LACTOFERRIN
Lactoferrin (LF) Lactoferrin (Lf), first reported almost 50 years ago (and referred as "red milk protein") is a non-hemic iron-binding protein. It is a member of the transferrin family, along with serum transferrin, ovotransferrin, and melanotransferrin, all of which function in iron transport, and inhibitors of carbonic anhydrase.
Lf is produced by mucosal epithelial cells in various mammalian species including humans, cows, goats, horses, dogs, and rodents, and it is also produced by fish. This multifunctional glycoprotein is found in mucosal secretions including tears, saliva, vaginal fluids, semen, nasal and bronchial secretions, bile, gastrointestinal fluids, urine, milk and colostrum. The most abundant antimicrobial proteins include lysozyme, collectin and and lactoferrin. Lf possesses a greater iron-binding affinity, and it is the only transferrin with the ability to retain this metal over a wide range of pH values, including resistance to proteolysis. The most striking physicochemical feature of Lf is its very high affinity for iron. In both Lf and related transferrins (Tfs), two Fe3+ ions are bound very tightly (K ~ 1022 M) but reversibly to LF, with two synergistically bound CO32− ions. Because of its wide distribution in various tissues, Lf is a highly multifunctional protein.
THE GENE
The nucleotide sequence of human milk Lf (hLf) was first determined in 1990 and compared with the amino acid sequence of human lactotransferrin determined previously. Lf genes are highly conserved among species, with an almost identical organization and an mRNA of about 1900–2600 bp. A homology search in sequence databases revealed nucleotide sequences for the Lfs of 13 species: 3 primates, 7 even-toed ungulates, 1 pig, 1 cat, and 1 mouse. Pairwise sequence identities ranged from a minimum of ~ 78% to nearly 100%. The main outliers in this group were Lfs from primates, in which human and chimpanzee are quite similar (95–98%) when compared to orangutan (79%). On the other hand, horse and camel; pig, cow, yak, water buffalo, bull, and goat Lfs share 81 and 78% of sequence identity respectively when compared to human Lf as outgroup. The sequence relationships given above show that the Lfs form a highly conserved sequence family, and also sequence identity between Lfs and other Tfs is relatively high at 60–65%.
THE STRUCTURE
Lf is an 80 kDa glycosylated protein of ~ 700 amino acids, with a high homology among species. It is comprised of a simple polypeptide chain folded into two symmetrical lobes (the N-lobe and C-lobe), which are highly homologous with one another (33–41% homology). The two lobes are connected via a hinge region containing parts of an α-helix between amino acids 333 and 343 in human Lf (hLf), which confers flexibility to the molecule. The polypeptide chain includes amino acids 1–332, comprising the N-lobe, and 344–703, comprising the C-lobe, and it is made up of α-helix and β-pleated sheet structures that create two domains within each lobe (domains I and II). Each lobe can bind a metal atom in synergy with the carbonate ion (CO3−2). Lf is capable of binding Fe2+ or Fe3+ ions, but it has also been observed to be bound to Cu2+, Zn2+ and Mn2+ ions. Because of its ability to reversibly bind Fe3+, Lf can exist free of Fe3+ (apo-Lf) or associated with Fe3+ (holo-Lf), and it has a different three-dimensional conformation depending on whether or not it is bound to Fe3+.
Apo-Lf has an open conformation, while holo-Lf is a closed molecule with greater resistance to proteolysis. Because of the common structural framework among Lfs, it is possible to model their conformations using crystallographic data from other Lf species (Fig. a). The amino acids directly involved at the iron-binding site in each lobe are Asp60, Tyr92, Tyr192 and His253, while Arg121 is involved in binding the CO32- ion (Fig. b). Lf is a basic, positively charged protein with a pI of 8.0–8.5. The primary structure of Lf shows the number and position of Cys residues that allow the formation of intramolecular disulfide bridges; Asn residues in the N- and C-terminal lobes provide several potential N-glycosylation sites.
Protein Aminoacids Percentage
As shown in the graph, lactoferrin shows a higher percentage of Alanin, Cystein, Leucin (which are non-polar aminoacids) and Arginine (a basic aminoacid).
Isoleucin and Methionin are the two non-polar aminoacids that are less present in lactoferrin. Threonin (neutral) and Histidin are also less frequent in the percentage if compared to the average composition of proteins.
