Collagen-induced arthritis is reduced in 5-lipoxygenase-activating protein-deficient mice.

Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.

Regulation of tumour necrosis factor production by adrenal hormones in vivo: insights into the antiinflammatory activity of rolipram.

1. The role of adrenal hormones in the regulation of the systemic and local production of tumour necrosis factor (TNF alpha) was examined in male Balb/c mice. 2. Intraperitoneal injection of 0.3 mg E. coli lipopolysaccharide (LPS, 0111:B4) led to high levels of circulating TNF alpha without stimulating TNF alpha production in the peritoneal cavity. Systemic production of TNF alpha in response to LPS was increased in adrenalectomized animals and in normal animals treated with the beta-adrenoceptor antagonist, propranolol. The glucocorticoid antagonist, RU 486, did not modify systemic TNF alpha production. These results indicate that systemic TNF alpha production is regulated by adrenaline but not by corticosterone. 3. When mice were primed with thioglycollate, TNF alpha was produced in the peritoneal cavity in response to low dose LPS (1 micrograms). The levels of TNF alpha in the peritoneal cavity were not enhanced by adrenalectomy or by treatment with either propranolol or RU 486, indicating local production of TNF alpha in the peritoneal cavity is not regulated by adrenaline or corticosterone. 4. The phosphodiesterase type IV (PDE-IV) inhibitor, rolipram, inhibited both the systemic production of TNF alpha in response to high dose endotoxin (ED50 = 1.3 mg kg-1) and the local production of TNF alpha in the peritoneal cavity in response to low dose endotoxin (ED50 = 9.1 mg kg-1). In adrenalectomized mice there was a slight reduction in the ability of rolipram to inhibit the systemic production of TNF alpha (ED50 = 3.3 mg kg-1) while the ability of rolipram to inhibit the local production of TNF alpha in the peritoneal cavity was virtually abolished (24% inhibition at 30 mg kg-1). The glucocorticoid antagonist, RU 486, also reduced the ability of rolipram to inhibit local TNF alpha production while propranolol was without effect. 5. Systemic treatment with rolipram increased the plasma concentrations of corticosterone in normal mice but not in adrenalectomized mice indicating that rolipram can cause adrenal stimulation in vivo. 6. In summary, these data indicate that systemic production of TNF alpha in response to high dose endotoxin is controlled differently from the local production of TNF alpha in response to low dose endotoxin. The systemic production of TNF alpha is regulated by catecholamines, but not by corticosterone, while the local production of TNF alpha in the peritoneal cavity is not regulated by basal levels of either catecholamines or corticosterone. 7. These data also show that the ability of rolipram to inhibit the local production of TNF alpha is dependent on the release of corticosterone from the adrenal glands.

ATP induces the release of IL-1 from LPS-primed cells in vivo.

The secretion of IL-1 from murine macrophages in vitro is an inefficient process that is distinct from those of other cytokines such as IL-6. We have therefore studied this process in vivo to see if these differences are maintained. Intraperitoneal injection of LPS in mice induced production and release of IL-6 into the extracellular fluid (peritoneal lavage). Although induction of intracellular IL-1 alpha and IL-1 beta was readily detected, these cytokines were not detected extracellularly. Injection of ATP 2 h after LPS led to the rapid extracellular release of IL-1 beta, IL-1 alpha, lactate dehydrogenase, and beta-N-acetylglucosaminidase. Western blot analysis revealed that a large proportion of the IL-1 beta was released as the 17-kDa form, whereas IL-1 alpha was unprocessed. Adenosine 5'-O-(3-thiotriphosphate) was also effective in causing IL-1 release but not UTP or ADP. This suggests that the ATP-mediated release of IL-1 is a receptor-mediated phenomenon that is associated with cell lysis.

GM-CSF rapidly primes mice for enhanced cytokine production in response to LPS and TNF.

Subcutaneous administration of Granulocyte-macrophage-colony stimulating factor (GM-CSF) to mice enhanced LPS-induced cytokine production in the circulation (TNF, IL-6) and peritoneal cavity (IL-1 beta, IL-6). This effect was induced rapidly (within 1 h) and persisted for 4 h. Mice made leukopenic with cyclophosphamide retained the ability to respond to GM-CSF. In addition, TNF induced IL-6 production was also enhanced. These results demonstrate that GM-CSF has a pronounced priming effect on cytokine producing cells in vivo and raises the possibility that this cytokine may contribute to the pathogenesis of septic shock.

Design and synthesis of 2-oxo-imidazolidine-4-carboxylic acid hydroxyamides as potent matrix metalloproteinase-13 inhibitors.

A novel series of imidazolidinone-based matrix metalloproteinase (MMP) inhibitors was discovered by structural modification of pyrrolidinone la. Potent inhibition of MMP-13 was exhibited by the analogues having 4-(4-fluorophenoxy)phenyl (4a, IC50 = 3 nM) and 4-(naphth-2-yloxy)phenyl (4h, IC50 = 4 nM) as P1' groups.