Paul Proost

Gelatinase B functions as regulator and effector in leukocyte biology

Matrix metalloproteinases (MMPs) form a family of enzymes with major actions in the remodeling of extracellular matrix (ECM) components. Gelatinase B (MMP‐9) is the most complex family member in terms of domain structure and regulation of its activity. Gelatinase B activity is under strict control at various levels: transcription of the gene by cytokines and cellular interactions; activation of the pro‐enzyme by a cascade of enzymes comprising serine proteases and other MMPs; and regulation by specific tissue inhibitors of MMPs (TIMPs) or by unspecific inhibitors, such as α2‐macroglobulin. Thus, remodeling ECM is the result of the local protease load, i.e., the net balance between enzymes and inhibitors. Glycosylation has a limited effect on the net activity of gelatinase B, and in contrast to the all‐or‐none effect of enzyme activation or inhibition, it results in a higher‐level, fine‐tuning effect on the ECM catalysis by proteases in mammalian species. Fast degranulation of considerable amounts of intracellularly stored gelatinase B from neutrophils, induced by various types of chemotactic factors, is another level of control of activity. Neutrophils are first‐line defense leukocytes and do not produce gelatinase A or TIMP. Thus, neutrophils contrast sharply with mononuclear leukocytes, which produce gelatinase A constitutively, synthesize gelatinase B de novo after adequate triggering, and overproduce TIMP‐1. Gelatinase B is also endowed with functions other than cleaving the ECM. It has been shown to generate autoimmune neo‐epitopes and to activate pro‐IL‐1β into active IL‐1β. Gelatinase B ablation in the mouse leads to altered bone remodeling and subfertility, results in resistance to several induced inflammatory or autoimmune pathologies, and indicates that the enzyme plays a crucial role in development and angiogenesis. The major human neutrophil chemoattractant, IL‐8, stimulates fast degranulation of gelatinase B from neutrophils. Gelatinase B is also found to function as a regulator of neutrophil biology and to truncate IL‐8 at the aminoterminus into a tenfold more potent chemokine, resulting in an important positive feedback loop for neutrophil activation and chemotaxis. The CXC chemokines GRO‐α, CTAP‐III, and PF‐4 are degraded by gelatinase B, whereas the CC chemokines MCP‐2 and RANTES are not cleaved.

A Potent Antimicrobial Protein from Onion Seeds Showing Sequence Homology to Plant Lipid Transfer Proteins

An antimicrobial protein of about 10 kD, called Ace-AMP1, was isolated from onion (Allium cepa L.) seeds. Based on the near-complete amino acid sequence of this protein, oligonucleotides were designed for polymerase chain reaction-based cloning of the corresponding cDNA. The mature protein is homologous to plant nonspecific lipid transfer proteins (nsLTPs), but it shares only 76% of the residues that are conserved among all known plant nsLTPs and is unusually rich in arginine. Ace-AMP1 inhibits all 12 tested plant pathogenic fungi at concentrations below 10 [mu]g mL-1. Its antifungal activity is either not at all or is weakly affected by the presence of different cations at concentrations approximating physiological ionic strength conditions. Ace-AMP1 is also active on two Gram-positive bacteria but is apparently not toxic for Gram-negative bacteria and cultured human cells. In contrast to nsLTPs such as those isolated from radish or maize seeds, Ace-AMP1 was unable to transfer phospholipids from liposomes to mitochondria. On the other hand, lipid transfer proteins from wheat and maize seeds showed little or no antimicrobial activity, whereas the radish lipid transfer protein displayed antifungal activity only in media with low cation concentrations. The relevance of these findings with regard to the function of nsLTPs is discussed.

HUMAN MONOCYTE CHEMOTACTIC PROTEINS-2 AND -3 : STRUCTURAL AND FUNCTIONAL COMPARISON WITH MCP-1

Structurally, the monocyte chemotactic proteins MCP‐1, ‐2, and ‐3 form a subfamily of the C‐C or β‐chemokines. Like other chemokines, MCPs are produced by a variety of cells on stimulation with cytokines (interleukin‐1, tumor necrosis factor‐α, interferon‐γ), bacterial and viral products or mitogens. MCP‐1 levels are enhanced during infection and inflammation, which are characterized by leukocyte infiltration. In vitro, MCPs are chemotactic for a distinct spectrum of target cells and show different specific biological activities depending on the cell type and the chemokine tested. MCP‐3 has the broadest range in that it activates monocytes, dendritic cells, lymphocytes, natural killer cells, eosinophils, basophils, and neutrophils. The most sensitive cells to all three MCPs are lymphocytes and monocytes. MCP‐1 is a potent basophil activator but does not attract eosinophils, whereas, at higher concentrations, MCP‐2 also stimulates both eosinophils and basophils. The signal transduction of MCPs on monocytes involves at least two G protein‐linked C‐C chemokine receptors: C‐C CKR‐1 binds MCP‐3 and C‐C CKR‐2 binds MCP‐1 and MCP‐3 but not MCP‐2. Receptor binding leads to enhanced [Ca2+]i for all chemokines except for MCP‐2.