SYNTHESIS AND TURNOVER
Biosynthesis
Lactoferrin is produced in neutrophils and stored, in the iron depleted state, in the specific granules (also known as secundary granules) and possibly in the tertiary granules. This protein, unlike myeloperoxidase and some other granular products, is not synthesized as a larger precursor and was found to be unphosphorylated. Lactoferrin transfer to its storage granules is dependent on acidification mechanisms and occurs through the medial and transcisternae of the Golgi apparatus. It therefore appears to be processed like proteins destined for secretion.
Turnover
The neutrophil lactoferrin within these granules has two destinations: it can either be secreted into the surrounding tissues or blood, or the granules can fuse with phagosomes. Secretion from polymorphonuclear cells into the circulation is dependent on degranulation factors, which in turn appear to be dependent on the activation of guanylate cyclase, cGMP and proteinkinase C (calcium dependent). This occurs in both aerobic and anaerobic conditions, is unaffected by the presence of hydrogen sulphide and is stimulated by IL-8 and surface bound IgG.
Distribution
Lactoferrin is present in plasma in relatively low concentrations (<1 mg/mL), with substantially higher levels being found in colostrum, human breast milk, and seminal plasma. Markedly higher levels occur in cord blood, tears, and vaginal mucus.
Lactoferrin levels in amniotic fluid were found to be undetectable before the 20th week of pregnancy. A significant increase occurs around week 30, whereafter it remains high until term.
Catabolism
Lactoferrin removal from circulation appears to occur in one of two ways: first, lactoferrin can be removed from the circulation, as well as from the interstitial spaces, by what would appear to be receptor-mediated endocytosis into phagocytic cells such as macrophages, monocytes and other cells of the RES, with subsequent transfer of the iron to ferritin. The alternative way of lactoferrin removal would be its direct uptake by the liver through an iron saturation-independent, clathrin-dependent, calcium-dependent process of endocytosis.
REGULATION
Lf has been identified in several tissues in both humans and animals. The amount of Lf synthesized in the mammary gland is controlled by prolactin. Its mRNA levels vary by tissue, suggesting tissue- or cell-specific regulation. The Lf full coding regions of 60 different species were analyzed, and it was found that the length of the gene varies from species to species (from 2055 to 2190 residues) due to deletions, insertions and mutations in the stop codon. Lf is expressed both constitutively and inducibly. It is constitutively expressed on mucosal surfaces, while in some tissues it is induced by external agents.
The Lf promoter contains an estrogen responsive element (REF), and is consequently positively regulated by estrogen. The chicken ovalbumin upstream promoter transcription factor (COUP-TF) binding element overlaps the ERE of the lactoferrin gene. COUP-TF binds to this element and competitively inhibits binding of the estrogen receptor to the lactoferrin ERE, thereby inhibiting transcription of the lactoferrin gene. Another negative regulator of LF gene transcription is repressor of estrogen receptor activity (REA). The absence of REA increases the expression of estrogen-induced Lf by up to 100-fold.
Lf can also be induced by compounds other than estrogens, such as retinoic acid, which stimulates gene expression in embryonic cells. Lf expression is upregulated by estrogen with a magnitude of response that is cell-type-specific, and it is also upregulated by retinoic acids. Transcription factors such as Ets, PU.1, C/EBP, CDP, and KLF5 also modulate Lf gene expression, mainly in myeloid cells. Lf expression is also modulated by oxidative stress, in response to infection, or during the early steps of embryogenesis.
CELLULAR FUNCTIONS
Lactoferrin shows a huge amount of different functions because of its several properties:
Antimicrobial activities
Several functions have been attributed to Lf. It is considered to be a key component of the innate host defense system because it can respond to a variety of physiological and environmental changes. The structural features of Lf provide additional functionalities beyond the Fe homeostasis function common to all transferrins. Specifically, Lf exhibits strong antimicrobial activity against a broad spectrum of bacteria (Gram+ and Gram−), fungi, yeasts, viruses and parasites, although it seems to promote the growth of beneficial bacteria like Lactobacillus and Bifidobacteria. One of the first antimicrobial properties discovered for Lf was its role in sequestering iron from bacterial pathogens. This was believed to be the sole antimicrobial action of lactoferrin because apo-lactoferrin possessed antibacterial activity. It was later demonstrated that lactoferrin can also kill microorganisms through an iron-independent mechanism in which lactoferrin directly interacts with the bacterial cell surface.
