René Conings

Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation

Biological functions of proteins are influenced by posttranslational modifications such as on/off switching by phosphorylation and modulation by glycosylation. Proteolytic processing regulates cytokine and chemokine activities. In this study, we report that natural posttranslational citrullination or deimination alters the biological activities of the neutrophil chemoattractant and angiogenic cytokine CXCL8/interleukin-8 (IL-8). Citrullination of arginine in position 5 was discovered on 14% of natural leukocyte-derived CXCL8(1–77), generating CXCL8(1–77)Cit5. Peptidylarginine deiminase (PAD) is known to citrullinate structural proteins, and it may initiate autoimmune diseases. PAD efficiently and site-specifically citrullinated CXCL5, CXCL8, CCL17, CCL26, but not IL-1β. In comparison with CXCL8(1–77), CXCL8(1–77)Cit5 had reduced affinity for glycosaminoglycans and induced less CXCR2-dependent calcium signaling and extracellular signal-regulated kinase 1/2 phosphorylation. In contrast to CXCL8(1–77), CXCL8(1–77)Cit5 was resistant to thrombin- or plasmin-dependent potentiation into CXCL8(6–77). Upon intraperitoneal injection, CXCL8(6–77) was a more potent inducer of neutrophil extravasation compared with CXCL8(1–77). Despite its retained chemotactic activity in vitro, CXCL8(1–77)Cit5 was unable to attract neutrophils to the peritoneum. Finally, in the rabbit cornea angiogenesis assay, the equally potent CXCL8(1–77) and CXCL8(1–77)Cit5 were less efficient angiogenic molecules than CXCL8(6–77). This study shows that PAD citrullinates the chemokine CXCL8, and thus may dampen neutrophil extravasation during acute or chronic inflammation.

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.

Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

Leukocyte infiltration during acute and chronic inflammation is regulated by exogenous and endogenous factors, including cytokines, chemokines and proteases. Stimulation of fibroblasts and human microvascular endothelial cells with the inflammatory cytokines interleukin-1β (IL-1β) or tumour necrosis factor alpha (TNF-α) combined with either interferon-α (IFN-α), IFN-β or IFN-γ resulted in a synergistic induction of the CXC chemokine CXCL10, but not of the neutrophil chemoattractant CXCL8. In contrast, simultaneous stimulation with different IFN types did not result in a synergistic CXCL10 protein induction. Purification of natural CXCL10 from the conditioned medium of fibroblasts led to the isolation of CD26/dipeptidyl peptidase IV-processed CXCL10 missing two NH2-terminal residues. In contrast to intact CXCL10, NH2-terminally truncated CXCL10(3–77) did not induce extracellular signal-regulated kinase 1/2 or Akt/protein kinase B phosphorylation in CXC chemokine receptor 3-transfected cells. Together with the expression of CXCL10, the expression of membrane-bound CD26/dipeptidyl peptidase IV was also upregulated in fibroblasts by IFN-γ, by IFN-γ plus IL-1β or by IFN-γ plus TNF-α. This provides a negative feedback for CXCL10-dependent chemotaxis of activated T cells and natural killer cells. Since TNF-α and IL-1β are implicated in arthritis, synovial concentrations of CXCL8 and CXCL10 were compared in patients suffering from crystal arthritis, ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. All three groups of autoimmune arthritis patients (ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis) had significantly increased synovial CXCL10 levels compared with crystal arthritis patients. In contrast, compared with crystal arthritis, only rheumatoid arthritis patients, and not ankylosing spondylitis or psoriatic arthritis patients, had significantly higher synovial CXCL8 concentrations. Synovial concentrations of the neutrophil chemoattractant CXCL8 may therefore be useful to discriminate between autoimmune arthritis types.

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.

Characterization of granulocyte chemotactic activity from human cytokine-stimulated chondrocytes as interleukin 8

Human articular chondrocytes, when stimulated with interleukin 1 beta (IL 1 beta), tumor necrosis factor-alpha (TNF-alpha), or with the double stranded RNA poly (rI).poly (rC), produce a chemotactic activity for granulocytes. The induction with IL 1 beta could be abolished by an antibody to IL 1 beta but not by an antibody to interleukin 6 (IL 6), indicating that the latter is not a mediator for the production of chemotactic activity. The inducers had no direct chemotactic effect on granulocytes. The granulocyte chemotactic factor from chondrocytes was characterized with a specific antibody against leukocyte-derived interleukin 8 (IL 8). The specificity of this antibody was demonstrated by immunochemical and biological criteria such that it could immunoprecipitate only the 6-7 kDa IL 8 protein from fibroblasts, and that it did not neutralize a structurally related monocyte chemotactic protein. This antibody against IL 8 completely neutralized the granulocyte chemotactic activity from stimulated chondrocytes. This demonstrates the identity of chondrocyte IL 8 with leukocyte- and fibroblast-derived IL 8. Our data show that leukocyte chemotaxis into the inflamed joint can be mediated by IL 8, induced in both synovial fibroblasts and chondrocytes by the inflammatory cytokines IL 1 and TNF-alpha.

Stimulation of angiostatic platelet factor-4 variant (CXCL4L1/PF-4var) versus inhibition of angiogenic granulocyte chemotactic protein-2 (CXCL6/GCP-2) in normal and tumoral mesenchymal cells

Chemokines affect inflammation and cancer through leukocyte attraction and angiogenesis. Here, we demonstrate that CXCL4L1/platelet factor‐4 variant (PF‐4var), a highly angiostatic chemokine, is poorly chemotactic for phagocytes and is inducible in monocytes by inflammatory mediators but remained undetectable in macrophages and neutrophils. In addition, CXCL4L1/PF‐4var production by mesenchymal tumor cells was evidenced in vitro and in vivo by specific ELISA and immunohistochemistry. CXCL4L1/PF‐4var, but not CXCL4/PF‐4, was coinduced with the angiogenic chemokine CXCL6/granulocyte chemotactic protein‐2 (GCP‐2) by cytokines, e.g., IL‐1β and IL‐17, in sarcoma cells, but not in diploid fibroblasts. Furthermore, the induction of CXCL6/GCP‐2 in endothelial cells by IL‐1β was enhanced synergistically by TNF‐α but inhibited by IFN‐γ, which synergized with IL‐1β to produce the angiostatic CXCL10/IFN‐γ‐induced protein‐10. These findings indicate that the equilibrium between angiostatic and angiogenic factors during inflammation and tumor progression is rather complex and differs depending on the chemokine, cell type, and stimulus. Selective intervention in the chemokine network may drastically disturb this delicate balance of angiogenesis and tissue repair. Application of angiostatic CXCL4L1/PF‐4var without attraction of protumoral phagocytes may be beneficial in cancer therapy.

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.

Monocyte Chemotactic Proteins Related to Human MCP-1

This study describes the isolation and identification of two novel monocyte chemotactic factors from human tumor cells. Since the corresponding 7.5 kD and 11 kD proteins show high structural and functional similarity with MCP-1 (1) they are designated MCP-2 and MCP-3, respectively. Based on the conservation of four cysteine residues, these molecules can be classified in the chemokine family.