Annick Pierce

Lactoferrin and host defence: an overview of its immuno-modulating and anti-inflammatory properties.

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins that is abundantly expressed and secreted from glandular epithelial cells. In secretions, such as milk and fluids of the intestinal tract, lactoferrin is an important component of the first line of host defence. During the inflammatory process, lactoferrin, a prominent component of the secondary granules of neutrophils (PMNs), is released in infected tissues and in blood and then it is rapidly cleared by the liver. In addition to the antimicrobial properties of lactoferrin, a set of studies has focused on its ability to modulate the inflammatory process and the overall immune response. Though many in vitro and in vivo studies report clear regulation of the immune response and protective effect against infection and septic shock by lactoferrin, elucidation of all the cellular and molecular mechanisms of action is far from being achieved. 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.

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 α or 1-methyl-4-phenylpyridinium treatment

The presence of the iron-binding protein lactoferrin (Lf) in some specific areas of the central nervous system and particularly in the normal human substantia nigra, where it is found in dopaminergic (DA) neurons and some glial cells, led us to investigate Lf synthesis in this area. Lf mRNA were identified using in situ hybridization and found in small ameboid cells. These cells were identified using immunocytochemistry as activated microglia since they exhibited macrophage markers such as the CD68 and the CR1 antigens. Double immunofluorescent labeling confirmed that the two Lf immunostained cell populations were activated microglia and DA neurons. Since activated microglia contained both Lf and its messenger, these cells are the Lf producing cells. The presence of Lf in DA neurons in which no Lf messengers were visible, might be due to an endocytosis mechanism, DA neurons probably internalizing Lf produced in microglial cells located in their neighborhood. In neuropathological disorders, such as Alzheimers and Parkinsons diseases, inflammatory process and oxidative stress are events that contribute to neuronal death. Since Lf concentration increases during these pathologies, we studied the level of Lf expression under these different stresses and showed, using RT-PCR, that the immortalized human embryonic microglial CHME cell line produced Lf transcripts under tumor necrosis factor alpha or 1-methyl-4-phenylpyridinium treatment whereas untreated cells did not. These data confirm that Lf is produced only when microglia are activated.

Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer

We investigated the expression levels of human lactoferrin (Lf), a steroid hormone‐inducible gene product the expression of which is often altered during oncogenesis, and of Δ‐lactoferrin (ΔLf), its alternative isoform, which has been shown to be absent from tumor cell lines in commonly used human breast epithelial cell lines, using semiquantitative RT‐PCR. Both mRNAs were detected but with levels of expression lower than those found in normal breast epithelial cells. This downregulation was much more visible for ΔLf since its expression was either significantly diminished (BT‐20, MCF‐7 cell lines) or practically absent (MDA‐MB‐231, T‐47D, HBL 100 cell lines). In order to determine whether Lf gene products are useful prognosic tools, we further analyzed their expression levels in 99 primary breast cancer biopsies. ΔLf transcripts were found in all of the samples, whereas Lf transcripts were found in 88% of them. Lf and ΔLf expression levels were positively correlated (p = 0.003). Lf expression was related to tumor type with a higher recovery in lobular‐type tumors (p = 0.04). ΔLf expression was related to the histoprognostic grading (p = 0.02). In univariate analyses, ΔLf and Lf expressions were prognosis parameters, high concentrations being associated with a longer overall survival.

Lactoferrin is synthesized by mouse brain tissue and its expression is enhanced after MPTP treatment

The biological role and origin of human lactoferrin (Lf) within the brain in normal and disease processes are as yet uncharted. In this context the origin and expression of brain Lf in normal and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice were investigated using immunohisto chemistry, PCR amplification and in situ hybridization. Lf immunostaining was observed both on sections of mouse lactating mammary gland, which was used as a positive control, and brains from young, adult and aged mice. Lf immunoreactivity was present in the pituitary gland, the hippocampus and the cortex of mouse brains and to a greater extent in older mice. After reverse transcription, Lf transcripts were also found in these brain sections. Lf distribution and expression in the MPTP-induced parkinsonian mouse model were next investigated. A marked depletion of dopamine and its metabolites: dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxy indole acetic acid (5-HIAA) occurs in the high dose MPTP-treated mice. The level of Lf expression was found to be greatly increased in the same animals but Lf immunoreactivity detected in the same brain region was not found increased in the affected areas.