Immunomodulatory activity
Lf displays immunological properties that influence both innate and acquired immunities. Its relationship with the immune system is evident from the fact that people with congenital or acquired Lf deficiency have recurring infections. Oral administration of bLf seems to influence mucosal and systemic immune responses in mice. Lf can modulate both specific and non-specific expression of antimicrobial proteins, pattern recognition receptors and lymphocyte movement related proteins. The role that Lf plays in regulating innate immune responses confirms its importance as a first line host defense mechanism against invading pathogens, modulating both acute and chronic inflammation. Most intriguing is the ability of Lf to induce mediators from innate immune cells that subsequently impact adaptive immune cell function. Lf's positive charge allows it to bind to negatively charged molecules on the surface of various cells of the immune system, and it has been suggested that this association can trigger signaling pathways that lead to cellular responses such as activation, differentiation and proliferation. Lf can be transported into the nucleus, where it can bind DNA and activate different signaling pathways.
In addition to inducing systemic immunity, Lf can promote skin immunity and inhibit allergic responses. It activates the immune system against skin allergens, causing dose-dependent inhibition of Langerhans cell migration and the accumulation of dendritic cells in lymph nodes. Leukocytes exposed to Lf modulate their cytokine production; proinflammatory cytokines, TNF-α, IL-6, and IL-1β can be modulated by Lf to increase or decrease. Production of these factors is dependent upon the type of signal recognized by the immune system. At the cellular level, Lf increases the number of natural killer (NK) and adaptative (T strain CD4+ and CD8+) cells, boosts the recruitment of polymorphonuclear cells (PMNs) in the blood, induces phagocytosis, and can modulate the myelopoietic process. It is well documented that IL-12 plays an important role in driving development of helper T-cell type 1 immunity. Therefore, the role of Lf in the regulation of proinflamatory cytokines and IL-12 clearly demonstrates communication between innate and adaptative immune responses.
Anticarcinogetic activity
The anti-tumor properties of Lf were discovered about a decade ago and have been confirmed by numerous laboratory studies which have shown that bovine lactoferrin (bLf) significantly reduces chemically induced tumorigenesis when administered orally to rodents. Since then, human clinical studies are showing that ingestion of LF can have a beneficial effect against progression of cancer. bLf possesses antimetastatic effects and inhibits the growth of transplanted tumors. Similar to its role in inflammation, Lf has the ability to modulate the production of cytokines in cancer. Lf can induce apoptosis and arrest tumor growth in vitro; it can also block the transition from G1 to S in the cell cycle of malignant cells. Additionally, treatment of tumors in mice with recombinant human Lf (rhLf) inhibits their growth, increases the levels of anticarcinogenic cytokines such as IL-18, and activates NK cells and CD8+ T lymphocytes. Recently, colorectal cancer was inhibited by bLf in animal models, and human Lf reduced the risk of colon carcinogenesis as demonstrated by a clinical trial. Increasing evidence suggests that inhibition of the Akt signaling pathway might be a promising strategy for cancer treatment. In breast cancer, Lf is able to limit the growth of tumor cells, and addition of exogenous Lf to the culture media of breast cancer cell lines (MDA-MB-231) induced cell cycle arrest at the G1/S transition. Additionally, Lf induced growth arrest and nuclear accumulation of Smad-2 in HeLa cells. The ability of bovine Lfcin to induce apoptosis in THP-1 human monocytic leukemic cells has also been demonstrated. Although the results achieved by several researchers point to a clear anti-tumor role for Lf, the mechanisms by which it exerts these effects are not fully understood. Thus, further work on this subject is required.
Enzimatic activity
Lf has the ability to function as an enzyme in some catalytic reactions. A remarkable similarity of certain motifs between Lf and ribonuclease A has been observed. Lf has DNA binding properties and can act in transcriptional activation of specific DNA sequences or as a mediator of signal transduction. Lf has the highest levels of amylase and ATPase activities of all the milk proteins; however, these are not its only enzymatic activities. Indeed, Lf exhibits a wide variety of activities, which can be attributed to variations in the nature of its protein characteristics; Lf has multiple isoforms, degrees of glycosylation, variations in tertiary structure (holo- or apo-Lf) and degrees of oligomerization. The discovery of Lf's enzymatic properties has helped to elucidate its many physiological functions.
