D. A. Willoughby

Possible new role for NF-κB in the resolution of inflammation

Inflammation involves the sequential activation of signaling pathways leading to the production of both pro- and anti-inflammatory mediators. Although much attention has focused on pro-inflammatory pathways that initiate inflammation, relatively little is known about the mechanisms that switch off inflammation and resolve the inflammatory response. The transcription factor NF-κB is thought to have a central role in the induction of pro-inflammatory gene expression and has attracted interest as a new target for the treatment of inflammatory disease. We show here that NF-κB activation in leukocytes recruited during the onset of inflammation is associated with pro-inflammatory gene expression, whereas such activation during the resolution of inflammation is associated with the expression of anti-inflammatory genes and the induction of apoptosis. Inhibition of NF-κB during the resolution of inflammation protracts the inflammatory response and prevents apoptosis. This suggests that NF-κB has an anti-inflammatory role in vivo involving the regulation of inflammatory resolution.

Screens for anti‐inflammatory drugs

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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.

Resolution of inflammation

Acute inflammatory reactions, in contrast to chronic inflammatory reactions, are usually self-limiting and resolve. We have investigated the resolving phase of a number of immune and non-immune inflammatory reactions induced in the pleural cavity of rats. COX-2 is expressed during resolution of these models. Using carrageenan pleurisy, we showed that this enzyme has a proinflammatory role as the reaction develops but an antiinflammatory role as the lesion resolves. This antiinflammatory role is associated with production of cyclopentenone prostaglandins and the absence of PGE2. Dual COX-1/COX-2 inhibitors or COX-2 inhibitors when given at the peak of the inflammatory response delay resolution, an effect reversed by replacing CyPGs into the pleural space. PGF2alpha like the CyPGs appears to have a role in resolving this reaction. Stress proteins are also induced in a variety of acute inflammatory models during resolution. Heme oxygenase-1 (HO-1) is one such protein so too are members of the hsp70 family. An inducer of HO-1 promotes resolution whereas an inhibitor is proinflammatory. In most cases it appears to be the macrophage that is the source of proteins necessary for resolution to occur. Understanding how proinflammatory pathways switch to the antiinflammatory pathways necessary for resolution to take place may eventually allow the exploitation of endogenous antiinflammatory pathways in the treatment of chronic inflammation.

A novel role for phospholipase A2 isoforms in the checkpoint control of acute inflammation

Acute inflammation can be considered in terms of a series of checkpoints where each phase of cellular influx, persistence, and clearance is controlled by endogenous stop and go signals. It is becoming increasingly apparent that in addition to initiating the inflammatory response, eicosanoids may also mediate resolution. This suggests there are two phases of ara‐chidonic acid release: one at onset for the generation of proinflammatory eicosanoids and one at resolution for the synthesis of proresolving eicosanoids. What is unclear is the identity of the phospholipase (PLA2) isoforms involved in this biphasic release of arachidonic acid. We show here that type VI iPLA2 drives the onset of acute pleurisy through the synthesis of PGE2, LTB4, PAF, and IL‐1β. However, during resolution there is a switch to a sequential induction of first sPLA2 (types IIa and V) that mediates the release of PAF and lipoxin A4, which, in turn, are responsible for the subsequent induction of type IV cPLA2 that mediates the release of arachidonic acid for the synthesis of proresolving pros‐taglandins. This study is the first of its kind to address the respective roles of PLA2 isoforms in acute resolving inflammation and to identify type VI iPLA2 as a potentially selective target for the treatment of inflammatory diseases.—Gilroy, D. W., Newson, J., Sawmynaden, P., Willoughby, D. A., Croxtall, J. D. A novel role for phospholipase A2 isoforms in the checkpoint control of acute inflammation.

Differential effects of inhibitors of cyclooxygenase (cyclooxygenase 1 and cyclooxygenase 2) in acute inflammation

The anti-inflammatory activity of drugs more selective for cyclooxgenase isoform inhibition (cyclooxygenase 1, cyclooxygenase 2), were compared in rat carrageenin-induced pleurisy. Suppression of inflammation by cyclooxygenase 2-selective inhibitors, NS-398 (N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide) and nimesulide (4-nitro-2-phenoxy-methanesulfonanilide), and by piroxicam and aspirin, more selective for cyclooxygenase 1, was measured. Piroxicam and aspirin significantly inhibited inflammatory cell influx, exudate and prostaglandin E2 formation, 6 h after carrageenin injection. Cyclooxygenase 2 inhibitors had little effect on these parameters with NS-398 alone reducing prostaglandin E2 levels, but increasing levels of leukotriene B4. In contrast, at 3 h after carrageenin injection, cyclooxygenase 2 inhibitors significantly inhibited all inflammatory parameters however suppression with piroxicam and aspirin was greater, and more pronounced than at 6 h. NS-398 and nimesulide dosing did not reduce thromboxane B2 production from platelets isolated from rats with carrageenin-induced pleurisy, demonstrating that at the doses used, cyclooxygenase 2 inhibitors did not inhibit cyclooxygenase 1, as platelets contain only this isoform. Therefore, in the rat carrageenin-induced pleurisy, drugs more selective for the inhibition of cyclooxygenase 1 attenuate inflammation over a wider time frame than cyclooxygenase 2-selective drugs, suggesting a significant role for cyclooxygenase 1 in this model. Inhibition of cyclooxygenase 2 by NS-398 however, resulted in an increase in the potent chemoattractant leukotriene B4.

