Interferon-gamma-inducing factor (IGIF, interleukin-18) is a recently described cytokine that shares structural features with the interleukin-1 (IL-1) family of proteins and functional properties with IL-12. Like IL-12, IGIF is a potent inducer of interferon (IFN)-gamma from T cells and natural killer cells. IGIF is synthesized as a biologically inactive precursor molecule (proIGIF). The cellular production of IL-1beta, a cytokine implicated in a variety of inflammatory diseases, requires cleavage of its precursor (proIL-1beta) at an Asp-X site by interleukin-1beta-converting enzyme (ICE, recently termed caspase-1). The Asp-X sequence at the putative processing site in proIGIF suggests that a protease such as caspase-1 might be involved in the maturation of IGIF. Here we demonstrate that caspase-1 processes proIGIF and proIL-1beta with equivalent efficiencies in vitro. A selective caspase-1 inhibitor blocks both lipopolysaccharide-induced IL-1beta and IFN-gamma production from human mononuclear cells. Furthermore, caspase-1-deficient mice are defective in lipopolysaccharide-induced IFN-gamma production. Our results thus implicate caspase-1 in the physiological production of IGIF and demonstrate that it plays a critical role in the regulation of multiple proinflammatory cytokines. Specific caspase-1 inhibitors would provide a new class of anti-inflammatory drugs with multipotent action.

Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.

Objective. To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases.Methods. A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys.Results. LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its

SummaryConsiderable evidence has associated the expression of matrix metalloproteinases (MMPs) with the degradation of cartilage and bone in chronic conditions such as arthritis. Direct evaluation of MMPs' role in vivo has awaited the development of MMP inhibitors with appropriate pharmacological properties. We have identified butanediamide, N4-hydroxy-2-(2-methylpropyl)-as a potent MMP inhibitor with sufficient solubility and stability to permit evaluation in an experimental model of chronic destructive arthritis (adjuvant-induced arthritis) in rats. In this model, pronounced acute and chronic synovial inflammation, distal tibia and metatarsal marrow hyperplasia associated with osteoclasia, severe bone and cartilage destruction, and ectopic new bone growth are well developed by 3 wk after adjuvant injection. Rats were injected with Freund's adjuvant on day 0. GI168 was was administered systemically from days 8 to 21 by osmotic minipumps implanted subcutaneously. GI168 at 6, 12, and 25 mg/kg per d reduced ankle swelling in a dose-related fashion. Radiologicai and histological ankle joint evaluation on day 22 revealed a profound dose related inhibition of bone and cartilage destruction in treated rats relative to rats receiving vehicle alone. A significant reduction in edema, pannus formation, periosteal new bone growth and the numbers of adherent marrow osteoclasts was also noted. However, no significant decrease in polymorphonuclear and mononuclear leukocyte infiltration of synovium and marrow hematopoietic cellularity was seen. This unique profile of antiarthritic activity indicates that GI168 is osteo-and chondro-protective, and it supports a direct role for MMP in cartilage and bone damage and pannus formation in adjuvant-induced arthritis.

No abstract

SUMMARYElevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a /Jj-agonist which activates adenylate cyclase, its ability lo inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol. did not inhibit IL-l/i produclion in vitro, both drugs did inhibit tumour necrosis factoralpha (TNF-a) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar ICjoS of approximately OlyiM. This inhibition was effectively reversed by the /i2-antagonist oxprenolol, indicating that the inhibition was mediated through the ,tf2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human Tcelis. as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-a production, and was not reversed by a /32-antagonist, indicating thai a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-a inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with EDsos of approximately 01 mg/kg. This inhibition could be abrogated by dosing orally with the /^blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3h. while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-a production. Though satmeterol inhibited serum TNF-a levels by up to 94% in this assay, it protected less than 50% ofthe animals from the lethal effects ofthe LPS/gal actosa mine mixture. This observation suggests that functional levels of TNF-a localized in tissues may not be accurately reflected by serum levels.

SUMMARYInhibitors of cyclic nucleotide phosphodiesterases are known to suppress lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-Q) production in vitro In human monocytes. The most potent of these have selectivity for type IV PDEs, suggesting that this class of PDE is the major type involved in the regulation of human TNF-« production. Using compounds of two distinct chemical structural classes, a quinazolincdionc (CP-77 059) and a 4 arylpyrrolidinone (rolipram). we show here that PDE-IV-specific inhibitors are also potent in stippressing LPSinduced TNF-rv production in vitro in sodium periodate-clicited murine macrophages (IC'syS of 1 and 33, respectively). We then report the in vivo anti-inflammatory effect of PDE-IV inhibition in five murine models of inflammation: (i) elevation of serum TNF-a induced by a subtethal LPS injection; {ii) LPS-indueed endotoxic shock; (iii) LPS/galactosamine-induced endotoxic shock; (iv) carrageenan-induced paw oedema; and (v) adjuvant arthritis. Following a sublcthal {5/(g/mouse) injection of LPS, serum TNF-a levels in miee peaked sharply, reaching concentrations of 3-12ng/ ml 90 min after injection. In this sublethal LPS assay. CP-77 059 was about 30 times more potent than rolipram, with a minimum effective dose of 01 mg/kg versus 3 mg/kg for rolipram. This rank order is in keeping with the relative in vitro ICsoS for CP-77059 and rolipram, as well as their relative Ki against the human PDE-IV enzyme (46 nM and 220 nM, respectively). In LPS-induced endotoxic shock, rolipram and CP-77 059 at relatively high doses of 30 and 10 mg/kg. respectively. significantly reduced serum TNF-ft levels, and also inhibited mortality 66%. In the LPS/ galactosamine shock model, in which mice are rendered exquisitely sensitive to LPS by coinjection with galaetosamine. oniy 01 ^ig of LPS/mouse Is necessary for serum TNF-cv elevation and death. Both rolipram and the CP-77059 caused dose-dependent reduction of serum TNF-rt and lethality. In the carrageenan-induced paw oedema model, in which there is a pronounced local TNF-(v response {without a serum TNF-a elevation), rolipram significantly inhibited paw swelling as well as localized TNF-cv levels in the paw. In the adjuvant arthritis model, a chronic model of inflammation also possessing localized TNF-a elevation in the inflamed paw, rolipram and CP-77059 suppressed ankle swelling and radiological evidence of joint damage. These data are consistent with a major role for PDE-IV in regulation of TNF-a production and inflammatory responses in murine systems. It suggests a potential therapeutic use for PDE-IV-specific inhibitors in inflammatory disease such as rheumatoid arthritis, septic shock and other inflammatory diseases where TNF-a has been postulated to be a contributing factor in the pathology ofthe disease.

No abstract