Lactoferricins
It has been identified a region of amino acids at the N-terminus that retains its biological activity when separated from the full molecule; this was termed lactoferricin (Lfc-B) and was shown to exhibit greater antimicrobial activity than Lf. The activity that is exerted by this region corresponds to residues 17–41 of bLf. The region also includes two Cys residues linked by a disulfide bridge that contains many hydrophobic and positively charged residues.
The tertiary structure of Lfcin is markedly different when compared to its homologous region of intact Lf. An in situ model of hLfcin and a solvated version show structural differences at their N- and C-termini. A single β-sheet strand replaces the long α-helix observed in the Lf structure; such a structure may be better suited to recognize bacterial membrane topology. Additionally, the bLfcin peptide contains an alpha-helix and this region retains high homology among other species of mammals and corresponds to amino acids 12–48.
CLINICAL USE
Large scale production
The physiological capabilities of Lf in host defense combined with current pharmaceutical and nutritional needs have led to the classification of Lf as a nutraceutical protein, and for several decades, investigators have looked for the most convenient way to produce it.
Thus, the development of commercial production strategies for the production of recombinant Lf as a safe, effective drug and nutraceutical protein is one of the major goals in both research and industry. Purification of Lf depends on the intrinsic properties and features of the molecule, such as its net positive charge, its ability to bind Fe3+ and its glycosylation state. However, the need for larger amounts of Lf has led to the development of strategies to obtain a recombinant form of the protein (rLf).
In global biotechnology, there are now three major competing approaches for the production of rLf: production in transgenic animal milk, production in microscopic fungi, and production in plants. To date, several rLf expression systems have been developed that utilize both prokaryotic and eukaryotic organisms. The first expression systems utilized Bay Hamster Kidney Cells to express human lactoferrin.
The transformation of the filamentous fungi Aspergillus awamory allowed for the expression of hLf. Expression systems were developed in yeasts, bacteria, insects and plants, which have produced human recombinant Lf (rhLf), as well as Lf peptides, including Lfc, which reached expression levels of 0.1 mg/L in a plant model and 1200 mg/L in P. pastoris. Use of viral vectors has allowed for the expression of Lf by insect infection, either in cell culture or directly in the organism, where the expression of both hLf and pLf has been successful. This practice has resulted in the production of transgenic Spodoptera frugiperda and Bombyx mori, which express 205 μg of pLf per infected pupa and up to 65 mg/L in larvae. Lf has also been expressed in higher eukaryotic organisms, including both plants and animals. Using microinjection and direct infection with viral vectors in the mammary gland, transgenic animals have been created that produce milk containing recombinant Lf. These animals include goats, mice, rabbits and cows. Levels of up to 2 g/L have been achieved in transgenic goat milk.
Molecular farming, which involves the utilization of plants as bioreactors, is well depicted as a tool for the production of valuable therapeutic and industrial proteins. This process is advantageous because of the lack of contamination from human or animal pathogens and endotoxins in the final product. Furthermore, higher plants are able to synthesize proteins with the proper folding, glycosylation and functional activity, and plant cells can direct the protein of interest to environments that reduce its degradation. hLf was expressed in plant expression models and bLf was only expressed in tobacco plants. However, none of these nuclear transformation methods are able to promote the accumulation of a large amount of rLf. Therefore, new promoters or regulatory elements as well as other transformation methods should be tested to increase accumulation and stability of recombinant proteins. The expression of Lf in plant models could significantly improve crop quality by increasing its resistance to some diseases; additionally, it provides a source for high quality Lf protein.
Clinical use
Lactoferrin, thanks to its properties, is already used in the treatment of several pathologies such as wound closure, gastric wound healing, sepsis, viral infection.
Because of its property as a new anti-carcinogetic molecule it has been used in clinical trials in cancer, in particular in solid tumours, breast cancer, lung carcinoma (NSCLC), renal cell carcinoma.
