Leonid L. Reznikov

Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response.

An interleukin-18 binding protein (IL-18BP) was purified from urine by chromatography on IL-18 beads, sequenced, cloned, and expressed in COS7 cells. IL-18BP abolished IL-18 induction of interferon-gamma (IFNgamma), IL-8, and activation of NF-kappaB in vitro. Administration of IL-18BP to mice abrogated circulating IFNgamma following LPS. Thus, IL-18BP functions as an inhibitor of the early Th1 cytokine response. IL-18BP is constitutively expressed in the spleen, belongs to the immunoglobulin superfamily, and has limited homology to the IL-1 type II receptor. Its gene was localized on human chromosome 11q13, and no exon coding for a transmembrane domain was found in an 8.3 kb genomic sequence. Several Poxviruses encode putative proteins highly homologous to IL-18BP, suggesting that viral products may attenuate IL-18 and interfere with the cytotoxic T cell response.

The antitumor histone deacetylase inhibitor suberoylanilide hydroxamic acid exhibits antiinflammatory properties via suppression of cytokines

Suberoylanilide hydroxamic acid (SAHA) is a hydroxamic acid-containing hybrid polar molecule; SAHA specifically binds to and inhibits the activity of histone deacetylase. Although SAHA, like other inhibitors of histone deacetylase, exhibits antitumor effects by increasing expression of genes regulating tumor survival, we found that SAHA reduces the production of proinflammatory cytokines in vivo and in vitro. A single oral administration of SAHA to mice dose-dependently reduced circulating TNF-α, IL-1-β, IL-6, and IFN-γ induced by lipopolysaccharide (LPS). Administration of SAHA also reduced hepatic cellular injury in mice following i.v. injection of Con A. SAHA inhibited nitric oxide release in mouse macrophages stimulated by the combination of TNF-α plus IFN-γ. Human peripheral blood mononuclear cells stimulated with LPS in the presence of SAHA released less TNF-α, IL-1-β, IL-12, and IFN-γ (50% reduction at 100–200 nM). The production of IFN-γ stimulated by IL-18 plus IL-12 was also inhibited by SAHA (85% at 200 nM). However, SAHA did not affect LPS-induced synthesis of the IL-1-β precursor, the IL-1 receptor antagonist, or the chemokine IL-8. In addition, IFN-γ induced by anti-CD3 was not suppressed by SAHA. Steady-state mRNA levels for LPS-induced TNF-α and IFN-γ in peripheral blood mononuclear cells were markedly decreased, whereas IL-8 and IL-1-β mRNA levels were unaffected. Because SAHA exhibits antiinflammatory properties in vivo and in vitro, inhibitors of histone deacetylase may stimulate the expression of genes that control the synthesis of cytokines and nitric oxide or hyperacetylate other targets.

Overview of interleukin-18: more than an interferon-gamma inducing factor.

Initially described in 1989 as interferon‐γ (IFN‐γ) inducing factor (IGIF), interleukin‐18 (IL‐18) is a novel proinflammatory cytokine that is clearly more than an inducer of IFN‐γ The cytokine possesses several biological properties such as activation of nuclear factor‐κB (NF‐κB), Fas ligand expression, the induction of both CC and CXC chemokines, and increased production of competent human immunodeficiency virus. Most activities are due to a receptor complex that recruits the IL‐1 receptor‐activating kinase (IRAK), leading to translocation of NF‐κB. This property and others support the concept that IL‐18 is related to the IL‐1 family. Indeed, one of the IL‐18 receptor chains is the IL‐1 receptor‐related protein, a member of the IL‐1R family. In addition, IL‐18 is structurally similar to IL‐1β and like IL‐1β is first synthesized as a leaderless precursor requiring the IL‐1β converting enzyme for cleavage into an active molecule. The biology of IL‐18 is reviewed in the overview and the implication for a role for this cytokine in disease is presented. J. Leukoc. Biol. 63: 658–664; 1998.

