Ghislain Opdenakker

Amino-terminal Truncation of Chemokines by CD26/Dipeptidyl- peptidase IV

Chemokines are key players in inflammation and infection. Natural forms of the C-X-C chemokine granulocyte chemotactic protein-2 (GCP-2) and the C-C chemokine regulated on activation normal T cell expressed and secreted (RANTES), which miss two NH2-terminal residues, including a Pro in the penultimate position, have been isolated from leukocytes or tumor cells. In chemotaxis and intracellular calcium mobilization assays, the truncation caused a reduction in the specific activity of RANTES but not of GCP-2. The serine protease CD26/dipeptidyl-peptidase IV (CD26/DPP IV) could induce this observed NH2-terminal truncation of GCP-2 and RANTES but not that of the monocyte chemotactic proteins MCP-1, MCP-2 and MCP-3. No significant difference in neutrophil activation was detected between intact and CD26/DPP IV-truncated GCP-2. In contrast to intact natural RANTES(1–68), which still chemoattracts monocytes at 10 ng/ml, CD26/DPP IV-truncated RANTES(3–68) was inactive at 300 ng/ml and behaved as a natural chemotaxis inhibitor. Compared with intact RANTES, only a 10-fold higher concentration of RANTES(3–68) induced a significant Ca2+ response. Furthermore, RANTES(3–68) inhibited infection of mononuclear cells by an M-tropic HIV-1 strain 5-fold more efficiently than intact RANTES. Thus, proteolytic processing of RANTES by CD26/DPP IV may constitute an important regulatory mechanism during anti-inflammatory and antiviral responses.

Natural Haemozoin Induces Expression and Release of Human Monocyte Tissue Inhibitor of Metalloproteinase-1

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.

Transcriptional control of the human MCP-2 gene promoter by IFN-gamma and IL-1beta in connective tissue cells.

Human monocyte chemotactic protein‐2 (MCP‐2) is a member of the CC chemokine family. It is produced by mononuclear leukocytes, diploid fibroblasts, and tumor cells after induction with IL‐1β or IFN‐γ. To understand the transcriptional regulation of the gene, we have analyzed the structure and function of the promoter region. The sequence of the 5′‐flanking region was determined and the transcription start site was found to be located at 68 nucleotides upstream of the ATG translation start codon. 5′‐Deletion mutants were generated and transfected into E6SM diploid fibroblasts and MG‐63 osteosarcoma cells. Expression was measured by luciferase assay in transfected unstimulated cells and after stimulation with IL‐1β, IFN‐γ, or a combination. The region between nucleotides ‐143 and ‐73 (relative to the transcription initiation site), containing putative cis‐elements for GATA‐1, H‐APF1, AP‐1, and GAS, is important for basal transcription levels in both cell lines. Stimulation for 18 h with IL‐1β alone failed to affect expression of any of the constructs both in diploid fibroblasts and in osteosarcoma cells. In both cell lines IFN‐γ increased the activity of all mutants that possessed the region between ‐340 and ‐301. In MG‐63 cells, stimulation with the combination of IL‐1β and IFN‐γ caused an additional increase in expression of the constructs from ‐340 onward. Finally, the presence of transcription factors in nuclear extracts of MG‐63 cells and their specificity to bind to various oligonucleotide probes in this [‐340; ‐301] region were evidenced by electromobility shift assays. These results show that IFN‐γ, produced by lymphocytes and NK cells, induces the transcription of the MCP‐2 gene in fibroblasts and thereby can indirectly contribute to recruitment of various leukocyte cell types to inflammatory sites. J. Leukoc. Biol. 66: 502–511; 1999.

Enzymatic processing by MMP-2 and MMP-9 of wild-type and mutated mouse β-dystroglycan

Dystroglycan (DG) is a membrane‐associated protein complex formed by two noncovalently linked subunits, α‐DG, a highly glycosylated extracellular protein, and β‐DG, a transmembrane protein. The interface between the two DG subunits, which is crucial to maintain the integrity of the plasma membrane, involves the C‐terminal domain of α‐DG and the N‐terminal extracellular domain of β‐DG. It is well known that under both, physiological and pathological conditions, gelatinases (i.e. MMP‐9 and/or MMP‐2) can degrade DG, disrupting the connection between the extracellular matrix and the cytoskeleton. However, the molecular mechanisms and the exact cleavage sites underlying these events are still largely unknown. In a previous study, we have characterized the enzymatic digestion of the murine β‐DG ectodomain by gelatinases, identifying a main cleavage site on the β‐DG ectodomain produced by MMP‐9. In this article, we have deepened the pattern of the β‐DG ectodomain digestion by MMP‐2 by using a combined approach based on SDS‐PAGE, Orbitrap, and HPLC‐ESI‐IT mass spectrometry. Furthermore, we have characterized the kineticparameters of the digestion of some β‐DG ectodomain mutants by gelatinases.

