Lipoprotein transport across the blood–brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.

Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (K d ‫؍‬ 37.5 nM; n ‫؍‬ 90,000/cell) and low (K d ‫؍‬ 2 M; n ‫؍‬ 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport. Lactoferrin (Lf)1 (1) is a mammalian cationic iron-binding glycoprotein belonging to the transferrin (Tf) family. Despite some striking differences, mainly in the glycan moiety, there are marked sequence and conformational homologies among Lfs from different species, as well as similar general functions (for review, see Ref.2). Many physiological roles have been ascribed to Lf, particularly in the host defense against infection and severe inflammation (for review, see Ref. 3). This broad spectrum of biological functions relies on the interaction of Lf with numerous cells. The binding of Lf to cells is independent of its degree of iron saturation and is mediated mainly via interaction of the cluster of basic amino acids at its NH 2 terminus with sulfated molecules (4, 5). However, Lf is also targeted to specific cell receptors, and only a few of these involved in its uptake have been clearly identified. The 105-kDa Lf receptor characterized on activated human T-cells (6) is expressed at the cell surface of platelets (7), megacaryocytes (8), dopaminergic neurons, and mesencephalon microvessels (9). Lf receptor internalizes Lf, which is subsequently degraded (30 -40%), whereas the remaining fraction is recycled (10). In addition, the low density lipoprotein receptor-related protein (LRP) displays a high affinity for Lf and is responsible for its clearance (11)(12)(13)(14). This is inhibited by RAP, the receptor-associated protein known to be an antagonist for LRP (15). Transcytosis of Lf was described for HT29 cells (16) and was a minor pathway, up-regulated during iron deprivation (17).Lf is produced by exocrine glands (1, 18) and is widely distributed in the body fluids. It is stored in specific granules of neutrophilic leukocytes (19) and is relea...

Lactoferrin (Lf) is an iron binding glycoprotein of the transferrin family that is expressed in most biological fluids and is a major component of mammals' innate immune system. Its protective effect ranges from direct antimicrobial activities against a large panel of microorganisms, including bacteria, viruses, fungi, and parasites, to anti-inflammatory and anticancer activities. This plethora of activities is made possible by mechanisms of action implementing not only the capacity of Lf to bind iron but also interactions of Lf with molecular and cellular components of both host and pathogens. This chapter summarizes our current understanding of the Lf structure-function relationships that explain the roles of Lf in host defense.

The screening of a bovine submaxillary gland cDNA library yielded 25 clones coding for bovine lactotransferrin. The nucleotide sequence of the longest insert contained a protein-coding region of 21 15 nucleotides and a 3' non-coding region of 194 nucleotides followed by a poly(A) tract of about 55 nucleotides. The predicted peptide sequence included a 16-amino-acid signal sequence upstream of the first amino acid of the native protein.The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the N-terminal and tryptic peptide sequences derived from purified bovine milk lactotransferrin, and also by similarity with human and murine lactotransferrins. The cDNA described corresponds to a 705-amino-acid-long preprotein that lacks the start methionine. The sequence of the secreted protein is 689 amino acids long and contains five potential glycosylation sites. Bovine lactotransferrin is 69% and 64% identical to human and murine lactotransferrins, respectively.The transferrins are a family of non-haem, iron-binding proteins which includes serum transferrin, lactotransferrin and melanotransferrin. They are monomeric glycoproteins with a molecular mass of about 80 kDa constituted by two lobes, each possessing one iron-binding site with the capacity to bind reversibly one ferric ion (Fe3+) (reviewed in [l, 21). Lactotransferrins (also called lactoferrins) are present in various biological fluids such as human milk [3 -51, bovine milk [6], saliva [7, 81 and mucous secretions [8, 91. They are also present in leucocytes [S, 10, 111. Although lactotransferrins were first isolated in 1960 from both human and bovine milk, most of the studies concerning their structure and biological roles (reviewed in [l, 21 and [12 -141) were performed on the human protein. The sequence of human lactotransferrin was resolved using chemical methods [15] and cDNA analysis [16] (and cited by Anderson et al. in [17]). It consists of a 691-amino-acid polypeptide chain to which two biantennary glycans of the N-acetyllactosaminic type are linked [18]. This sequence is 70% identical to the sequence of murine lactotransferrin recently determined by cDNA sequencing [19] and resolved as a 688-amino-acid polypeptide chain to which a single glycan of the N-acetyllactosaminic type is conjugated 1201.The only information known about the primary structure of bovine lactotransferrin concerned its N-terminal amino acid sequence APRKNVRWXTISQPE [21] and its carboCorrespondence to A.

The degeneration of nigral dopaminergic neurons in Parkinson disease is believed to be associated with oxidative stress. Since iron levels are increased in the substantia nigra of parkinsonian patients and this metal catalyzes the formation of free radicals, it may be involved in the mechanisms of nerve cell death. The cause of nigral iron increase is not understood. Iron acquisition by neurons may occur from iron-transferrin complexes with a direct interaction with specific membrane receptors, but recent results have shown a low density of transferrin receptors in the substantia nigra. To investigate whether neuronal death in Parkinson disease may be associated with changes in a pathway supplementary to that of transferrin, lactoferrin (lactotransferrin) receptor expression was studied in the mesencephalon. In this report we present evidence from immunohistochemical staining of postmortem human brain tissue that lactoferrin receptors are localized on neurons (perikarya, dendrites, axons), cerebral microvasculature, and, in some cases, glial cells. In parkinsonian patients, lactoferrin receptor immunoreactivity on neurons and microvessels was increased and more pronounced in those regions of the mesencephalon where the loss of dopaminergic neurons is severe. Moreover, in the substantia nigra, the intensity of immunoreactivity on neurons and microvessels was higher for patients with higher nigral dopaminergic loss. These data suggest that lactoferrin receptors on vulnerable neurons may increase intraneuronal iron levels and contribute to the degeneration of nigral dopaminergic neurons in Parkinson disease.

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.

The existence of a functional receptor for secretory component (SC) on the eosinophil membrane might explain the preferential degranulation induced by secretory IgA (sIgA) when compared to serum IgA. Indeed, flow cytometry analysis revealed that purified human SC could bind to a subpopulation (4-59%) of blood eosinophils purified from 19 patients with eosinophilia. Binding of radiolabeled human SC could be competitively inhibited using unlabeled SC or secretory IgA but not with serum IgA or IgG. Immunoprecipitation and immunosorbent chromatography using human SC revealed the presence of a major component at 15 kDa in eosinophil extracts as well as in culture supernatants but not in neutrophils. The 15-kDa protein eluted from the human SC immunosorbent was able to bind to SC or to sIgA but not to serum IgA. Eosinophils preincubated with human SC or sIgA released eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) after addition of anti-SC or anti-IgA monoclonal antibody as respective cross-linking reagents. These results indicated that binding of free or complexed SC to human eosinophils could induce eosinophil degranulation. Furthermore, the dose-dependent inhibition by SC of mediator release induced by sIgA but not by serum IgA, suggested that the receptor for SC could be involved in the preferential degranulation mediated by sIgA. These results indicate a novel pathway of eosinophil activation and its potential involvement in mucosal immunity, particularly in inflammatory diseases associated with infiltration of eosinophils and the enhanced production of sIgA.

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