Cisplatin-induced acute renal failure is associated with an increase in the cytokines interleukin (IL)-1beta, IL-18, IL-6, and neutrophil infiltration in the kidney.

We have demonstrated that caspase-1-deficient (caspase-1–/–) mice are functionally and histologically protected against cisplatin-induced acute renal failure (ARF). Caspase-1 exerts proinflammatory effects via the cytokines interleukin (IL)-1β, IL-18, IL-6, and neutrophil recruitment. We sought to determine the role of the cytokines IL-1β, IL-18, and IL-6 and neutrophil recruitment in cisplatin-induced ARF. We first examined IL-1β; renal IL-1β increased nearly 2-fold in cisplatin-induced ARF and was reduced in the caspase-1–/– mice. However, inhibition with IL-1 receptor antagonist (IL-1Ra) did not attenuate cisplatin-induced ARF. Renal IL-18 increased 2.5-fold; however, methods to inhibit IL-18 using IL-18 antiserum and transgenic mice that overproduce IL-18-binding protein (a natural inhibitor of IL-18) did not protect. Renal IL-6 increased 3-fold; however, IL-6-deficient (IL-6–/–) mice still developed cisplatin-induced ARF. We next examined neutrophils; blood neutrophils increased dramatically after cisplatin injection; however, prevention of peripheral neutrophilia and renal neutrophil infiltration with the neutrophil-depleting antibody RB6-8C5 did not protect against cisplatin-induced ARF. In summary, our data demonstrated that cisplatin-induced ARF is associated with increases in the cytokines IL-1β, IL-18, and IL-6 and neutrophil infiltration in the kidney. However, inhibition of IL-1β, IL-18, and IL-6 or neutrophil infiltration in the kidney is not sufficient to prevent cisplatin-induced ARF.

Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1β

The proinflammatory cytokine IL-18 was investigated for its role in human myocardial function. An ischemia/reperfusion (I/R) model of suprafused human atrial myocardium was used to assess myocardial contractile force. Addition of IL-18 binding protein (IL-18BP), the constitutive inhibitor of IL-18 activity, to the perifusate during and after I/R resulted in improved contractile function after I/R from 35% of control to 76% with IL-18BP. IL-18BP treatment also preserved intracellular tissue creatine kinase levels (by 420%). Steady-state mRNA levels for IL-18 were elevated after I/R, and the concentration of IL-18 in myocardial homogenates was increased (control, 5.8 pg/mg vs. I/R, 26 pg/mg; P < 0.01). Active IL-18 requires cleavage of its precursor form by the IL-1β-converting enzyme (caspase 1); inhibition of caspase 1 also attenuated the depression in contractile force after I/R (from 35% of control to 75.8% in treated atrial muscle; P < 0.01). Because caspase 1 also cleaves the precursor IL-1β, IL-1 receptor blockade was accomplished by using the IL-1 receptor antagonist. IL-1 receptor antagonist added to the perifusate also resulted in a reduction of ischemia-induced contractile dysfunction. These studies demonstrate that endogenous IL-18 and IL-1β play a significant role in I/R-induced human myocardial injury and that inhibition of caspase 1 reduces the processing of endogenous precursors of IL-18 and IL-1β and thereby prevents ischemia-induced myocardial dysfunction.

Interleukin-1β-regulating antibody XOMA 052 (gevokizumab) in the treatment of acute exacerbations of resistant uveitis of Behcet's disease: an open-label pilot study.

