Jean-Pierre Lenaerts

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.

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.

Citrullination of CXCL10 and CXCL11 by peptidylarginine deiminase: a naturally occurring posttranslational modification of chemokines and new dimension of immunoregulation.

Interactions between chemokines and enzymes are vital in immunoregulation. Structural protein citrullination by peptidylarginine deiminase (PAD) has been associated with autoimmunity. In this report, we identified a novel naturally occurring posttranslational modification of chemokines, that is, the deimination of arginine at position 5 into citrulline of CXC chemokine ligand 10 (CXCL10) by rabbit PAD and human PAD2. Citrullination reduced (>/= 10-fold) the chemoattracting and signaling capacity of CXCL10 for CXC chemokine receptor 3 (CXCR3) transfectants; however, it did not affect CXCR3 binding. On T lymphocytes, though, citrullinated CXCL10 remained active but was again weaker than authentic CXCL10. PAD was also able to convert CXCL11, causing an impairment of CXCR3 signaling and T-cell activation, though less pronounced than for CXCL10. Similarly, receptor binding properties of CXCL11 were not altered by citrullination. However, deimination decreased heparin binding properties of both CXCL10 and CXCL11. Overall, chemokines are the first immune modulators reported of being functionally modified by citrullination. These data provide new structure-function dimensions for chemokines in leukocyte mobilization, disclosing an anti-inflammatory role for PAD. Additionally because citrullination has severe consequences for chemokine biology, this invites to reassess the involvement and impact of PAD and citrullinated peptides in inflammation, autoimmunity, and hematologic disorders.

Diverging binding capacities of natural LD78beta isoforms of macrophage inflammatory protein-1alpha to the CC chemokine receptors 1,3 and 5 affect their anti-HIV-1 activity and chemotactic potencies for neutrophils and eosinophils

Recently, the LD78β isoform of the CC chemokine macrophage inflammatory protein (MIP)‐1α was shown to efficiently chemoattract lymphocytes and monocytes and to inhibit infection of mononuclear cells by R5 HIV‐1 strains. We have now demonstrated that after cleavage of the NH2‐terminal Ala‐Pro dipeptide by CD26, LD78β(3 – 70) became the most potent chemokine blocking HIV‐1. LD78β(3 – 70) competed tenfold more efficiently than LD78β(1 – 70) with [125I] RANTES for binding to the CC chemokine receptors CCR5 and CCR1. Contrary to LD78α, LD78β(1 – 70) at 30 ng / ml efficiently competed with [125I] RANTES for binding to CCR3 and mobilized calcium in CCR3 transfectants, whereas LD78β(3 – 70) showed a 30‐fold decrease in CCR3 affinity compared to LD78β(1 – 70). This demonstrates the importance of the penultimate proline in LD78β(1 – 70) for CCR3 recognition. Both LD78β isoforms efficiently chemoattracted eosinophils from responsive donors. In contrast, only the CCR3 agonist LD78β(1 – 70) and not LD78β(3 – 70), induced calcium increases in eosinophils with low levels of CCR1. In responder neutrophils, LD78β(3 – 70) elicited calcium fluxes at a 30‐fold lower dose (10 ng / ml) compared to intact LD78β and LD78α, whereas the three MIP‐1α isoforms were equipotent neutrophil chemoattractants. Taken together, both LD78β isoforms are potent HIV‐1 inhibitors (CCR5) and activators for neutrophils (CCR1) and eosinophils (CCR1, CCR3), affecting infection and inflammation.

Synergistic induction of MCP-1 and -2 by IL-1beta and interferons in fibroblasts and epithelial cells.

