Willy Put

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.

Monocyte Chemotactic Protein-1 in Proliferative Vitreoretinal Disorders

PURPOSE To investigate whether the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) are involved in the pathogenesis of proliferative vitreoretinal disorders and to study their possible interaction with IL-6. METHODS In a prospective study of 125 consecutive patients (125 eyes), vitreous and paired serum samples were obtained and were assayed for MCP-1 and IL-8. Levels of IL-6 were determined by proliferation of the IL-6-dependent hybridoma cell line 7TD1. RESULTS Monocyte chemotactic protein-1 was detected in 13 (48%) of 27 vitreous samples from patients with retinal detachment, in five (63%) of eight samples from patients with macular pucker, in 31 (72%) of 43 samples from patients with proliferative vitreoretinopathy, and in 32 (76%) of 42 samples from patients with proliferative diabetic retinopathy, but not in samples from five patients with idiopathic epiretinal membrane. There was a significant (P = .049) correlation between the incidence of MCP-1 detection in retinal detachment, macular pucker, and proliferative vitreoretinopathy groups and the severity of proliferation. Interleukin-8 was detected in two vitreous samples from eyes with retinal detachment, in two samples from eyes with proliferative vitreoretinopathy, and in three samples from eyes with proliferative diabetic retinopathy. Monocyte chemotactic protein-1 levels in the vitreous samples were positively correlated with IL-6 levels (r = .31, P = .01). Interleukin-6 levels were significantly (P = .0097) greater in vitreous samples with than without detectable levels of MCP-1. CONCLUSION Monocyte chemotactic protein-1 is present in a substantial percent of vitreous samples from eyes with proliferative vitreoretinal disorders and may help in stimulating the infiltration of monocytes and macrophages into eyes with these disorders.

Microbial Toll‐like receptor ligands differentially regulate CXCL10/IP‐10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN‐γ and provide a mechanism for enhanced synovial chemokine levels in septic arthritis

The CXC chemokine IFN‐γ‐inducible protein‐10 (IP‐10/CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T cells and natural killer cells. Peripheral blood mononuclearcells (PBMC) produce low but significant amounts of IP‐10/CXCL10 protein upon stimulation with double‐stranded (ds) RNA, the Toll‐like receptor 3 (TLR3) ligand. IFN‐γ is a superior IP‐10/CXCL10inducer. The bacterial TLR4 and TLR2 ligands, LPS and peptidoglycan (PGN), inhibit IFN‐γ‐ or dsRNA‐dependent IP‐10/CXCL10 production in PBMC, whereas IL‐8/CXCL8 production was enhanced. In fibroblasts a different picture emerges with IFN‐γ inducing moderate and dsRNA provoking strong IP‐10/CXCL10 production. Furthermore, treatment of fibroblasts with IFN‐γ in combination with bacterial LPS or PGN results in a synergistic production of IP‐10/CXCL10 and IL‐8/CXCL8. The synergistic induction of IP‐10/CXCL10 in fibroblasts is reflected by significantly enhanced IP‐10/CXCL10 concentrations in synovial fluids of septic compared to osteoarthritis patients to reach on average higher levels than those of IL‐8/CXCL8. These high amounts of IP‐10/CXCL10 produced by connective tissue fibroblasts not only attract CXCR3 expressing activated Th1 cells and natural killer cells to sites of infection but may also antagonize the CCR3 dependent attraction of Th2 lymphocytes and exert CXCR3‐independent, defensin‐like antibacterial activity.

TLR ligands and cytokines induce CXCR3 ligands in endothelial cells: enhanced CXCL9 in autoimmune arthritis

CXC chemokines are potent attractants of neutrophil granulocytes, T cells or natural killer cells. Toll-like receptors (TLR) recognize microbial components and are also activated by endogenous molecules possibly implicated in autoimmune arthritis. In contrast to CXC chemokine ligand 8 (CXCL8), no CXC chemokine receptor 3 (CXCR3) ligand (ie CXCL9, CXCL10 and CXCL11) was induced by bacterial TLR ligands in human microvascular endothelial cells (HMVEC). However, peptidoglycan (PGN), double-stranded (ds) RNA or lipopolysaccharide (LPS) (TLR2, TLR3 or TLR4 ligands, respectively) synergized with interferon-γ (IFN-γ) at inducing CXCL9 and CXCL10. In contrast, enhanced CXCL11 secretion was only obtained when IFN-γ was combined with TLR3 ligand. Furthermore, flagellin, loxoribine and unmethylated CpG oligonucleotide (TLR5, TLR7 and TLR9 ligands, respectively) did not enhance IFN-γ-dependent CXCR3 ligand production in HMVEC. In analogy with TLR ligands, tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β), in combination with IFN-γ, synergistically induced CXCL9 and CXCL11 in HMVEC and human fibroblasts, two fundamental cell types delineating the joint cavity. Etanercept, a humanized soluble recombinant p75 TNF-receptor/IgG1Fc fusionprotein, neutralized synergistic CXCL9 production induced by TNF-α plus IFN-γ, but not synergy between IFN-γ and the TLR ligands PGN or LPS. Synovial chemokine concentrations exemplify the fysiopathological relevance of the observed in vitro chemokine production patterns. In synovial fluids of patients with spondylarthropathies (ie ankylosing spondylitis or psoriatic arthritis) or rheumatoid arthritis, significantly enhanced CXCL9, but not CXCL11 levels, were detected compared to concentrations in synovial fluids of patients with metabolic crystal-induced arthritis. Thus, CXCL9 is an important chemokine in autoimmune arthritis.