Diagnostic use
Lf can also be used as a molecular marker; detection of urinary Lf via electrochemical immunosensors aids in the diagnosis of urinary tract infections. Increased levels of Lf serve as a clinical marker of inflammatory Severe Acute Respiratory Syndrome or septicemia.
Lactoferrin is released in regulation response to inflammation, any such event will increase its levels through neutrophil activation and degranulation. The diagnostic application of these levels is similar to that of several different indicators of immune stimulation, such as neopterin and elastase-a1-proteinase inhibitor complex and others, rendering lactoferrin levels relatively nonspecific. A number of clinical applications are nonetheless described in the literature. These are mostly of diagnostic or prognosticpredictive value and include plasma lactoferrin determination as an index of the total blood neutrophil pool or neutrophil kinetics as a tool in the diagnosis of chronic myeloid leukemia, granulocytic leukemia, chronic calcifying pancreatitis, cystic fibrosis ,septicemia,congenital aplasia of the vasa deferentia and seminal vesicles, schizophrenia,joint inflammation and cartilage degradation,psoriasis and rheumatoid arthritis.
Lactoferrin antibodies have been demonstrated in patients with Felty’s syndrome, and the detection of these antibodies may prove useful in its diagnosis. It has further been suggested that b-lactoferrin/RNase and g-lactoferrin/RNase may be of value in the detection of breast cancer.
LIPOPOLYSACCHARIDES
Lipopolysaccharides (LPS) , also known as lipoglycans, a major constituent of the Gram-negative bacteria outer membrane, is one of the most potent inducer of the innate immune response. Recognition of various form of LPS from different strains of Gram-negative bacteria triggers a signaling cascade that results in the release of pro-inflammatory mediators, such as cytokines and chemokines, as well as small molecules, such as lipid mediators and reactive oxygen species. LPS is known to initiate the morbidity and mortality associated with Gram-negative sepsis, as well as to modulate a myriad other host innate inflammatory responses. Specifically, LPS has been characterized as the ‘prototypical stimuli’ for host activation through myeloid cells (neutrophils, monocytes, macrophages, dendritic cells) and non-myeloid cells (fibroblasts, platelets), as well as other innate host defense mechanisms, such as serum complement, and specific components within the intrinsic coagulation pathway.
LPS is ubiquitous within our environment, in vivo and in vitro, and can express potent bioactivity in extremely small amounts. This bacterial component is a complex molecule consisting of three parts: a core oligosaccharide, a distal hydrophilic O side chain, and a highly conserved lipid A portion. The lipid A moiety is the main pathogen-associated molecular pattern of LPS, and is responsible for its toxic pro-inflammatory properties.
TOLL-LIKE RECEPTORS
Toll-like receptors patways are major regulators of the innate immune response. Perharps the most widely studied TLR interaction involves LPS through the TLR4 complex in which LPS initiates gene activation via distinct signaling pathways. Co-expression of glycosylphosphatidylinositol-anchored proteins MD-2, CD-14 and CD11b/CD18 (Mac-1, CR3) with TLR4 are essential to the functional integrity of the LPS receptor complex. In addition LPS-binding protein (LBP) mediates the interaction of LPS with CD14 and CD11b/CD18. This event induces activation of the toll/IL-1 receptor (TIR) domains associated with either Toll/IL-1 receptor domain-containing adapter protein (TIRAP) and myeloid differentiation marker 88 (MyD88) or TIR domain-containing adaptor molecule (TRAM).
INFLAMMATORY PROCESS
- bacterial entry (LPS)
- iron release from necrotic cells and granulocytes
- ROS production
- tissutal damage
- release of pro-inflammatory cytokines (IL-1, IL-6, TNF-α) and nitric oxide (NO)
- expression of adhesion molecules (ICAM, Selectin) and chemokines (IL-8)
- adhesion of APC and granulocytes
Lactoferrin can interfere with this physiological pathway at different stages.
INTERACTIONS
Some of the possible mechanisms of interaction are summarized here:
Thus, lactoferrin can have both an immuno-stimulator and immuno-suppressor function depending on the microenviroment. As well, it can fulfil its role by a direct action with LPS or just activating different LPS-independent pathways. (Puddu et al, 2010)
1. LACTOFERRIN RECEPTORS
Since lactoferrin is a multifunctional protein, it is quite hard to evaluate all its functions and to discover all receptors. It has been proved that lactoferrin is able to ligate many receptors in different districts of the body.
