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.

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 Alzheimer's and Parkinson's 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.

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

The presence of iron in brain tissue in increased concentrations in Parkinson's disease cases, where it might be responsible for oxidative stress, and the parallel observation that the iron transporter lactoferrin (Lf) was present in increased amounts in surviving neurons, led us to study the synthesis of Lf in a mouse model of Parkinson's disease. In this context, the origin and expression of brain Lf in normal, aged and MPTP (1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine)-treated mice were investigated. Lf immunostaining was observed mainly on microvessels in the cerebral cortex of the adult mice and to a greater extent in older mice. Lf immunoreactivity was also present in the hippocampus only in the aged mouse brains, associated with structures which seemed to be pyramidal neurons and fibers. After RT-PCR (polymerase chain reaction), Lf transcripts were found in mouse brain tissue whatever the age of the animals studied but the level of their expression was very low. No up-regulation of Lf was detectable during aging. Lf distribution and expression in the MPTP-induced Parkinsonian mouse model were also investigated. A marked depletion of dopamine (DA) occurred in the high dose MPTP-treated mice. The level of Lf expression was found to be markedly increased in the same animals and this up-regulation occurred on the first day after MPTP administration. When the brain was stressed by the neurotoxin MPTP, Lf expression increased in line with antioxidant enzymes such as catalase and gamma-glutamylcysteine synthetase, which may permit the protection of brain tissue from oxidative damage induced by the drug.

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.

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

The main biological properties of lactoferrin are thought to concern inflammation and immunomodulation processes, including maturation of immature B and T cells. Lactoferrin accelerates T-cell maturation by inducing the expression of the CD4 surface marker. In this report, using the Jurkat T-cell line, we have shown that lactoferrin upregulates the expression of CD4 antigen through the activation of a transduction pathway. Using an anti-phosphotyrosine antibody, lactoferrin was demonstrated to induce a cascade of phosphorylation of numerous proteins on their tyrosine residues. This tyrosine-phosphorylation was transient, reaching maxima between 5 and 10 min. We also identified the mitogen-activated protein kinase (MAP kinase) which presented an enhanced catalytic activity, reaching a maximum at 10 min of incubation with lactoferrin. Moreover, the use of inhibitors such as genistein and PD98059, tyrosine kinases and MAP kinase kinase (or MEK) inhibitors respectively, allowed us to correlate the activation of MAP kinase with the upregulation of CD4 expression. Finally, using Lck-defective Jurkat cells, our results showed that the p56(lck) (Lck) kinase is necessary for MAP kinase activity and CD4 expression. This paper demonstrates that lactoferrin activates transduction pathway(s) in lymphoblastic T-cells, and that Lck and the Erk2 isoform of MAP kinase are implicated in the upregulation of CD4, induced by lactoferrin in these cells.

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

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-alpha (TNF-alpha). As TNF-alpha 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-alpha, 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.

Receptor-mediated transcytosis of lactoferrin through the blood-brain barrier.

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 (Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 microM; 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.