Adrian Richard Moore

Cyclo-oxygenase and nitric oxide synthase isoforms in rat carrageenin-induced pleurisy.

1 The profiles of cyclo‐oxygenase (COX) and nitric oxide synthase (NOS) isoforms were determined in the rat carrageenin‐induced pleurisy model of acute inflammation. 2 The enzymes were assessed in peripheral blood leucocyte (PBL) cell pellets taken from untreated animals and at 2, 6 and 24 h after injection of the irritant in pleural exudate cell pellets and lung homogenates. 3 COX activity was assessed by the generation of prostacyclin (PGI2, measured as the stable metabolite, 6‐keto prostaglandin F1α) and prostaglandin E2 (PGE2). Western blot analysis and immunohistochemistry were also carried out. 4 NOS activity was based on the conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline in the presence (total NOS activity) or absence of Ca2+ (inducible NOS; iNOS). 5 Peripheral blood leucocyte samples contained low levels of COX activity. In pleural exudate cell pellets, COX activity peaked at 2 to 6 h after injection of the carrageenin. At 24 h, COX activity was significantly reduced. 6 Western blot analysis demonstrated that the inducible isoform of COX (COX‐2), was the predominant enzyme at all time points. Low levels of COX‐2 were seen in PBLs. In pleural exudate cell pellets maximal COX‐2 protein levels were seen at 2h. 7 Immunohistochemistry confirmed the findings of Western blot studies. Approximately 10% of polymorphonuclear neutrophils (PMNs) in PBLs from untreated animals were immunopositive for COX‐2. In cell pellet smears from carrageenin‐induced pleurisy taken 2 h after injection of the irritant, PMNs were also the major source of COX‐2 immunoreactivity. A small proportion of macrophages and mesothelial cells were also immunolabelled for COX‐2. 8 Low levels of NOS activity were seen in PBLs. In pleural exudates NOS activity was maximum at 6h and greatly reduced by 24 h. This activity was solely attributable to iNOS. 9 The present results illustrated a similar profile of COX and NOS activity in the carrageenin‐induced pleurisy model of acute inflammation. It was demonstrated that COX‐2 and iNOS were the predominant isoforms of their respective enzymes.

Heme oxygenase isoform expression in cellular and antibody-mediated models of acute inflammation in the rat

Heme oxygenase (HO) is the rate‐limiting enzyme in the catabolism of heme to biliverdin, carbon monoxide (CO), and free iron. The enzyme exists as a constitutive isoform (HO‐2) and an inducible isoform (HO‐1), which is also a stress protein (HSP32). HO‐1 has previously been shown to be associated with the resolution phase of a non‐immune model of acute inflammation. In addition, elevation of the enzyme was markedly anti‐inflammatory. In the present study, these observations have been extended to two pleural models of immune‐driven inflammation in the rat, an immediate type III hypersensitivity (Arthus) reaction and a delayed type IV hypersensitivity reaction. Whilst these models have differing inflammatory mechanisms and time courses, they both showed HO activity to be maximal during the resolution phase. This activity was associated with increases in exudate bilirubin (a breakdown product of biliverdin) and increased expression of HO‐1. Immunocytochemical analysis of inflammatory cell smears from the two models showed that HO‐1 and HO‐2 expression was restricted to mononuclear cells in the type IV hypersensitivity reaction, but included the polymorphonuclear cell population in the type III hypersensitivity reaction. Thus, irrespective of the pathogenesis of the lesion, evidence is accumulating to suggest that HO‐1 has a universal role in the resolution of inflammation. Copyright

Diphenylene iodonium, an inhibitor of free radical formation, inhibits platelet aggregation

Diphenylene iodonium is an inhibitor of the enzyme NADPH-oxidase and prevents the generation of oxygen-derived free radicals in neutrophils (Cross and Jones, 1986). Here we show that diphenylene iodonium (0.25-2 microM) inhibited, according to the dose, thrombin-induced platelet-aggregation in human washed platelets and ADP-induced platelet aggregation in platelet-rich plasma. At the concentrations which inhibited platelet aggregation diphenylene iodonium did not alter platelet concentrations of cAMP or cGMP but enhanced the anti-platelet activity of iloprost, sodium nitroprusside or cultured endothelial cells. These findings highlight the importance of free radicals as platelet pro-aggregatory agents.

Interaction between neutrophil-derived elastase and reactive oxygen species in cartilage degradation

The role of proteases and reactive oxygen species (ROS) in polymorphonuclear neutrophil (PMN) induced cartilage degradation in vitro were studied. ONO-5046, a novel synthetic elastase inhibitor, significantly and dose dependently protected cartilage from degradation induced by PMNs stimulated with phorbol myristate acetate (PMA), opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine plus cytochalasin-B, or A-23187. The degradation by PMA-stimulated PMNs was unaffected by protease inhibitors which lack anti-elastase activity. However, the hydrogen peroxide (H2O2) reducing agent catalase afforded significant protection. Measurement of elastase activity following PMN activation by PMA showed that antioxidants which reduce H2O2 and/or hypochlorous acid decreased elastase activity. Thus, it is suggested that an indirect interaction between ROS and elastase activity may exist in PMN induced cartilage degradation. Furthermore, the possible implication of an endogenous elastase inhibitor(s) is discussed.

Cyclopentenone prostaglandins-new allies in the war on inflammation.

Although the effects of anti-inflammatory drugs have been well studied, little is known about the signaling pathways involved in the inflammatory response. The recent finding that cyclopentenone prostaglandins inhibit IκB kinase provides a new explanation for the anti-inflammatory effects of these molecules.

Prostaglandin F2α produced by inducible cyclooxygenase may contribute to the resolution of inflammation

Cyclooxygenase-2 may play a role in resolution of carrageenan-induced pleurisy in rats by generating anti-inflammatory prostanoids. Here, we show exudate prostaglandin F2α concentrations rise during resolution of this model. These were reduced by the selective cyclooxygenase-2 inhibitor NS-398, which exacerbated inflammation. Concomitant treatment with NS-398 and the synthetic FP receptor agonist fluprostenol reversed this exacerbation. This suggests prostaglandin F2α produced by cyclooxygenase-2 contributes to resolution of this inflammatory reaction.

Marked Enhanced Efficacy of Cyclosporin When Combined with Hyaluronic Acid

SummaryUsing two models of chronic inflammation (both T-cell-mediated) in the Wistar rat, we have shown that, when given intravenously, the efficacy of cyclosporin can be markedly enhanced by combining it with sodium hyaluronate 7.5 mg/kg (HA; 500–800 kDa). When tested against avridine polyarthritis and cotton pellet granuloma formation, a marginally effective dose of cyclosporin 2.5 mg/kg could be transformed into a highly effective dose by combining it with HA 7.5 mg/kg. The latter combination appeared to mimic a highly efficacious dose of cyclosporin (10 mg/kg). Although the mechanism of this effect is as yet unclear, the potential clinical benefits are great.

Coronary vasoconstriction in vitro in the hearts of polyarthritic rats: effectiveness of in vivo treatment with the endothelin receptor antagonist SB 209670

Here we demonstrate that perfused hearts removed from polyarthritic rats develop a pronounced coronary vasoconstriction ex vivo. This vasoconstriction is almost entirely blocked by in vivo pretreatment of the rats with the endothelin receptor antagonist, SB 209670. Thus, inflammatory states may be associated with an increased activity of the endothelin system, leading to vascular dysfunction and vasoconstriction.