Denis J. Schrier

Role of chemokines and cytokines in a reactivation model of arthritis in rats induced by injection with streptococcal cell walls.

Intraarticular injection of streptococcal cell wall (SCW) antigen followed by intravenous challenge results in a T cell‐mediated monoarticular arthritis in female Lewis rats. Initial studies showed that this reactivation response to intravenous SCW antigen is dependent on the presence of interleukin‐1 (IL‐1) and tumor necrosis factor α (TNF‐α) and that the early phase of swelling is neutrophil‐dependent. Neutrophil depletion or passive immunization with antibodies to P‐selectin or macrophage inflammatory protein‐2 reduced the intensity of ankle edema and the influx of neutrophils. After the first few days, however, the arthritic response is mediated primarily by mononuclear cells. Joint tissues showed up‐regulation of mRNA for monocyte chemotactic protein‐1 (MCP‐1), which could be inhibited in part by anti‐IL‐4; treatment of rats with antibodies to IL‐4 or MCP‐1 significantly suppressed development of ankle edema and histopathological evidence of inflammation. Antibodies to interferon‐γ or IL‐10 had no effect. Treatment with anti‐MCP‐1 also suppressed influx of 111In‐labeled T cells into the ankle joint. These data suggest that the late, mononuclear‐dependent phase of SCW‐induced arthritis in female Lewis rats requires cytokines that up‐regulate MCP‐1, which in turn may facilitate recruitment and extravasation of mononuclear cells into the joint. J. Leukoc. Biol. 63: 359–363; 1998.

Anti-inflammatory properties of the protein kinase C inhibitor, 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H- pyrrole-2,5-dione monohydrochloride (GF109203X) in the PMA-mouse ear edema model.

Protein kinase C (PKC) mediates a number of intracellular signal transduction pathways implicated in the pathogenesis of inflammation, including phospholipase A2-dependent arachidonic acid release and eicosanoid production. Recent studies demonstrate that the PKC inhibitor GF109203X significantly reduces a number of inflammatory processes resulting from PKC activation by the topical application of phorbol myristate acetate (PMA) to mouse ears. In this model, GF109203X significantly reduced edema at doses similar to the PKC inhibitor staurosporine, and more effectively than indomethacin, zileuton, or sodium meclofenamate. Histological and biochemical analysis of biopsies from control and drug-treated ears revealed a marked reduction in edema, infiltrating neutrophils, and levels of the neutrophil-specific marker, myeloperoxidase, in GF109203X-treated mice. Prostaglandin E2 levels were also reduced in ears treated with GF109203X. These data suggest that GF109203X is an effective antiinflammatory agent as evaluated in the PMA model of edema, and implicates PKC as a potential target in the development of novel anti-inflammatory agents.

Effects of the phosphodiesterase inhibitor rolipram on streptococcal cell wall-induced arthritis in rats.

The phosphodiesterase-IV (PDE-IV) inhibitor, rolipram, is antiinflammatory in a number of animal models and inhibits the release of a variety of cytokines, including TNFalpha. Arthritis induced in rats by systemic reactivation with streptococcal cell walls (SCW) following intraarticular sensitization is a TNFalpha-dependent, delayed-type hypersensitivity (DTH) reaction. Rolipram administered during the reactivation phase dose-dependently inhibited hind paw edema through day 24, the day of peak swelling. PMN and T-cell recruitment to the arthritic joint were also attenuated in rolipram-treated rats. Histologic examination of ankle sections from rolipram-treated animals showed a marked attenuation of synovial inflammation. Mechanistic studies to determine the role of glucocorticoids in mediating rolipram action showed that the inhibitory effect of rolipram on swelling was not reversed by RU 486, a glucocorticoid antagonist. In contrast, RU 486 reversed the inhibitory effects of rolipram on TNFalpha secretion. To further evaluate the role of cAMP in the model, the beta-adrenergic receptor (betaAR) agonist isoproterenol was tested, and found to inhibit swelling but not the release of TNFalpha. These results are consistent with the view that the inhibitory effects of rolipram may be partially mediated by cAMP-dependent, but TNFalpha-independent, mechanisms. The betaAR antagonists propranolol and nadolol had no appreciable affect on the antiinflammatory effect of rolipram. However, rolipram reversed the lethal effects of the antagonists observed when either was administered alone. Apparently, beta-adrenergic mechanisms moderate the response to challenge, and rolipram treatment, presumably as a result of its effects on cAMP levels, reverses the toxic effect of the antagonists.

Oxazole, thiazole, and imidazole derivatives of 2,6-di-tert-butylphenol as dual 5-lipoxygenase and cyclooxygenase inhibitors

Benzylidene di-tert-butylphenols containing oxazole, thiazole, and imidazole substituents are dual inhibitors of 5-lipoxygenase and cyclooxygenase with IC50 values <5 μM. The oxazole and thiazole analogs exhibit oral antiinflammatory activity.