Processing by CD26/dipeptidyl-peptidase IV reduces the chemotactic and anti-HIV-1 activity of stromal-cell-derived factor-1α

The chemokine stromal‐cell‐derived factor‐1α (SDF‐1α) chemoattracts lymphocytes and CD34+ haematopoietic progenitors and is the ligand for CXCR4 (CXC chemokine receptor 4), the main co‐receptor for T‐tropic HIV‐1 strains. SDF‐1α was NH2‐terminally cleaved to SDF‐1α(3‐68) by dipeptidyl‐peptidase IV (CD26/DPP IV), which is present in blood in soluble and membrane‐bound form. SDF‐1α(3‐68) lost both lymphocyte chemotactic and CXCR4‐signaling properties. However, SDF‐1α(3‐68) still desensitized the SDF‐1α(1‐68)‐induced Ca2+ response. In contrast to CD26/DPP IV‐processed RANTES(3‐68), SDF‐1α(3‐68) had diminished potency to inhibit HIV‐1 infection. Thus, CD26/DPP IV impairs the inflammatory and haematopoietic potency of chemokines but plays a dual role in AIDS.

cDNA cloning and molecular analysis of two self-incompatibility alleles from apple

Complementary DNA clones representing two alleles of the self-incompatibility (S) locus of apple (Malus × domestica Borkh.) have been isolated and characterised. One of the alleles corresponds to a 29 kDa ribonuclease (S-RNase) that was purified from pistil tissue. On northern blots, both cDNAs hybridized to a transcript that was only present in pistils and not in the other plant tissues analysed. Corresponding genomic sequences, amplified by PCR, were found to contain a single intron of 138 bp and 1100 bp respectively. Comparison of both sequences shows that the cDNAs encode mature proteins containing 65% of identical residues. Eight invariable cysteine residues, conserved regions around two histidines thought to play a role in RNA catalysis, and a number of other distinct residues are conserved between the apple S-RNases and similar proteins in the family Solanaceae. As this is the first report of sequences of S-alleles from a species belonging to a family that is not related with the Solanaceae, the structural features of S-RNase deduced from a comparison of their sequences are discussed.

IL-1β and IFN-γ induce the expression of diverse chemokines and IL-15 in human and rat pancreatic islet cells, and in islets from pre-diabetic NOD mice

Aims/hypothesisCytokines and chemokines are important mediators of immune responses due to their ability to recruit and activate leukocytes. Using microarray analysis we observed that rat beta cells exposed to IL-1β and IFN-γ have increased mRNA levels of chemokines and IL-15. The aim of this study was to characterize the expression of IP-10, MIP-3α, fractalkine and IL-15 in rat beta cells, human pancreatic islets, and in islets isolated from NOD mice, both during the pre-diabetic period and following islet transplantation.MethodsFACS-purified rat beta cells and human islets were cultured with IL-1β, IFN-γ and/or TNF-α. Islets were isolated from NOD or BALB/c mice at different ages. For syngeneic islet transplantation, 2- or 3-week-old NOD islets were grafted under the kidney capsule of spontaneously diabetic NOD recipients. Chemokine and IL-15 mRNA expression and protein release were evaluated, respectively, by RT-PCR and ELISA.ResultsHuman islets and rat beta cells express IP-10, MIP-3α, fractalkine and IL-15 mRNAs upon exposure to cytokines. The expression of IL-15, IP-10 and fractalkine is regulated by IFN-γ, while the expression of MIP-3α is IL-1β-dependent. Moreover, cytokines induced IL-15, IP-10, Mig, I-TAC and MIP-3α protein accumulation in culture medium from human islets. In vivo, there was an age-related increase in IL-15, IP-10 and MIP-3α expression in islets isolated from NOD mice. Following syngeneic islet transplantation, increased expression of IL-1β, IFN-γ, fractalkine, IP-10, MCP-1 and MIP-3α mRNAs were observed in the grafts.Conclusion/interpretationCytokine-exposed islets or beta cells express chemokines and IL-15. This could contribute to the recruitment and activation of mononuclear cells and development of insulitis in early Type 1 diabetes and during graft destruction.