Objective Uveitis and retinal vasculitis are sight-threatening manifestations of Behçets disease with limited treatment options. This pilot study aimed to evaluate the safety, pharmacokinetics and clinical activity of XOMA 052 (gevokizumab), a recombinant humanised anti-interleukin 1β antibody, in Behçets disease patients with uveitis. Methods Patients with acute posterior or panuveitis, and/or retinal vasculitis, resistant to azathioprine and/or ciclosporin, and receiving 10 mg/day or less of prednisolone, were enrolled into the 98-day study. Immunosuppressive agents were discontinued at baseline. Patients received a single infusion of XOMA 052 (0.3 mg/kg). The safety and uveitis status and pharmacokinetics of XOMA 052 were evaluated. Results Seven patients enrolled and completed the study. No treatment-related adverse event was observed. XOMA 052 treatment was associated with rapid and durable clinical response in all patients. Complete resolution of intraocular inflammation was achieved in 4–21 days (median 14 days), with a median duration of response of 49 days (range 21–97 days); one patient remained exacerbation free throughout the study. Conclusions Well tolerated, XOMA 052 resulted in a rapid onset and sustained reduction in intraocular inflammation in patients with resistant uveitis and retinal vasculitis. Moreover, the effect was observed despite discontinuation of immunosuppressive agents and without the need to increase corticosteroid dosages.

A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity.

IL-1F7 was discovered in expressed sequence tag databases as a member of the increasing family of proteins sharing sequence homology to IL-1α/β, IL-1Ra, and IL-18. In the present study using immunohistochemical staining, IL-1F7 was localized in human peripheral monocytic cells, suggesting its role in immune regulation. Recombinant human IL-1F7b was shown to bind to the IL-18Rα but without IL-18 agonistic or antagonistic function. Using chemical cross-linking, we observed that, unlike IL-18, IL-1F7b fails to recruit the IL-18Rβ chain to form a functionally active, ternary complex with the IL-18Rα chain. IL-1F7b shares two conserved amino acids with IL-18 (Glu-35 and Lys-124), which participate in the interaction of IL-18 with the IL-18Rα chain as well as the IL-18-binding protein (IL-18BP), a secreted protein that neutralizes IL-18 activity. In testing whether IL-1F7b interacts with IL-18BP, we unexpectedly observed that IL-1F7b enhanced the ability of IL-18BP to inhibit IL-18-induced IFNγ by 25–30% in a human natural killer cell line. This effect was observed primarily at limiting concentrations of IL-18BP (3.12–12.5 ng/ml) and at a 50- to 100-fold molar excess of IL-1F7b. Similar results were obtained by using isolated human peripheral blood mononuclear cells. To study the molecular basis of this effect we performed binding studies of IL-1F7b and IL-18BP. After cross-linking, a high molecular weight complex consisting of IL-1F7b and IL-18BP was observed on SDS/PAGE. We propose that after binding to IL-18BP, IL-1F7b forms a complex with IL-18Rβ, depriving the β-chain of forming a functional receptor complex with IL-18Rα and thus inhibiting IL-18 activity.

Ischemia alone is sufficient to induce TNF-alpha mRNA and peptide in the myocardium.

Over-production of tumor necrosis factor-alpha (TNF-&agr;) following myocardial ischemia-reperfusion contributes to cardiac dysfunction, and anti-TNF-&agr; has therapeutic potential for myocardial protection in cardiac surgery with obligatory ischemia. It remains unclear, however, whether myocardial TNF-&agr; production occurs during ischemia and whether cardiac myocytes constitute a source of myocardial TNF-&agr;. Ischemia alone has been shown to activate myocardial NF-&kgr;B. We hypothesized that ischemia alone is sufficient to induce myocardial TNF-&agr; gene expression and peptide synthesis. We examined TNF-&agr; production and NF-&kgr;B activation in the isolated rat heart subjected to global normothermic ischemia. Myocardial ischemia resulted in rapid I&kgr;B-&agr; degradation and NF-&kgr;B activation. Immunofluorescence staining detected NF-&kgr;B intranuclear translocation primarily in myocardial interstitial cells. Ischemia alone induced a time-dependent increase in myocardial TNF-&agr;. TNF-&agr; peptide increased to 20.3 ± 3.0 pg/mg after 25 min of ischemia (P < 0.05 vs 8.9 ± 2.0 pg/mg in perfusion control). TNF-&agr; was also localized to myocardial interstitial cells. Increased TNF-&agr; peptide level correlated with TNF-&agr; mRNA expression. We conclude that ischemia alone induces TNF-&agr; gene expression and peptide synthesis in the myocardium that are associated with NF-&kgr;B activation. Non-myocytes constitute the main source of myocardial TNF-&agr; following ischemia. The results suggest that therapeutic strategies attempting to decrease myocardial TNF-&agr; production need to be applied before or in the early phase of ischemia.

