Yolande Chvatchko

Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis.

Two distinct IL-18 neutralizing strategies, i.e. a rabbit polyclonal anti-mouse IL-18 IgG and a recombinant human IL-18 binding protein (rhIL-18BP), were used to treat collagen-induced-arthritic DBA/1 mice after clinical onset of disease. The therapeutic efficacy of neutralizing endogenous IL-18 was assessed using different pathological parameters of disease progression. The clinical severity in mice undergoing collagen-induced arthritis was significantly reduced after treatment with both IL-18 neutralizing agents compared to placebo treated mice. Attenuation of the disease was associated with reduced cartilage erosion evident on histology. The decreased cartilage degradation was further documented by a significant reduction in the levels of circulating cartilage oligomeric matrix protein (an indicator of cartilage turnover). Both strategies efficiently slowed disease progression, but only anti-IL-18 IgG treatment significantly decreased an established synovitis. Serum levels of IL-6 were significantly reduced with both neutralizing strategies. In vitro, neutralizing IL-18 resulted in a significant inhibition of TNF-alpha, IL-6, and IFN-gamma secretion by macrophages. These results demonstrate that neutralizing endogenous IL-18 is therapeutically efficacious in the murine model of collagen-induced arthritis. IL-18 neutralizing antibody or rhIL-18BP could therefore represent new disease-modifying anti-rheumatic drugs that warrant testing in clinical trials in patients with rheumatoid arthritis.

p38 Mitogen-Activated Protein Kinase Downregulates Endothelial Progenitor Cells

Background—Transplantation of endothelial progenitor cells (EPCs) improves neovascularization after ischemia, but patients with coronary artery disease (CAD) or diabetes mellitus show a reduced number of EPCs and impaired functional activity. Therefore, we investigated the effects of risk factors, such as glucose and TNF-&agr;, on the number of EPCs in vitro to elucidate the underlying mechanisms. Methods and Results—EPCs of patients or healthy subjects were isolated from peripheral blood. Incubation with glucose or TNF-&agr; dose-dependently reduced the number of EPCs (79.9±1.3% and 74.3±8.1% of control; P<0.05, respectively). This reduction was not caused by apoptosis. TNF-&agr; and glucose induced a dose- and time-dependent activation of the p38 MAP kinase, the downstream kinase mitogen- and stress-activated kinase 1, and the transcription factor cAMP-responsive element–binding protein (CREB), in EPCs. Moreover, EPCs from CAD patients had significantly higher basal p38-phosphorylation levels (1.83±0.2-fold increase; P<0.05) compared with healthy subjects. The inhibition of the p38-kinase by SB203580 or infection with a dominant negative p38 kinase adenovirus significantly increased basal number of EPCs (136.7±6.3% and 142.9±18% versus control, respectively). Likewise, ex vivo cultivation of EPCs from patients with CAD with SB203580 significantly increased the number of EPCs and partially reversed the impaired capacity for neovascularization of EPCs in vivo (relative blood flow: 0.40±0.03 versus 0.64±0.08, P<0.05). The increased numbers of EPCs by SB203580 were associated with an augmentation of EPC proliferation and a reduction of cells expressing the monocytic marker proteins CD14 and CD64, suggesting that p38 regulates proliferation and differentiation events. Conclusions—These results demonstrate that p38 MAP kinase plays a pivotal role in the signal transduction pathways regulating the number of EPCs ex vivo. SB203580 can prevent the negative effects of TNF-&agr; and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy.

Evidence for altered interleukin 18 (IL)-18 pathway in human heart failure

Interleukin (IL)‐18 is the interferon‐γ‐inducing factor and has potent proinflammatory activities. IL‐18 has been recently implicated in atherosclerotic plaque instability and myocardial ischemia‐reperfusion injury. However, it is unknown whether IL‐18 expression is increased in human myocardium or if it has any role in heart failure. We analyzed the expression of IL‐18, its receptor IL‐18Rα, and its endogenous inhibitor, IL‐18 binding protein (IL‐18BP) in myocardial tissue from patients with end‐stage heart failure (ischemic or dilated cardiomyopathy) and controls by use of quantitative real‐time reverse transcriptase polymerase chain reaction, Western blot or immunohistochemical techniques. Plasma levels of IL‐18 were also determined in 48 patients with heart failure. IL‐18 mRNA and protein levels were up‐regulated in the myocardium of patients with ischemic cardiomyopathy. Both ischemic and dilated myocardium showed increased IL‐18Rα levels, suggesting potential biological effects. In addition, mRNA levels of IL‐18 BP were down‐regulated in the failing myocardium. Finally, plasma IL‐18 levels were significantly elevated in patients with heart failure and were higher in those who died at follow‐up than in survivors. The results suggest a potential role for the immunoinflammatory IL‐18 signaling pathway in the pathophysiology of heart failure and identify novel therapeutic targets for future testing.