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.

Synergistic induction of CXCL9 and CXCL11 by Toll‐like receptor ligands and interferon‐γ in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids

The synovial cavity constitutes the ideal stage to study the interplay between microbial Toll‐like receptor (TLR) ligands and cytokines. Infiltrated leukocytes and synovial fibroblasts produce cytokine‐ and chemokine‐induced proteases for remodeling the extracellular matrix. The regulation of chemokine function for attraction and activation of leukocytes constitutes a key feature in host immunity and resolution of inflammation after infection. Enhanced levels of the CXC chemokine ligand (CXCL9)/monokine induced by interferon‐γ (IFN‐γ) and CXCL11/IFN‐inducible T cell α chemoattractant, two chemoattractants for activated T cells and natural killer cells, and ligands for CXC chemokine receptor 3 (CXCR3) were detected in the synovial fluid of septic arthritis compared with osteo‐ and crystal arthritis patients. In vitro, IFN‐γ and TLR3 ligation by double‐stranded RNA (dsRNA) induced the expression of CXCL9 and CXCL11 in leukocytes and skin‐muscle fibroblasts, whereas ligation of TLR2, TLR4, TLR5, and TLR9 by peptidoglycan (PGN), lipopolysaccharide (LPS), flagellin, and unmethylated CpG oligonucleotides, respectively, did not. PGN and LPS, but not unmethylated CpG oligonucleotides, even inhibited IFN‐γ‐induced CXCL9 and CXCL11 expression in leukocytes. In sharp contrast, in fibroblasts, the TLR ligands PGN, dsRNA, LPS, and flagellin synergized with IFN‐γ for the production of CXCL9 and CXCL11. Although TLR ligands stimulate leukocytes to produce CXCL8/interleukin‐8 during the early innate defense, they contribute less to the production of CXCR3 ligands, whereas fibroblasts are important sources of CXCR3 ligands. These results illustrate the complex interaction between cytokines and TLR ligands in infection.

Selective induction of CCL18/PARC by staphylococcal enterotoxins in mononuclear cells and enhanced levels in septic and rheumatoid arthritis

Chemokines are mediators of innate and acquired immunity. CCL18, also designated pulmonary and activation‐regulated chemokine (PARC), dendritic cell‐derived CC chemokine‐1 (DC‐CK1), alternative macrophage activation‐associated CC chemokine‐1 (AMAC‐1) and macrophage inflammatory protein‐4 (MIP‐4), was for the first time isolated from peripheral blood mononuclear cells (PBMC) and biochemically characterized. We found that CCL18/PARC protein is spontaneously secreted by PBMC and is selectively induced in PBMC by staphylococcal enterotoxins (SEA, SEB) and IL‐4, but not by IFN‐γ andthe CXCL8/IL‐8 inducers lipopolysaccharide (LPS) or Concanavalin A. Human fibroblasts, chondrocytes and endothelial cells did not produce CCL18/PARC in response to inflammatory mediators such as measles virus, double‐stranded RNA, LPS or IL‐1β, whereas up to 150 ng/ml of CCL2/MCP‐1 was induced under these conditions. In synovial fluids from septic and rheumatoid arthritis patients, fourfold‐enhanced CCL18/PARC levels (150 ng/ml) were detected compared to those in crystal‐induced arthritis and osteoarthritis. In septic arthritis, the synovial levels of CCL18/PARC were fivefold higher than those of CXCL8/IL‐8. Immunochemistry revealed CD68+ monocytes/macrophages as the main CCL18/PARC‐producing cell type in both PBMC and arthritic synovial tissue. In addition, CD1a+ blood dendritic cells expressed CCL18/PARC. These findings suggest that monocytic cells respond to Gram‐positive bacterial infection by the production of CCL18/PARC in the synovial cavity.