Maryse Masson

Lipomannans, But Not Lipoarabinomannans, Purified from Mycobacterium chelonae and Mycobacterium kansasii Induce TNF-α and IL-8 Secretion by a CD14-Toll-Like Receptor 2-Dependent Mechanism

Lipoarabinomannans (LAMs) are glycolipids from the mycobacterial cell wall that exhibit various biological activities, including proinflammatory and anti-inflammatory responses. However, little is known about the properties of lipomannans (LMs), considered to be precursors of LAMs. In this study, we provide evidence that LMs purified from Mycobacterium chelonae and a clinical strain of Mycobacterium kansasii stimulated mRNA expression and secretion of TNF-α and IL-8 from human macrophage-like differentiated THP-1 cells. In contrast to LMs, LAMs were not able to induce a significant cytokine-inducing effect. The mechanism of activation by LMs was investigated using various Abs raised against surface receptors for multiple bacterial products. The presence of anti-CD14 or anti-Toll-like receptor 2 (TLR2) Abs profoundly affected production of TNF-α and IL-8, suggesting that both CD14 and TLR2 participate in the LM-mediated activation process. Furthermore, stimulation of cells was dependent on the presence of the LPS-binding protein, a plasma protein that transfers glycolipids to CD14. Chemical degradation of the arabinan domain of mannose-capped LAM from M. kansasii, which presented no cytokine-eliciting effect, restored the cytokine-inducing activity at a level similar to those of LMs. These results support the hypothesis that the presence of an arabinan in LAMs prevents the interaction of these glycolipids with TLR2/CD14 receptors. In addition, we found that phosphatidylinositol dimannosides isolated from M. kansasii did not induce cytokine secretion. This study suggests that LMs isolated from different mycobacterial species participate in the immunomodulation of the infected host and that the d-mannan core of this glycolipid is essential for this function.

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

Lactoferrin is an iron-binding glycoprotein implicated in particular in the control of immune functions and cell proliferation. We have investigated its involvement, at inflammatory concentrations, in cancer progression. We report that lactoferrin has a significant effect on natural killer (NK) cell cytotoxicity against haematopoietic and breast epithelial cell lines. Lactoferrin increases cytolysis at a low concentration (10 micrograms/ml) while at a high concentration (100 micrograms/ml) it modulates cytolysis depending on the target cell phenotype. By pre-treatment of either NK cells or target cells with lactoferrin, we have demonstrated that the lactoferrin effect is due both to a modulation of NK cell cytotoxicity and the target cell sensitivity to lysis. Lactoferrin binds to 91% of the naturally heterogeneous CD56dim/bright NK cell population and increases the NK cell cytotoxic activity at low concentrations. High concentrations of lactoferrin seem to be toxic for the CD56bright NK cells and decrease NK cell cytotoxicity. Lactoferrin also exerts an effect on target cells depending on the cell phenotype. It does not modify the susceptibility to lysis of haematopoietic cells such as Jurkat and K-562 cells, but does significantly increase that of the breast and colon epithelial cells. We have also demonstrated that lactoferrin inhibits epithelial cell proliferation by blocking the cell cycle progression.

Lactoferrin Inhibits the Lipopolysaccharide-Induced Expression and Proteoglycan-Binding Ability of Interleukin-8 in Human Endothelial Cells

ABSTRACT Interleukin-8 (IL-8), a C-X-C chemokine bound to endothelium proteoglycans, initiates the activation and selective recruitment of leukocytes at inflammatory foci. We demonstrate that human lactoferrin, an antimicrobial lipopolysaccharide (LPS)-binding protein, decreases both IL-8 mRNA and protein expression induced by the complex Escherichia coli 055:B5 LPS/sCD14 in human umbilical vein endothelial cells. The use of recombinant lactoferrins mutated in the LPS-binding sites indicates that this inhibitory effect is mediated by an interaction of lactoferrin with LPS and CD14s that suppresses the endotoxin biological activity. Furthermore, since dimeric IL-8 and lactoferrin are both proteoglycan-binding molecules, the competition between these proteins for heparin binding was investigated. Lactoferrin strongly inhibited the interaction of radiolabeled IL-8 to immobilized heparin, whereas a lactoferrin variant lacking the amino acid residues essential for heparin binding was not inhibitory. Moreover, this process is specific, since serum transferrin, a glycoprotein whose structure is close to that of lactoferrin, did not prevent the interaction of IL-8 with heparin. These results suggest that the anti-inflammatory properties of lactoferrin during septicemia are related, at least in part, to the regulation of IL-8 production and also to the ability of lactoferrin to compete with chemokines for their binding to proteoglycans.

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 p56lck (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. J. Cell. Biochem. 79:583–593, 2000.

Mycobacterial Lipomannan Induces Matrix Metalloproteinase-9 Expression in Human Macrophagic Cells through a Toll-Like Receptor 1 (TLR1)/TLR2- and CD14-Dependent Mechanism

ABSTRACT Lipomannans (LM) from various mycobacterial species were found to induce expression and secretion of the matrix metalloproteinase 9 (MMP-9) both in human macrophage-like differentiated THP-1 cells and in primary human macrophages. Inhibition studies using antireceptor-neutralizing antibodies are indicative of a Toll-like receptor 1 (TLR1)/TLR2- and CD14-dependent signaling mechanism. Moreover, LM was shown to down-regulate transcription of the metalloproteinase inhibitor TIMP-1, a major endogenous MMP-9 regulator.

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.

LACTOFERRIN STIMULATES THE MITOGEN-ACTIVATED PROTEIN KINASE IN THE HUMAN LYMPHOBLASTIC T JURKAT CELL LINE

Lactoferrin11 (also called lactotransferrin) has been shown to be involved in numerous inflammatory and immune response functions such as regulation of granulocyte monocyte colony stimulating factor6,7,12,17 and of interleukin9 synthesis, activation of NK cell activity13, inhibition of metastasis2 and maturation of T- and B-cells15,16. Recently, it has also been demonstrated the activation of a nuclear reporter gene harbouring the spe-ci fic DNA sequence that lactoferrin recognizes8. Therefore, lactoferrin could be considered as a transcriptional factor1. It requires that lactoferrin is internalized and imported into the nucleus of target cells where it acts as a transcriptional factor1. However, there is no evidence that cell-surface lactoferrin receptors can transport lactoferrin either directly or via a new trafficking pathway to the nucleus. In order to investigate how lactoferrin affects the immune system, we have developed an experimental model using the human lymphoblastic T Jurkat cell line which upon lactoferrin treatment slowly undergoes a maturation process5. Jurkat cells express the lactoferrin receptor10 at the cell surface in coated-pit like domains, associated into clusters, and in different subsets of intracellular vesicles3 . Therefore, we have investigated the first intracellular events following the binding of lactoferrin to lymphocytes: internalization process of lactoferrin and activation of a transduction pathway.