The interaction between LF and its receptors can trasduce intracellular signals in order to evoke LF-dependent/LPS-independent biological responses.
- Intelectin
Intelectin, also called SI-hLfR (small intestine human lactoferrin receptor) is a receptor which has been purified from brush-border membranes of the small intestine using affinity chromatography assays.
It has a molecular weight of approximately 130 kDa and consists of a single polypeptide chain.
Recent studies have shown that intelectin works as a GPI-anchored protein and is able to mediate different functions such as transmembrane signaling and endocytosis.
The SI-LfR might facilitate iron absorption and act as a transcriptional factor to enhance biosyntesis of some signal proteins such as caspase 1 and IL-18. (A)
Orally administered LF has been shown to enhance mucosal CD4, CD8 and NK cells, which is likely to be mediated by the SI-LfR. (B)
Lactoferrin can, in fact, enhance the immunology activity by stimulating the syntesis of pro-inflammatory cytokines (IL-18, TNF-α, IL-1β and IFN-γ), useful in the anti-bacterial response caused by LPS.
Other important properties concern the inhibition of angiogenesis and metastatization processes.
- Nucleolin
Nucleolin is a 105kDa nuclear protein that has also been descrived as a cell-surface receptor for several ligands, such as matrix laminin 1 and midkin. However it is unlikely that nucleolin directly transduces the intracellular signals in response to LF, because nucleolin lacks the membrane-spanning region and the cytoplasmatic domain responsible for signal transduction.
It has been suggested that nucleolin might bind lactoferrin and act as a carrier to take LF inside the nucleus where it might activate the trascription of related genes.
- Low-density lipoprotein receptor-related protein (LRP/LRP1)
LRP is a membrane glycoprotein that is abundantly expressed on hepatocytes, neurons, smooth muscle cells and fibroblasts.
LF covalently binds domains I and IV. LRP is known as an endocytotic receptor and partecipates in hepatocyte uptake of lipoproteins containing triglycerides and cholesterol. It has also been proved that removal of bovine lactoferrin (bLF) from plasma is mediated by LRP which may have a fundamental role (mediated by the liver) in the decreasing bioavailability of lactoferrin when administered intravenously.
- CD14
Monocytes and dendritic cells (DCs) are known to express CD14, a LPS receptor that activated the immune system (mCD14).
Lactoferrin directly interacts with the solube form of CD14 (sCD14) and protects animal from septic shock induced by LPS.
(Suzuki et al, 2005)
2. IMMUNOSUPPRESSIVE PATHWAYS
DC-SIGN is is a C-type lectin receptor present on both macrophages and dendritic cells. DC-SIGN recognises and binds to mannose type carbohydrates, a class of pathogen associated molecular patterns (PAMPs) commonly found on viruses, bacteria and fungi, activating phagocytosis.
DC-SIGN directly interferes with the TLR-mediated pro-inflammatory signaling by the inhibition of NF-κB transcription.
Although the direct LF binding to C-type lectin receptors has been demonstrated for DC-SIGN in HIV transmission, we cannot exclude the possibility that some LF inhibitory effects concerning LPS could be due to its activation of this class of receptors. (Groot et al, 2005)
Since glycans with polymannosidic structures are specific to bLF and not found on hLF, this example clearly illustrates a typical non-specific effect of exogenous LF in in vivo experiments.
3. LF-LPS BINDING
At the N-terminus of the molecule, LF contains a highly basic arginin-rich region which binds to a variety of anionic biological molecules.
- Arg2-Arg3-Arg4-Arg5: binds to polyanions such as Lipid A, heparin, lysozime and DNA
- Arg28-Lys29-Val30-Arg31(-Gly32-Pro33-Pro34): binds to E.coli LPS 055B5
These two regions bind LPS with a very high affinity (Kd=3.6±1nM)
There is also a C-lobe region which is able to bind LPS with a lower affinity (Kd=390±20nM) and it is exposed at high protein concentrations. (Baveye et al, 2000)
DIRECT LACTOFERRIN-LPS INTERACTION
It has been shown that bovine lactoferrin inhibits the production of TNF-α. Bovine lactoferrin shows 60% of homology with human lactoferrin (Van Berkel et al, 2002) and so it can be assumed that hLF might play the same role.