Monocyte chemotactic protein (MCP)‐1 and MCP‐2, two closely related CC chemokines, are important mediators of monocyte and lymphocyte migration. These chemokines are secreted by various normal cell types, including fibroblasts, epithelial cells, and leukocytes, as well as by tumor cells. After stimulation with different cytokines and cytokine inducers the MCP‐2 production levels are always lower than those of MCP‐1. In human diploid fibroblasts cytokines differentially regulate chemokine induction, interleukin (IL)‐1β and interferon (IFN)‐γ being potent stimuli of MCP‐1 and MCP‐2, respectively. Co‐stimulation of fibroblasts by 10 U/mL IL‐1β and 20 ng/mL IFN‐γ resulted in a synergistic induction of MCP‐2, whereas the combined effect on MCP‐1 and IL‐6 production was rather additive. These findings were confirmed at the mRNA level by Northern blot analysis. In contrast, in human MG‐63 fibroblastoid cells and HEp‐2 epithelial cells, selected for their poor responsiveness to IL‐1β and IFN‐γ, MCP‐2 as well as MCP‐1 and IL‐6 were synergistically induced, yielding protein levels that were increased 3‐ to 30‐fold above the additive levels. When IFN‐β was used as a co‐stimulant of IL‐1β, a similar synergistic induction of MCP‐1 and MCP‐2 was measured both at the protein and the mRNA level. It can be concluded that, when synergy occurred, the MCP‐1 and MCP‐2 expression levels reached a comparable maximum, indicative for an equal contribution of these chemokines in normal and pathological conditions. J. Leukoc. Biol. 63: 364–372; 1998.

The CXC chemokine GCP-2/CXCL6 is predominantly induced in mesenchymal cells by interleukin-1beta and is down-regulated by interferon-gamma: comparison with interleukin-8/CXCL8.

Human granulocyte chemotactic protein-2 (GCP-2)/CXCL6 is a CXC chemokine that functionally uses both of the IL-8/CXCL8 receptors to chemoattract neutrophils but that is structurally most related to epithelial cell–derived neutrophil attractant-78 (ENA-78)/CXCL5. This study provides the first evidence that GCP-2 protein is, compared with IL-8, weakly produced by some sarcoma, but less by carcinoma cells, and is tightly regulated in normal mesenchymal cells. IL-1β was the predominant GCP-2 inducer in fibroblasts, chondrocytes, and endothelial cells, whereas IL-8 was equally well up-regulated in these cells by TNF-α, measles virus, or double-stranded RNA (dsRNA). In contrast, lipopolysaccharide (LPS) was a relatively better stimulus for GCP-2 versus IL-8 in fibroblasts. IFN-γ down-regulated the GCP-2 production in fibroblasts induced by IL-1β, TNF-α, LPS, or dsRNA. The kinetics of GCP-2 induction by IL-1β, LPS, or dsRNA in fibroblasts differed from those of IL-8. Freshly isolated peripheral blood mononuclear leukocytes, which are a good source of IL-8 and ENA-78, failed to produce GCP-2. However, lung macrophages and blood monocyte–derived macrophages produced GCP-2 in response to LPS. Quantitatively, secretion of GCP-2 always remained inferior to that of IL-8, despite the fact that the ELISA recognized all posttranslationally modified GCP-2 isoforms. The expression of GCP-2 was confirmed in vivo by immunohistochemistry. The patterns of producer cell types, inducers and kinetics and the quantities of GCP-2 produced, suggest a unique role for GCP-2 in physiologic and pathologic processes.

Regulated production and molecular diversity of human liver and activation-regulated chemokine/macrophage inflammatory protein-3 alpha from normal and transformed cells.

Liver and activation-regulated chemokine (LARC), also designated macrophage inflammatory protein-3α (MIP-3α), Exodus, or CCL20, is a C-C chemokine that attracts immature dendritic cells and memory T lymphocytes, both expressing CCR6. Depending on the cell type, this chemokine was found to be inducible by cytokines (IL-1β) and by bacterial, viral, or plant products (including LPS, dsRNA, and PMA) as measured by a specific ELISA. Although coinduced with monocyte chemotactic protein-1 (MCP-1) and IL-8 by dsRNA, measles virus, and IL-1β in diploid fibroblasts, leukocytes produced LARC/MIP-3α only in response to LPS. However, in myelomonocytic THP-1 cells LARC/MIP-3α was better induced by phorbol ester, whereas in HEp-2 epidermal carcinoma cells IL-1β was the superior inducer. The production levels of LARC/MIP-3α (1–10 ng/ml) were, on the average, 10- to 100-fold lower than those of IL-8 and MCP-1, but were comparable to those of other less abundantly secreted chemokines. Natural LARC/MIP-3α protein isolated from stimulated leukocytes or tumor cell lines showed molecular diversity, in that NH2- and COOH-terminally truncated forms were purified and identified by amino acid sequence analysis and mass spectrometry. In contrast to other chemokines, including MCP-1 and IL-8, the natural processing did not affect the calcium-mobilizing capacity of LARC/MIP-3α through its receptor CCR6. Furthermore, truncated natural LARC/MIP-3α isoforms were equally chemotactic for lymphocytes as intact rLARC/MIP-3α. It is concluded that in addition to its role in homeostatic trafficking of leukocytes, LARC/MIP-3α can function as an inflammatory chemokine during host defense.