The role of chemokines in inflammation

Chemokines, together with adhesion molecules, cytokines, and proteases, are essential for the directional migration of leukocytes during normal and inflammatory processes. Interleukin-8 and monocyte chemotactic protein-1 are the best-characterized members of the C-X-C and C-C chemokine subfamilies, respectively. However, more than 20 human chemokines have been identified but are only partially characterized at the biological level. Chemokines are involved in chemotaxis of monocytes, lymphocytes, neutrophils, eosinophils, basophils, natural killer cells, dendritic cells, and endothelial cells. This review describes the chemokine subfamilies, the chemokine producer and target cells, their receptors, singal transduction mechanisms, and the role of chemokines during physiological and pathological conditions. More and more evidence points to a role for chemokines in chemotaxis-related phenomena, such as the expression of adhesion molecules, the secretion of proteinases, inhibition of apoptosis, hematopoiesis, and angiogenesis. Chemokines are also involved in diseases such as cancer (tumor regression and tumor metastasis), autoimmune diseases, and bacterial or viral infection.

Natural truncation of RANTES abolishes signaling through the CC chemokine receptors CCR1 and CCR3, impairs its chemotactic potency and generates a CC chemokine inhibitor

Selective leukocyte trafficking towards sites of inflammation is mediated by chemokines. RANTES is a CC chemokine that attracts lymphocytes, monocytes, dendritic cells, eosinophils, basophils and NK cells. A natural form of human RANTES lacking two N‐terminal residues was isolated from stimulated sarcoma cells, fibroblasts, and leukocytes. RANTES(3 – 68) showed a more than tenfold reduction in chemotactic potency for monocytes and eosinophils. To elucidate the mechanism involved, receptor recognition studies were performed. In cells transfected with the CC chemokine receptor (CCR) 5, the major co‐receptor for macrophage‐tropic HIV‐1 strains, RANTES(3 – 68) mobilized calcium and desensitized RANTES(1 – 68)‐induced calcium fluxes equally well as RANTES(1 – 68). However, RANTES(3 – 68) was ineffective on CCR1 and CCR3 transfectants. The reduced potency of natural RANTES(3 – 68) by selective loss of receptor‐activating characteristics was confirmed with recombinant RANTES(3 – 68). In chemotaxis assays using monocytic cells, RANTES(3 – 68) inhibited RANTES(1 – 68), macrophage inflammatory protein‐1α (MIP‐1α), MIP‐1β or monocyte chemotactic protein‐3 (MCP‐3), but not MCP‐1‐ or MCP‐2‐induced chemotaxis. Thus, a minor post‐translational modification has a remarkable impact on the biological activities of RANTES and a pathophysiologically induced change in the relative amounts of intact and truncated RANTES might affect the outcome of inflammation or HIV infection.

Monocyte chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and in interleukin-1β-exposed human and rat islet cells

Aims/hypothesis. Monocyte chemoattractant protein-1 (MCP-1) attracts monocytes and T lymphocytes, and could thus contribute to mononuclear cell infiltration in Type I (insulin-dependent) diabetes mellitus. Cytokines induce MCP-1 mRNA expression in pancreatic rat beta cells. To investigate this issue, we analysed the signal transduction for IL-1β-induced MCP-1 expression in rat beta cells and in vitro MCP-1 mRNA expression and protein release by human islets as well as in vivo islet MCP-1 mRNA expression in prediabetic non-obese diabetic mice. Methods. Fluorescence-activated cell sorting-purified rat beta cells were cultured for 6 h with IL-1β (30 U/ml) or MAPK inhibitors or both. Human islets were cultured for 6–72 h with the cytokines IL-1β, IFN-γ or the inducible nitric oxide synthase (iNOS) inhibitor NG-methyl-l-arginine or both. We measured MCP-1 mRNA by RT-PCR and protein by ELISA. The MCP-1 mRNA expression in islets from male and female non-obese diabetic mice (2–12 weeks of age) was measured by real time reverse transcription-polymerase chain reaction (RT-PCR). Results. Interleukin-1β induced MCP-1 mRNA expression in rat beta cells, with a maximum induction after 6 h. A combination of p38 and ERK1/2 inhibitors decreased MCP-1 expression by 70 %. IL-1β induced both MCP-1 mRNA expression and a threefold increase in medium MCP-1 protein accumulation in human islet cells. This effect was not prevented by iNOS blockers. In vivo there was an age-related increase in MCP-1 mRNA expression in islets from male and female non-obese diabetic mice, reaching a peak at 8 weeks. Conclusion/interpretation. In rat and human islet cells MCP-1 mRNA is induced by IL-1β. Both ERK1/2 and p38 MAPK, but not nitric oxide, contribute to MCP-1 expression. In non-obese diabetic mice MCP-1 mRNA expression increases with age, peaking at the early phases of insulitis. The production of MCP-1 by pancreatic beta cells could contribute to the recruitment of mononuclear cells into pancreatic islets in early Type I diabetes. [Diabetologia (2001) 44: 325–332]

Platelets Release CXCL4L1, a Nonallelic Variant of the Chemokine Platelet Factor-4/CXCL4 and Potent Inhibitor of Angiogenesis

Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.