Cell proliferation and nitric oxide (NO) production were determined in the RAW 264.7 (murine macrophage) cell model.
These results show that bLF alone is not able to influence murine macrophage cell stress or production of NO. In contrast, exposure of RAW 264.7 cells to LPS (1 μg/ml) significantly increased NO produtcion and reduced cell proliferation. However bLF significantly down-regulates nitric oxide production or protects against LPS-induced immune cell stress, in a dose response manner. (Fig B)
To evaluate the endotoxin-neutralizing capability of LF by regulating production of pro-inflammatory cytokine TNF-α, bLF or bovin serum albumine (BSA) were added at different concentrations to LPS-challenged (100ng/ml) differentiated THP-1 cells.
Results show that bLF but not BSA significantly reduced TNF-α release from THP-1 activated cells in a dose-dependent way. (Fig A)
To judge whether bLF blocks LPS receptor on THP-1 cells (human acute monocytic leukemia cell line) or detoxifies LPS by binding it directly, pre-treated THP-1 cells that were incubated with bLF for 1h prior to challenge with LPS for 4 hours were compared with bLF and LPS co-treatment for 4h. The figure shows that a significant reduction in TNF-α production occurred during bLF+LPS co-treatment rather than during bLF pre-treatment.
These results confirm that the key reaction mediated by bLF was not directly on the THP-1 receptors; rather, the anti-inflammation actions were mediated by neutralization of LPS (Fig B).
(Tian et al, 2010)
4. ACTIVATION OF THE IMMUNE SYSTEM
When tissues are infected, reactive oxygen species are abundantly produced, either by generated by free iron released from necrosed tissues or overproduced by activated granulocytes. This oxydative burst, together with the excessive release of pro-inflammatory cytokines, mainly IL-1 and TNF-α, highly contributes to the pathogenesis of the septic shock. The protective anti-inflammatory activity of LF lies in its ability to bind free ferric ions but also exogenous pro-inflammatory bacterial components (such as LPS) and their receptors. Binding to pro-inflammatory molecules has down-regulating effects on both the activation and recruitment of immune cells in inflammed tissues. (Legrand and Mazurier, 2010)
Although a massive release of both pro and anti-inflammatory cytokines upon infection is regarded as the major cause of organ dysfunction and death, a significant decrease of the cytokine activity may be beneficial in avoiding lethal effects of endotoxemia but not bacteremia.
It has been studied the role of LF as a protective molecule in a LPS-induced bacteriemia in mice. The table below shows that pretreatment of mice with LF before i.v. E.coli administration (lethal dose of 5×10^8) is associated with a significant 60% decrease of TNF-α level, measured at 2h after E.coli injection. In contrast, 2h pretreatment with LF causes a very strong (15 fold) elevation of TNF-α activity in the circulation.
The protective role of LF against a lethal dose of LPS has been suggested to be due to granulocyte activation.
Authors measured then neutrophil recruitment and turnover estrablished by the content of cells of the neutrophil lineage in the bone marrow and circulating blood following 24h and 2h i.v. pretreatment of mice with LF.
Results show that LF causes an increase of all cell types of the lineage (both mature and bands).
These results give proof of the protective activity of LF against E.coli infection. This may results from a significant mobilization of the neutrophil pool in the bone marrow and circulating blood rather than a direct interaction with LPS.
Furthermore LF strongly elicit increasing IL-1 serum levels and this could represent an additional protective action since IL-1, given 24h before bacterial challenge, enhanced resistance to infection, probably by elicitation of acute phase protein synthesis. The protective effect of LF, given 24h before bacteria, may also be in part explained by a reduction of TNF-α levels.
(Zimecki et al, 2004)
5. INTERACTION WITH sCD14
The ability of LF to bind free LPS may account, in part, for the anti-inflammation activities of the protein. However, since optimal protection of animals against septic shock requires a 12 to 24h pre-injection it has been assumed that other mechanisms are involved in addiction to simple LPS scavenging.
Previously it has been illustrated the region of hLF responsible of the binding with LPS.