Introduction of the interferon γ gene into mouse T lymphoma cells with low MHC class I-expression results in selective induction of H-2Dk and concomitant enhanced metastasis

Abstract Interferon-γ(IFNγ)-induced up-regulation of MHC class I expression on tumor cells can induce a potent CD8-mediated antitumor response. Consequently, many investigators have proposed IFNγ gene transfection as a means to immunogenize tumor cells and to vaccinate against metastatic disease. In this study, we demonstrate that transfection of the IFNγ gene in a BW5147 variant (LiDlo) with low MHC class I expression results in a selective induction of H-2Dk but unaltered H-2Kk expression. In earlier reports we demonstrated a positive correlation between H-2Dk expression and enhanced metastatic potential of BW variants. In accordance with these observations, we observed that intravenous inoculation of LiDlo(IFNγ) variants into syngeneic AKR mice led to enhanced metastasis as compared to parental LiDlo and LiDlo(neo) control transfectants. Tumor cells, derived from local subcutaneous tumors or sporadic metastases from mice inoculated with LiDlo tumor cells, were found to up-regulate H-2Dk selectively. Anti-asialoGM1 treatment of AKR mice allowed rapid experimental metastasis formation by the LiDlo and LiDlo(neo) variants, indicating that natural killer (NK) cells control the metastatic behavior of these tumor cells. This was corroborated by in vitro cytotoxicity experiments, demonstrating that LiDlo and LiDlo(neo) tumor cells were NK-sensitive, while the BW IFNγ transfectants became resistant to lymphokine-activated killer cells and poly(I)·poly(C)-induced NK cells. We thus conclude that (a) IFNγ up-regulates selectively the MHC class I antigen H-2Dk, (b) H-2Dk governs susceptibility towards NK cells, and (c) NK susceptibility determines the experimental metastatic behavior of BW tumor cells.

Applications of glycobiology: biological and immunological effects of a chemically modified amylose-derivative

Carbohydrate chemistry, oligosaccharide sequencing, synthesis technologies and glyco-engineering have helped to establish glycobiology alongside molecular biology. However, examples of therapeutic implications of glycobiology are limited to oligosaccharides. These include engineered antibodies conta...

Cytokines, Chemokines and Proteinases in Autoimmune Diseases

Chemotactic cytokines or chemokines form a family of pro-inflammatory proteins that are functionally linked to various classes of proteases, including matrix metalloproteinases (MMPs). Both families of molecules are key players in the migration of inflammatory cells in autoimmune diseases. For example, the human chemokine interleukin- 8 acts as a fast secretagogue of gelatinase B in granulocytes and is increased in the synovial fluid of arthritis patients and may locally recruit and activate neutrophils. The latter leukocytes are the most abundant inflammatory cell type in the joints of patients with rheumatoid arthritis. In the inflamed joint, the activity of matrix remodeling enzymes contributes to the breakdown of cartilage constituents. For instance, gelatinase B (MMP-9) was documented to cleave type II collagen in 25 fragments. Several of these fragments contain the intact immunodominant epitopes that were discovered by epitope scanning experiments with lymphocytes from rheumatoid arthritis patients. Gelatinase B thus helps to generate immunodominant peptides that stimulate T lymphocytes and thus maintain the activation of the specific arm of immunity in arthritic diseases. Furthermore, with the use of highly specific monoclonal antibodies, the presence of gelatinase B was detected by immunohistochemistry in the lesions of patients with various autoimmune diseases. Studies in rheumatoid arthritis and multiple sclerosis led us to postulate the “Remnant Epitopes Generate Autoimmunity” or REGA model for autoimmunity. This model is based on the pathophysiological role of three major classes of molecules involved in a specific primary immune defense mechanisms: the cytokines, the chemokines and the proteases.