Differential induction of monocyte chemotactic protein‐3 in mononuclear leukocytes and fibroblasts by interferon‐α / β and interferon‐γ reveals MCP‐3 heterogeneity

Monocyte chemotactic protein‐3 (MCP‐3) is a pluripotent CC chemokine, attracting most leukocytic cell types. With the use of a sensitive and specific ELISA, MCP‐3 was found to be inducible in fibroblasts and peripheral blood mononuclear cells (PBMC) by cytokines and cytokine inducers. MCP‐3 production levels (1–10 ng/ml) were tenfold lower compared to those of MCP‐1. In diploid fibroblasts, synergistic induction of MCP‐3, but not of MCP‐1, mRNA and protein was observed by combined treatment with IL‐1β and IFN‐γ. In PBMC, IFN‐α and IFN‐β (but not IFN‐γ), as well as measles virus and double‐stranded RNA, were potent inducers of MCP‐3, which suggests a role for this chemokine in an early stage of viral infections. In contrast, endotoxin failed to induce MCP‐3 production in fibroblasts and PBMC. Purification of MCP‐3 from PBMC revealed biochemical heterogeneity. In monocyte chemotaxis and calcium mobilization assays, pure 11‐kDa MCP‐3 from PBMC showed similar potencies as MCP‐3 from tumor cells. It was concluded that the induction of MCP‐3 by IFN is regulated differently in fibroblasts and PBMC. In view of the multiple target cells for MCP‐3, local and strictly regulated chemokine production might be important to conduct selectively the immune response in infection or inflammation.

Identification of the monocyte chemotactic protein from human osteosarcoma cells and monocytes: Detection of a novel N-terminally processed form

The chemotactic activity for monocytes in culture supernatants from double-stranded RNA-stimulated human MG-63 osteosarcoma cells and from LPS-stimulated human monocytes was purified to homogeneity and characterized by amino acid sequence analysis. The chemotactic protein derived from the fibroblastoid osteosarcoma cells had a blocked N-terminus but sequencing of tryptic fragments showed that it was identical with a recently identified monocyte chemoattractant designated MCP-1 or MCAF isolated from glioma or myelomonocytic cells, respectively. Preparations of monocyte -derived chemotactic activity appeared to contain not only the blocked protein, but also a novel N-terminally processed form of this molecule, lacking 5 amino acid residues.

Carcinoma cell-derived chemokines and their presence in oral fluid.

Chemokines are important in inflammation and in carcinogenesis. We hypothesized that besides oro-laryngeal cancer, oral inflammatory states, such as periodontitis, may also influence the chemokine profile of oral fluid. The aim of this study was to characterize the chemokine isoforms in the oral fluid of patients with periodontitis and in the oral fluid of patients with head and neck cancer. Using enzyme-linked immunosorbent assays (ELISA), it was found that the concentrations of CXCL8, CXCL10, and CCL14 were significantly elevated in the oral fluids of the cancer patients. However, periodontitis did not significantly alter the chemokine levels in oral fluid. Identification of chemokine isoforms by a proteomic approach using a newly developed three-step purification procedure was applied on the oral fluid of head and neck cancer and periodontitis patients and on the conditioned medium from carcinoma cells. Carcinoma cells produced predominantly intact CXCL1, CXCL2, CXCL8, and CCL2, whereas CXCL8 also appeared in a truncated, more active, form. Unfortunately, the chemokine concentrations in oral fluids were too low to allow full biochemical identification of the modified isoforms. However, the chemokine profile of head and neck cancer significantly changed after therapy, indicating that it is a useful parameter in clinical practice.