Human sCD14 shows acidic residues 35AVEVE39 and 53RVDADADPRQY63 which are involved in the interactions with LPS, while amino acids residues 7ELDDEDF13 are essential for cell activation.
It has been demonstrated, that all or part of the basic or acidic amphipatic stretches present in hLF and sCD14 are responsible for the high-affinity interactions between these two glycoproteins, resulting in the formation of a stable sCD14-hLF complex (Kd=16±7nM).
Endothelial cells (HUVEC) were used to prove the postulated role of LF in the modulation of endothelial adhesion molecules such as E-selectin and ICAM-1 normally induced by the LPS-sCD14 complex.
HUVEC exhibit a low basal expression of ICAM-1, which was not significantly increased in the presence of sCD14, hLf, or both proteins (negative controls) or LPS. When compared to unstimulated cells, a fivefold-higher level of ICAM-1 was detected in the presence of sCD14-LPS. This demonstrated that the sCD14-LPS complex induced the expression of ICAM-1 on endothelial cells. Interestingly, the ICAM-1 expression induced by the sCD14-LPS complex on HUVEC was significantly decreased in the presence of hLf (50 μg/ml). Taking the expression level induced by sCD14-LPS as a reference, a 51% ± 2% inhibition of the ICAM-1 expression was calculated when hLf was incubated with sCD14 and LPS at the same time. This inhibition was similar to that detected when hLf was mixed with LPS 30 min prior to sCD14 (56% ± 14%) or when an sCD14-LPS complex was preformed before the addition to hLf (53% ± 19%). A higher inhibition (81% ± 5%) was measured when LPS was added after the preincubation of sCD14 with hLF. In the latter case the expression was equal to the LPS-alone induced ICAM-1 expression giving a further proof that hLF is able to ligate sCD14 taking it away from the microenvironment.
Similar results were obtained with the E-selectin expression.
Lf may compete with LBP for the binding of LPS to mCD14. Here it has been reported the ability of hLf to bind to sCD14 and to inhibit at least one of its cell activation functions, the expression of ICAM-1 and E-selectin, two molecules essential to the recruitment process of leukocytes. Since CD14 plays an essential role in the endotoxin-mediated inflammatory response, it is possible that hLf interferes with the expression and/or activation of other molecules involved in the leucocyte recruitment process such as VCAM-1, integrins, and chemokines. The interactions of hLf with CD14 may also account for its previously reported effects on the expression of proinflammatory cytokines tumor necrosis factor alpha, IL-1, and IL-6. Therefore, hLf may modulate the recruitment of immune cells on inflammatory sites.
(Baveye et al, 2000)
CONCLUSION
Lactoferrin is a really important protein in the modulation of the immune system.
It sometimes plays a role as an inducer of inflammatory mediators (TLR, cytokines) and triggers the maturation of granulocytes and dendritic cells.
In other cases (i.e. sepsis) it shows the opposite role acting as an immuno suppressive molecule.
Comparison of different articles can often be problematic because of all the differences in conditions, times, reagents and their concentrations.
It has been hypothesized, though, that the crucial factor that directs the response, rather than the LF or LPS itselves, is more likely the LF/LPS ratio at a particular site of inflammation or in a particular biologic fluid:
[LF]
-----
[LPS]
Lactoferrin possesses pleiotropic roles which turn it into either a weapon or a shield in the host defense system.
When LPS concentration is high (i.e. during sepsis) LF acts as an immuno-suppressive molecule removing LPS from the environment, thus reducing the activation of the TLR pathway.
On the contrary when the inflammation is localized and LF is released from PMNs, it shows a pro-inflammatory activity in order to trigger the response of the host system against bacterial infection.
In synthesis:
- High ratio: immunostimulation
- Low ratio: immunosuppression
Many mechanisms concerning LF still remain unknown. It is crucial, therefore, to better investigate such molecular pathways for a complete understanding of the functions of this protein.
Lastly, since only traces of LPS are sufficient to trigger activation of cells and significant amounts of LPS were detected in commercial preparations of LF, it is very likely that many studies actually reported the effect of LPS or the LF-LPS complex on immune cells rather than the role of LF itself.
Scheda realizzata da Gabriele Casule ed Elena De Amicis