Advances in lactoferrin research

Three-Dimensional Structure of Lactoferrin: Implications for Function, Including Comparisons with Transferrin E.N. Baker, et al. Structures of Buffalo and Mare Lactoferrins: Similarities, Differences, and Flexibility A.K. Sharma, et al. Direct Detection and Quantitative Determination of Bovine Lactoferricin and Lactoferrin Fragments in Human Gastric Contents by Affinity Mass Spectrometry H. Kuwata, et al. Analysis of Bovine Lactoferrin in Whey Using Capillary Electrophoresis (CE) and Micellar Electrokinetic Chromatography (MEKC) P. Riechel, et al. Structural and Immunochemical Studies on Bovine Lactoferrin Fragments K. Shimazaki, et al. Role of the First N-Terminal Basic Cluster of Human Lactoferrin (R2R3R4R5) in the Interactions with the Jurkat Human Lymphoblastic T-Cells D. Legrand, et al. Glycation Ligand Binding Motif in Lactoferrin: Implications in Diabetic Infection Y.M. Li. Mouse Lactoferrin Gene: Promoter-Specific Regulation by EGF and cDNA Cloning of the EGF-Response-Element Binding Protein C. Teng, et al. Cloning of Human Genomic Lactoferrin Sequence and Expression in the Mammary Glands of Transgenic Animals S.J. Kim, et al. 33 Additional Articles. Index.

Tumor Necrosis Factor-α Increases Lactoferrin Transcytosis Through the Blood-Brain Barrier

Abstract : Lactoferrin (Lf) is an iron‐binding protein involved in host defense against infection and severe inflammation, which accumulates in the brain during neurodegenerative disorders. Prior to determining Lf function in pathological brain tissues, we investigated its transport through the blood‐brain barrier (BBB) in inflammatory conditions. For this purpose, we used a reconstituted BBB model consisting of the coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes in the presence of tumor necrosis factor‐α (TNF‐α). As TNF‐α can be either synthesized by brain glial cells or present in circulating blood, BBCECs were exposed to this cytokine at their luminal or abluminal side. We have been able to demonstrate that in the presence of TNF‐α, whatever the type of exposure, BBCECs were activated and Lf transport through the activated BBCECs was markedly increased. Lf was recovered intact at the abluminal side of the cells, suggesting that increased Lf accumulation may occur in immune‐mediated pathophysiology. This process was transient as 20 h later, cells were in a resting state and Lf transendothelial traffic was back to normal. The enhancement of Lf transcytosis seems not to involve the up‐regulation of the Lf receptor but rather an increase in the rate of transendothelial transport.

Human delta-lactoferrin is a transcription factor that enhances Skp1 (S-phase kinase-associated protein) gene expression.

Delta‐lactoferrin is a cytoplasmic lactoferrin isoform that can locate to the nucleus, provoking antiproliferative effects and cell cycle arrest in S phase. Using macroarrays, the expression of genes involved in the G1/S transition was examined. Among these, Skp1 showed 2–3‐fold increased expression at both the mRNA and protein levels. Skp1 (S‐phase kinase‐associated protein) belongs to the Skp1/Cullin‐1/F‐box ubiquitin ligase complex responsible for the ubiquitination of cellular regulators leading to their proteolysis. Skp1 overexpression was also found after delta‐lactoferrin transient transfection in other cell lines (HeLa, MDA‐MB‐231, HEK 293) at comparable levels. Analysis of the Skp1 promoter detected two sequences that were 90% identical to those previously known to interact with lactoferrin, the secretory isoform of delta‐lactoferrin (GGCACTGTAC‐S1Skp1, located at − 1067 bp, and TAGAAGTCAA‐S2Skp1, at − 646 bp). Both gel shift and chromatin immunoprecipitation assays demonstrated that delta‐lactoferrin interacts in vitro and in vivo specifically with these sequences. Reporter gene analysis confirmed that delta‐lactoferrin recognizes both sequences within the Skp1 promoter, with a higher activity on S1Skp1. Deletion of both sequences totally abolished delta‐lactoferrin transcriptional activity, identifying them as delta‐lactoferrin‐responsive elements. Delta‐lactoferrin enters the nucleus via a short bipartite RRSDTSLTWNSVKGKK(417–432) nuclear localization signal sequence, which was demonstrated to be functional using mutants. Our results show that delta‐lactoferrin binds to the Skp1 promoter at two different sites, and that these interactions lead to its transcriptional activation. By increasing Skp1 gene expression, delta‐lactoferrin may regulate cell cycle progression via control of the proteasomal degradation of S‐phase actors.