The effects of (R)-N-(1-methyl-2-phenylethyl) adenosine (L-PIA), a standard A1-selective adenosine agonist on rat acute models of inflammation and neutrophil function

L-PIA, a standard A1-selective adenosine agonist, was evaluated orally in carrageenan (CRG)- and reverse passive arthus-pleurisy. White blood cell (WBC) and exudate accumulation were assessed four hours after induction of the inflammatory response. L-PIA inhibited WBC accumulation in both models with ID50s of 4.37 and 4.42 mg/kg, respectively. In contrast, exudate was inhibited by L-PIA only in the CRG pleurisy model (ID50=1.01 mg/kg). In mechanistic studies, L-PIA reversed the drop in circulating neutrophil count which occurred within 15 minutes after CRG injection, suggesting that L-PIA may inhibit adhesion of the cells to the endothelium. The effects of L-PIA on several parameters of rat neutrophil function were determined. Enzyme release, O2−, TXB2, and LTB4 production were monitored in response to FMLP and opsonized zymosan (SOZ) stimulation. At high concentrations L-PIA had a mild inhibitory effect on O2− release in response to FMLP and had a moderate effect on arachidonic acid metabolite production, in response to both stimuli. The other response were unaffected. These results suggest that L-PIA may prevent diapedisis or neutrophil adhesion to the endothelium, but has a minimal effect on enzyme release O2−, LTB4 and TXB2 production.

The pharmacologic effects of 5-[3,5-BIS(1,1-dimethylethyl)-4-hydroxyphenyl]-1,3,4-thiadiazole-2(3H)-thione, choline salt (CI-986), a novel inhibitor of arachidonic acid metabolism in models of inflammation, analgesia and gastric irritation

CI-986 is a potent inhibitor of 5-lipoxygenase and cyclooxygenase pathway product biosynthesis from rat basophilic leukemia (RBL) cells. Because metabolites from these pathways have proinflammatory properties, CI-986 was evaluated in several acute and chronic models of inflammation and hyperalgesia. The compound inhibited swelling in the carrageenan footpad edema, Mycobacterium foot-pad edema and adjuvant arthritis models of inflammation with ID40 values of 1.0, 7.7., and 7.2 mg/kg, respectively. It was roughly equivalent in potency to the standard selective cyclooxygenase inhibitor, naproxen (ID40 = 0.7, 6.3, and 3.8 mg/kg, respectively). CI-986 was also evaluated in the acetic acid induced writhing hyperalgesia assay (ID50 = 0.23 mg/kg) and was approximately equipotent with indomethacin (ID50 = 0.87 mg/kg). Although the effects of CI-986 were similar to those of standard nonsteroidal antiinflammatory drugs (NSAIDs) in the inflammation models, its gastrointestinal profile was unique. CI-986 caused no gastrointestinal irritation at doses up to 200 mg/kg in acute and chronic studies. In contrast, standard NSAIDs caused ulcers at doses of 3.7-37 mg/kg after a single dose. Moreover, CI-986 inhibited the release of LTC4 and PGE2 by gastric mucosa and reduced mucosal and vascular damage induced by oral administration of absolute ethanol to rats. These results indicate that CI-986 is a potent nonulcerogenic antiinflammatory agent with novel pharmacologic properties.

The topical anti-inflammatory effects of a topical preparation of meclofenamic acid on carrageenan-induced footpad swelling in mice

A topical preparation of meclofenamic acid (Meclomen) was tested for anti‐inflammatory activity in a murine model of carrageenan footpad oedema. The preparation significantly inhibited swelling when applied to the carrageenan‐injected paw. Maximum inhibition was observed 4–5 h after carrageenan injection. The topical effects could not be attributed to systemic absorption because the preparation was more inhibitory when applied topically to the carrageenan‐injected paw than to a distant site or orally.

Ribozymes as Tools for Therapeutic Target Validation in Arthritis

In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5′ untranslated region, the coding region, and the 3′ untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.

Cytoprotective Effects of CI-959 in the Rat Gastric Mucosa: Modulation of Leukocyte Adhesion

BACKGROUND & AIMSnCI-959 is an anti-inflammatory agent that inhibits neutrophil adhesion, respiratory burst, and mast cell histamine release in vitro. In view of the emerging role of neutrophils in gastric erosive damage, the goals of this study were to assess the gastric cytoprotective effects of CI-959 and identify the mechanism responsible for this action.nnnMETHODSnCytoprotective effects in the rat nonsteroidal anti-inflammatory drug and ethanol erosion models were assessed using image analysis. The in vivo effects of CI-959 on gastric acid secretion, arachidonic acid metabolism, and intracellular sulfhydryl and leukocyte adhesion were also examined.nnnRESULTSnCI-959 protected prophylactically against the erosive damage induced by aspirin, indomethacin, and ethanol with 50% effective doses (ED50s) of 0.05, 1.0, and 0.07 mg/kg administered orally, respectively. When administered after indomethacin or ethanol, CI-959 had no effect on the healing of erosive damage. CI-959 did not alter gastric acid secretion, arachidonic acid metabolism, or intracellular sulfhydryl levels. In vivo, CI-959 blocked leukocyte adhesion in intravital microscopy studies using indomethacin (ED50, < 5 mg/kg orally) or platelet-activating factor (50% inhibiting concentration, approximately 10 mumol/L) as the adhesion stimulus.nnnCONCLUSIONSnThe most likely mechanism responsible for the cytoprotective effects of CI-595 is its inhibitory effects on leukocyte trafficking and/or adhesion.

Hydroxylamine analogs of 2,6 di-t-butylphenols: Dual inhibitors of cyclooxygenase and 5-lipoxygenase or selective 5-lipoxygenase inhibitors

The preparation of hydroxylamine analogs of 2,6-di-tert-butylphenols (DTBP) and the inhibition of cyclooxygenase (CO) and 5-lipoxygenase (5-LO) by these compounds is discussed.