effects of orally administered bovine lactoferrin on the immune system of healthy volunteers

Phagocytosis-enhancing effect of lactoferrin on bovine peripheral blood monocytes in vitro and in vivo

P-selectin-dependent leukocyte recruitment and intestinal mucosal injury induced by lactoferrin

Activated human gamma delta T lymphocytes express functional lactoferrin receptors

Lactoferrin modulates immune and infl ammatory responses

Characterization of mammalian receptors for lactoferrin

Human lactoferrin induces phenotypic and functional changes in murine splenic B cells

Immunostimulatory activity of lactotransferrin and maturation of CD4-CD8-murine thymocytes

Lactoferrin-induced upregulation of x (zeta) chain expression in peripheral blood T lymphocytes from cervical cancer patients

Surface expression of lactoferrin by resting neutrophils

Lactoferrin binding to neutrophilic polymorphonuclear leucocytes

Expression of lactoferrin on human granulocytes: analysis with polyclonal and monoclonal antibodies

Infl uence of lactoferrin on the function of human polymorphonuclear leukocytes and monocytes

Effects of lactoferrin and lactoferricin on the release of interleukin 8 from human polymorphonuclear leukocytes

Activation of macrophages by lactoferrin: secretion of TNF-a, IL-8 and NO

Lactoferrin stimulates A Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland

Modulation of classical C3 convertase of complement by tear lactoferrin

Activation of the classical pathway of complement by binding of bovine lactoferrin to unencapsulated Streptococcus agalactiae

Anti-complement effects of lactoferrin-derived peptides

Bovine lactoferrin stimulates the phagocytic activity of human neutrophils: identifi cation of its active domain

Immunoregulatory activities of lactoferrin in the delayed type hypersensitivity in mice are mediated by a receptor with affi nity to mannose

Lactoferrin augments BCG vaccine effi cacy to generate T helper response and subsequent protection against challenge with virulent Mycobacterium tuberculosis

Lactoferrin prevents dendritic cell-mediated human immunodefi ciency virus type 1 transmission by blocking the DC-SIGN-gp120 interaction

Lactoferrin-lipopolysaccharide interactions. Effect on lactoferrin binding to monocyte/macrophage-differentiated HL-60 cells

Regulation of cytokine release from mononuclear cells by the iron-binding protein lactoferrin

Lactoferrin or a fragment thereof inhibits the endotoxin-induced interleukin-6 response in human monocytic cells

Bovine lactoferrin decreases histopathological changes in the liver and regulates cytokine production by splenocytes of obstructive jaundiced rats

Neutralization of endotoxin in vitro and in vivo by a human lactoferrinderived peptide

Lactoferrin inhibits the binding of lipopolysaccharides to L-selectin and subsequent production of reactive oxygen species by neutrophils

Lactoferrin inhibits the lipopolysaccharide-induced expression and proteoglycan-binding ability of interleukin-8 in human endothelial cells

The effects of local administration of lactoferrin on infl ammation in murine autoimmune and infectious arthritis

Oral administration of lactoferrin inhibits infl ammation and nociception in rat adjuvant-induced arthritis

Preventive effect of recombinant human lactoferrin on lipopolysaccharide-induced preterm delivery in mice

Lactoferrin and surfactant protein A exhibit distinct binding specifi city to F protein and differently modulate respiratory syncytial virus infection

Lactoferrin works as a new LPS-binding protein in infl ammatory activation of macrophages

Reconstitution of the cellular immune response by lactoferrin in cyclophosphamide-treated mice is correlated with renewal of T cell compartment

Effects of lactoferrin on IL-6 production by peritoneal and alveolar cells in cyclophosphamide-treated mice

Orally administered lactoferrin restores humoral immune response in immunocompromised mice

Oral lactoferrin prevents body weight loss and increases cytokine responses during herpes simplex virus type 1 infection of mice

Orally administered bovine lactoferrin induces caspase-1 and interleukin-18 in the mouse intestinal mucosa: a possible explanation for inhibition of carcinogenesis and metastasis

Bovine lactoferrin inhibits tumorinduced angiogenesis

Long-term follow-up of chronic hepatitis

C patients treated with oral lactoferrin for 12 months

Recombinant human lactoferrin prevents NSAIDinduced intestinal bleeding in rodents

Protective effects of lactoferrin in Escherichia coli-induced bacteremia in mice: relationship to reduced serum TNF alpha level and increased turnover of neutrophils

Lactoferrin is synthesized by activated microglia in the human substantia nigra and its synthesis by the human microglial CHME cell line is upregulated by tumor necrosis factor alpha or 1-methyl-4-phenylpyridinium treatment

Tumor necrosis factor-alpha increases lactoferrin transcytosis through the blood-brain barrier

Lactoferrin, a potent tryptase inhibitor, abolishes latephase airway responses in allergic sheep

Exogenous topical lactoferrin inhibits allergen-induced Langerhans cell migration and cutaneous infl ammation in humans

Release of lactoferrin and elastase in human allergic skin reactions

IL-1beta-induced Langerhans cell migration and TNF-alpha production in human skin: regulation by lactoferrin

Lactoferrin: infl uences on Langerhans cells, epidermal cytokines, and cutaneous infl ammation

The inhibition of mast cell activation by neutrophil lactoferrin: uptake by mast cells and interaction with tryptase

Inhibition of tryptase release from human colon mast cells by protease inhibitors

Modulation of histamine release from human colon mast cells by protease inhibitors

Increased levels of lactoferrin in synovial fl uid but not in serum from patients with rheumatoid arthritis

Expression profi le of immune response genes in patients with Severe Acute Respiratory Syndrome

Markers of infl ammation in bacterial diarrhea among travelers, with a focus on enteroaggregative Escherichia coli pathogenicity

Adaptive and infl ammatory immune responses in patients infected with strains of Vibrio parahaemolyticus

Fecal lactoferrin is a sensitive and specifi c marker in identifying intestinal infl ammation

Faecal calprotectin and lactoferrin as markers of acute radiation proctitis: a pilot study of eight stool markers

Fecal lactoferrin: a new parameter to monitor infl iximab therapy

Effects of lactoferrin on non-specifi c immune responses of gilthead seabream (Sparus auratus L.). Fish Shellfi sh Immunol

Liposomal lactoferrin induced signifi cant increase of the interferon-alpha (IFN-alpha) producibility in healthy volunteers

In vivo effects of bifi dobacteria and lactoferrin on gut endotoxin concentration and mucosal immunity in Balb/c mice

Persorption of bovine lactoferrin from the intestinal lumen into the systemic circulation via the portal vein and the mesenteric lymphatics in growing pigs

The mode of oral bovine lactoferrin administration infl uences mucosal and systemic immune responses in mice