Characterization of Two Kinds of Lactotransferrin (Lactoferrin) Receptors on Different Target Cells

Lactotransferrin (Lf), an iron-binding glycoprotein present as a major component in the specific granules of human neutrophilic granulocytes is released in the blood during the acute phase of infection and participates in the regulation of the host-defence mechanisms. Our previous observations (Mazurier et al., 1989) showing i) that the activation by PHA of T-lymphocytes induces the appearance at the cell surface of Lf-receptors which are absent from the membrane of resting lymphocytes and ii) that Lf becomes a growth factor for the activated lymphocytes, led us to undertake a series of researches on the presence of Lf receptors at the surface of different blood cells. Characterization of Lf receptors was performed by flow cytofluorimetry using either Lf labelled on its glycan moiety with fluorescein or purified anti-lymphocyte Lf receptor antibodies. High affinity receptors for Lf were characterized only at the surface of human activated lymphocytes and of non-activated platelets. These two receptors possess common physicochemical properties and antigenic epitopes. Low affinity receptors for Lf were characterized on monocytes, eosinophils and neutrophils. These receptors are immunologically different from those found on activated lymphocytes and on non-activated platelets. Cell-lines of human lymphocyte T and megakaryocyte possess lactotransferrin receptors whose properties are similar to those found on peripheral blood cells. The soluble form of the receptor identified in the lymphocytes T culture medium possesses a molecular mass close to that of the membrane receptor suggesting that the cytoplasmic tail of the receptor should be very short.

Expression of delta-lactoferrin induces cell cycle arrest

Delta-lactoferrin (ΔLf) mRNA is the product of alternative splicing of the Lf gene. It has been found in normal tissues and was reported to be absent from their malignant counterparts. Our recent investigations have shown that ΔLf expression is a good prognostic indicator in human breast cancer. However, ΔLf has up till now only been identified as a transcript, and in order to characterize the ΔLf protein and determine its function we have used a ΔLf cDNA construct to produce the protein in vitro and in vivo.A 73 kDa protein was immunoprecipitated from in vitro translation products and this molecular weight is in accordance with the use of the first in frame AUG start codon located in exon 2. We also produced a cell line expressing ΔLf under doxycycline induction. Using this model we have been able to show that ΔLf is mainly distributed in the cytoplasm. Its expression induces cell cycle arrest and inhibits cell proliferation. Our results suggest that ΔLf may play an important role in the regulation of normal cell growth.

O-GlcNAcylation/Phosphorylation Cycling at Ser10 Controls Both Transcriptional Activity and Stability of Δ-Lactoferrin

Δ-Lactoferrin (ΔLf) is a transcription factor that up-regulates DcpS, Skp1, and Bax genes, provoking cell cycle arrest and apoptosis. It is post-translationally modified either by O-GlcNAc or phosphate, but the effects of the O-GlcNAc/phosphorylation interplay on ΔLf function are not yet understood. Here, using a series of glycosylation mutants, we showed that Ser10 is O-GlcNAcylated and that this modification is associated with increased ΔLf stability, achieved by blocking ubiquitin-dependent proteolysis, demonstrating that O-GlcNAcylation protects against polyubiquitination. We highlighted the 391KSQQSSDPDPNCVD404 sequence as a functional PEST motif responsible for ΔLf degradation and defined Lys379 as the main polyubiquitin acceptor site. We next investigated the control of ΔLf transcriptional activity by the O-GlcNAc/phosphorylation interplay. Reporter gene analyses using the Skp1 promoter fragment containing a ΔLf response element showed that O-GlcNAcylation at Ser10 negatively regulates ΔLf transcriptional activity, whereas phosphorylation activates it. Using a chromatin immunoprecipitation assay, we showed that O-GlcNAcylation inhibits DNA binding. Deglycosylation leads to DNA binding and transactivation of the Skp1 promoter at a basal level. Basal transactivation was markedly enhanced by 2–3-fold when phosphorylation was mimicked at Ser10 by aspartate. Moreover, using double chromatin immunoprecipitation assays, we showed that the ΔLf transcriptional complex binds to the ΔLf response element and is phosphorylated and/or ubiquitinated, suggesting that ΔLf transcriptional activity and degradation are concomitant events. Collectively, our results indicate that reciprocal occupancy of Ser10 by either O-phosphate or O-GlcNAc coordinately regulates ΔLf stability and transcriptional activity.