Cutting Edge: A Murine, IL-12-Independent Pathway of IFN-γ Induction by Gram-Negative Bacteria Based on STAT4 Activation by Type I IFN and IL-18 Signaling

IFN-αβ is a potent immunoregulatory cytokine involved in the defense against viral and bacterial infections. In this study, we describe an as yet undefined IFN-αβ-dependent pathway of IFN-γ induction in mice. This pathway is based on a synergism of IFN-αβ and IL-18, and is independent of IL-12 signaling yet dependent on STAT4. In contradiction to current dogma, we show further that IFN-αβ alone induces tyrosine phosphorylation of STAT4 in murine splenocytes of different mouse strains. This pathway participates in the induction of IFN-γ by Gram-negative bacteria and is therefore expected to play a role whenever IFN-α or IFN-β and IL-18 are produced concomitantly during bacterial, viral, or other infections.

The Involvement of an ATP-Gated Ion Channel, P2X1, in Thymocyte Apoptosis

In the immune system, apoptosis is involved in intrathymic elimination of self-reactive thymocytes and in peripheral T cell tolerance to exogenous antigens. Here, we describe the role in T cell apoptosis of P(2x1), a nonselective cation channel activated by ATP. P(2X1) molecules are up-regulated in thymocytes during dexamethasone-induced apoptosis, and antagonists to these receptors protect thymocytes from cell death. Moreover, P(2X1) mRNA and protein levels increase in thymocytes induced to die in vivo by the superantigen staphylococcal enterotoxin B. In contrast, T cells undergoing apoptosis in the periphery do not express P(2X1). The demonstration that P(2X1) ion channels play a role in the apoptosis of thymocytes but not peripheral T cells illustrates a novel mechanism contributing to thymocyte cell death and opens new possibilities for investigating clonal deletion in the thymus.

The physiology of murine germinal center reactions.

Summary: Germinal center responses are the mechanism that the immune system uses normally to generate high affinity antigen‐specific B‐cell receptors and secreted immunoglobulins. Genetically altered mice have provided powerful toots for dissecting the physiology of these germinal center responses. In this review, we have attempted to summarize information from various sources and interpret the new observations based ON what was previously known. A section is included to review the basic anatomy of the relevant slim lures in lymph node and spleen. A summary of the mutant mice producing a phenotype where germinal center responses are altered is also furnished. This review is aimed at providing useful information to those working in this field as well as those wishing to understand more about in vivo immunology.

IL-18-binding protein expression by endothelial cells and macrophages is up-regulated during active Crohn's disease.

The pathogenesis of Crohn’s disease (CD) remains under intense investigation. Increasing evidence suggests a role for mature IL-18 in the induction of proinflammatory cytokines and Th1 polarization in CD lesions. The aim of this study was to investigate the contribution of the IL-18-neutralizing (a and c) and non-neutralizing (b and d) isoforms of IL-18-binding protein (IL-18BP) during active CD. Intestinal endothelial cells and macrophages were the major source of IL-18BP within the submucosa, and this IL-18BP production was also found to be relevant to other types of endothelial cells (HUVEC) and macrophages (peripheral monocytes). IL-18BP messenger transcript and protein were significantly increased in surgically resected specimens from active CD compared with control patients, correlating with an up-regulation of IL-18. Analysis of the expression of the four IL-18BP isoforms as well as being free or bound to IL-18 was reported and revealed that unbound IL-18BP isoforms a and c and inactive isoform d were present in specimens from active CD and control patients while isoform b was not detected. IL-18/IL-18BP complex was also detected. Interestingly, although most was complexed, free mature IL-18 could still be detected in active CD specimens even in the presence of the IL-18BP isoform a/c. These results demonstrate that the appropriate neutralizing isoforms are present in the intestinal tissue of patients with active CD and highlights the complexity of IL-18/IL-18BP biology.

