Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.

A new quantitative cytometric technique, termed the ArrayScan™, is described and used to measure NF-B nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-␣ (TNF␣). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-B was found to move to the nucleus with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes, a rate in each case about 4 -5 min slower than that of IB␣ degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNF␣ and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNF␣ stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNF␣ stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent IB␣ degradation and subsequent NF-B translocation could also be discriminated; LeuLeu-Leu aldehyde was only a partial inhibitor with an IC 50 of 2 M, while clastolactacystin ␤-lactone was a complete inhibitor with an IC 50 of 10 M. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNF␣ stimulation in both cell types with an IC 50 of 0.4 M. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNF␣ concentration measured after 17 h of stimulation. These results suggest that the ArrayScan™ technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-B. IL-11 and TNF␣ are two master cytokines that induce an almost identical proinflammatory response, including the production of chemotactic cytokines, adhesion molecules, and enzymes such as cyclooxygenase, nitric-oxide synthetase, and matrix metalloproteinases (1, 2). Many of these effects are a result of the activation by both IL-1 and TNF␣ of the NF-B transcription factor pathway, which is associated with the activation of many cellular defense genes (3, 4). Composed of p65 (RelA) and p50 proteins, NF-B is normally present in the cytoplasm in an inactive state in a complex with members of the IB inhibitor protein family, chiefly the 37-kDa IB␣ form. In this complexed form, a nuclear localization sequence found on NF-B is masked by the IB␣, preventing nuclear translocation of NF-B, DNA binding, and subsequent transcriptional activation (5-12). IL-1 or TNF␣ receptor activation induces within several minutes the specific phosphorylation of Ser 32 and Ser 36 on IB␣, the destruction of the phosphorylated IB␣ protein by proteasomes, and the translocation of NF-B to the nucleus (13-1...

A nonpeptidyl small molecule antagonist, compound A, to nonactivated very late antigen-4 (VLA4) was examined in lung inflammation induced by a single dose of ovalbumin challenge. Compound A presented a good pharmacokinetic property, when given intratracheally, and the blood cells from such pharmacokinetic study showed good receptor occupancy of the compound for approximately 8 hours. Compound A was then tested in an ovalbumin-induced airway inflammation model by intranasal or intravenous route of administration. There was a dose-dependent inhibition of eosinophilia in the bronchiolar lavage fluid, when compound A was given intranasally but not when it was given intravenously. For comparison, antibody to VLA4 and another compound, BIO1211, which reacts only with activated VLA4, were examined in this system. Immunohistochemical analyses of the lung tissue substantiated the findings in the bronchiolar lavage fluid. Specific staining of the major basic protein of eosinophils showed peribronchiolar infiltration of eosinophils. Some of these eosinophils were also positive for nitrotyrosine, suggesting activation of eosinophils in the lung interstitium. There was deposition of major basic protein and nitrotyrosine at the base of the perivascular endothelium, indicative of degranulation of eosinophils in the area. After intranasal treatment with compound A, eosinophils in the lungs and their activation products were substantially decreased, documenting its effectiveness in inhibiting lung inflammation.

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is implicated in the etiology of metabolic syndrome. We previously showed that pharmacological inhibition of 11β-HSD1 ameliorated multiple facets of metabolic syndrome and attenuated atherosclerosis in ApoE-/- mice. However, the molecular mechanism underlying the atheroprotective effect was not clear. In this study, we tested whether and how 11β-HSD1 inhibition affects vascular inflammation, a major culprit for atherosclerosis and its associated complications. ApoE-/- mice were treated with an 11β-HSD1 inhibitor for various periods of time. Plasma lipids and aortic cholesterol accumulation were quantified. Several microarray studies were carried out to examine the effect of 11β-HSD1 inhibition on gene expression in atherosclerotic tissues. Our data suggest 11β-HSD1 inhibition can directly modulate atherosclerotic plaques and attenuate atherosclerosis independently of lipid lowering effects. We identified immune response genes as the category of mRNA most significantly suppressed by 11β-HSD1 inhibition. This anti-inflammatory effect was further confirmed in plaque macrophages and smooth muscle cells procured by laser capture microdissection. These findings in the vascular wall were corroborated by reduction in circulating MCP1 levels after 11β-HSD1 inhibition. Taken together, our data suggest 11β-HSD1 inhibition regulates proinflammatory gene expression in atherosclerotic tissues of ApoE-/- mice, and this effect may contribute to the attenuation of atherosclerosis in these animals.