Functional Reconstitution and Regulation of IL-18 Activity by the IL-18Rβ Chain

IL-18 and IL-12 are major IFN-γ-inducing cytokines but the unique synergism of IL-18 and IL-12 remains unclear. In the human NK cell line NKO, IL-18Rα, and IL-18Rβ are expressed constitutively but IL-18 did not induce IFN-γ unless IL-12 was present. COS-1 fibroblasts, which produce the chemokine IL-8 when stimulated by IL-1β or TNF-α, do not respond to IL-18, despite abundant expression of the IL-18Rα chain. COS-1 cells lack expression of the IL-18Rβ chain. The IL-18Rβ cDNA was cloned from a human T-B lymphoblast cDNA library and COS-1 cells were transiently transfected with the IL-18Rβ chain and a luciferase reporter. In transfected COS-1 cells, IL-18 induced IL-8 and luciferase in the absence of IL-12 and independently of IL-1 and TNF. Ab against the IL-18Rα chain, however, prevented IL-18 responsiveness in COS-1 cells transfected with the IL-18Rβ chain, suggesting that both chains be functional. In NKO cells and PBMC, IL-12 increased steady-state mRNA levels of IL-18Rα and IL-18Rβ; the production of IFN-γ corresponded to IL-12-induced IL-18Rα and IL-18Rβ chains. We conclude that functional reconstitution of the IL-18Rβ chain is essential for IL-12-independent proinflammatory activity of IL-18-induced IL-8 in fibroblasts. The synergism of IL-18 plus IL-12 for IFN-γ production is, in part, due to IL-12 up-regulation of both IL-18Rα and IL-18Rβ chains, although postreceptor events likely contribute to IFN-γ production.

Liposomal Delivery of Purified Inhibitory-κBα Inhibits Tumor Necrosis Factor-α–Induced Human Vascular Smooth Muscle Proliferation

Vessel injury results in the elaboration of various cytokines, including tumor necrosis factor-alpha (TNF-alpha), which may influence vascular smooth muscle cell (VSMC) function and contribute to atherogenesis. We tested the hypothesis that TNF-alpha-induced VSMC proliferation requires activation of the transcription factor nuclear factor-kappaB (NF-kappaB), which could be prevented by delivery of the NF-kappaB inhibitory peptide, IkappaBalpha. TNF-alpha induced concentration-dependent human VSMC proliferation, and neutralizing antibody to interleukin-6 reduced TNF-alpha-induced VSMC proliferation by 65%. In TNF-alpha-stimulated VSMCs, there was a 3-fold increase in NF-kappaB-dependent luciferase reporter activity that was associated with degradation of IkappaBalpha. To determine an essential role for NF-kappaB in TNF-alpha-induced VSMC proliferation, recombinant IkappaBalpha was introduced into VSMCs via liposomal delivery. Under these conditions, TNF-alpha-induced NF-kappaB nuclear translocation and DNA binding were inhibited, NF-kappaB-dependent luciferase activity was reduced by 50%, there was no degradation of native IkappaBalpha detected, interleukin-6 production was reduced by 54%, and VSMC proliferation was decreased by 60%. In conclusion, the mitogenic effect of TNF-alpha on human arterial VSMCs is dependent on NF-kappaB activation and may be prevented by exogenously delivered IkappaBalpha. Furthermore, liposomal delivery of endogenous inhibitory proteins may represent a novel, therapeutically accessible method for selective transcriptional suppression in the response to vascular injury.