Interleukin-18/Interleukin-18 Binding Protein Signaling Modulates Ischemia-Induced Neovascularization in Mice Hindlimb

Abstract— Identification of factors that may stimulate ischemia-induced neovascularization without increasing atherosclerotic plaque progression is of major therapeutic importance. We hypothesized that interleukin-18 binding protein (IL-18BP), a major antiinflammatory protein with plaque-stabilizing activities, may affect the neovascularization in mice ischemic hindlimb. Ischemia was produced by artery femoral occlusion in mice that were subjected to in vivo intramuscular electrotransfer of either an empty plasmid or a murine IL-18BP plasmid. Angiographic score, capillary density (CD31 staining), and laser Doppler perfusion data at day 28 showed significant improvement in ischemic/nonischemic leg ratio by respectively 1.6-, 1.4-, and 1.5-fold in IL-18BP–treated mice compared with controls (P <0.01). This was associated with a significant 2-fold increase in both vascular endothelial growth factor (VEGF) and phospho-Akt protein content in the ischemic hindlimb of IL-18BP–treated mice (P <0.05). Similar results were obtained in IL-18–deficient mice. Because bone marrow–derived endothelial progenitor cells (BM-EPCs) are involved in postnatal vasculogenesis, EPCs were isolated and cultivated from bone marrow mononuclear cells. IL-18BP treatment led to a significant 1.8-fold increase in the percentage of BM-EPCs characterized as cells positive for both AcLDL-Dil and von Willebrand factor (P <0.001). In conclusion, IL-18BP stimulates ischemia-induced neovascularization in association with an activation of VEGF/Akt signaling and an increase in BM-EPCs mobilization and differentiation. Our findings strongly suggest a major antiangiogenic role of endogenous IL-18 in postischemic injury.

Inhibition of Airway Inflammation by Amino-Terminally Modified RANTES/CC Chemokine Ligand 5 Analogues Is Not Mediated through CCR3

Chemokines play a key role in the recruitment of activated CD4+ T cells and eosinophils into the lungs in animal models of airway inflammation. Inhibition of inflammation by N-terminally modified chemokines is well-documented in several models but is often reported with limited dose regimens. We have evaluated the effects of doses ranging from 10 ng to 100 μg of two CC chemokine receptor antagonists, Met-RANTES/CC chemokine ligand 5 (CCL5) and aminooxypentane-RANTES/CCL5, in preventing inflammation in the OVA-sensitized murine model of human asthma. In the human system, aminooxypentane-RANTES/CCL5 is a full agonist of CCR5, but in the murine system neither variant is able to induce cellular recruitment. Both antagonists showed an inverse bell-shaped inhibition of cellular infiltration into the airways and mucus production in the lungs following allergen provocation. The loss of inhibition at higher doses did not appear to be due to partial agonist activity because neither variant showed activity in recruiting cells into the peritoneal cavity at these doses. Surprisingly, neither was able to bind to the major CCR expressed on eosinophils, CCR3. However, significant inhibition of eosinophil recruitment was observed. Both analogues retained high affinity binding for murine CCR1 and murine CCR5. Their ability to antagonize CCR1 and CCR5 but not CCR3 was confirmed by their ability to prevent RANTES/CCL5 and macrophage inflammatory protein-1β/CCL4 recruitment in vitro and in vivo, while they had no effect on that induced by eotaxin/CCL11. These results suggest that CCR1 and/or CCR5 may be potential targets for asthma therapy.

IL-18 Binding Protein Protects Against Contact Hypersensitivity

Allergic contact dermatitis, the clinical manifestation of contact hypersensitivity, is one of the most common disorders of the skin. It is elicited upon multiple cutaneous re-exposure of sensitized individuals to the sensitizing agent. In this study, we demonstrate that using IL-18 binding protein (IL-18BP) to neutralize IL-18 significantly reduced clinical symptoms in a murine model of contact hypersensitivity. Furthermore, IL-18BP alleviated the relapses during established disease, as indicated by significant protection during re-exposure of mice that had previously undergone a contact hypersensitivity response without treatment. Although edema was not influenced, IL-18BP reduced the number of T cells homing to sites of inflammation, resulting in diminished local production of IFN-γ. Thus, by preventing the accumulation of effector T cells to the target tissue, IL-18BP appears to be a potent protective mediator to counter skin inflammation during contact hypersensitivity. Taken together with the evidence that IL-18 is present in tissue samples of the human disease, our data reinforces IL-18BP as a candidate for this therapeutic indication.