Inducible cyclooxygenase-derived 15-deoxy(Delta)12-14PGJ2 brings about acute inflammatory resolution in rat pleurisy by inducing neutrophil and macrophage apoptosis.

Failure of acute inflammation to resolve leads to persistence of the inflammatory response and may contribute to the development of chronic inflammation. Thus, an understanding of inflammatory resolution will provide insight into the etiology of chronic inflammation. In an acute pleurisy, polymorphonuclear leukocytes (PMNs) were found to predominate at the onset of the lesion but decreased in number by undergoing apoptosis, the principal mechanism by which PMNs died in this model. PMNs were progressively replaced by monocytes, which differentiated into macrophages. As with PMNs, macrophages also underwent programmed cell death leading to an abatement of the inflammatory response and eventual resolution. It was found that apoptosis of both these inflammatory cell types was mediated by pro‐resolving cyclooxygenase 2‐derived 15deoxy Δ 12‐14PGJ2, which is uniquely expressed during active resolution. Although PMN programmed cell death is well understood, the observation that macrophages apoptose during resolution of acute inflammation is less well described. These results provide insight into the mechanisms that switch off acute inflammation and prevent complications of wound healing and potentially the development of immune‐mediated chronic inflammation.

Induction of Cyclo-Oxygenase and Nitric Oxide Synthase in Inflammation

Publisher Summary This chapter focuses on the role of prostanoids and NO at each stage of the inflammatory response with particular emphasis given to the cellular source and the factors that may modulate the activity of their respective enzymes. Inhibition of prostanoid formation by use of nonsteroidal anti-inflammatory drugs (NSAIDs) ameliorates the classical signs of inflammation, indicating their pivotal role in the inflammatory response. Recently, a second isoform of cyclooxygenase (COX), the enzyme that liberates the prostanoids, has been identified. This discovery has given new impetus as to the role of COX isoforms in inflammation and has fueled the search for selective inhibitors free from side effects. Once liberated, arachidonic acid is converted to the biologically active PGs and thromboxanes (TXs), collectively termed prostanoids, by the enzyme COX, also known as prostaglandin H synthase or prostaglandin endoperoxidase synthase. COX has two functions: first, cyclo-oxygenase activity that catalyzes PGG 2 formation, and second, peroxidase activity that reduces the 15-hydroperoxyl group of PGG, to PGH 2 . The chapter keeps space for discussion on both COX-1 and COX-2. The biological activity of the elusive molecule, termed endothelium-derived relaxing factor (EDRF), involved in vascular relaxation, could be accounted for by nitric oxide (NO). It was found that NO, synthesized by endothelial cells (ECs), acted as an intercellular effector molecule, causing vasodilatation by activation of guanylate cyclase and the elevation of cGMP levels in vascular smooth muscle cells. Although NO is indubitably involved in inflammation its role cannot be defined as pro- or anti-inflammatory but rather depends on the prevailing circumstances. The products of the NOS and COX pathways as well as having potent effects on various cellular systems can also modulate the activity of their respective enzymes. Although the mutual effects between COX activity and action of NO effects are not clear cut as both stimulatory and inhibitory actions have been ascribed.

Differential effects of inhibition of isoforms of cyclooxygenase (COX-1, COX-2) in chronic inflammation

Abstract.Objective and Design: The anti-inflammatory effects of therapeutic dosing of drugs with greater selectivity for the inhibition of the constitutive (COX-1) or inducible isoform (COX-2) of cyclooxygenase were assessed in a model of chronic inflammation.¶Methods: The murine chronic granulomatous tissue air pouch model involves the subcutaneous injection of air into the dorsum of mice followed 24 h later by the intrapouch injection of an inflammatory stimulus (0.5 ml of Freunds complete adjuvant containing 0.1% croton oil). Aspirin, more selective in vitro for the inhibition of COX-1 (10,200 mg/kg) and nimesulide, a selective in vitro inhibitor of COX-2 (0.5, 5 mg/kg) were dosed p.o. daily from 3 days after injection of the inflammatory stimulus. Granuloma dry weight, vascularity and COX activity (measured as PGE2) were assessed at various time points throughout the inflammatory lesion to resolution at day 28. A second COX-2 inhibitor, NS 398 (0.1, 1, 10 mg/kg), was dosed p.o. daily from 3 days after the injection of the inflammatory stimulus and its effects on granuloma dry weight, vascularity and COX activity were measured at 7 days.¶Results: Aspirin (200 mg/kg) significantly inhibited levels of PGE2 throughout the time course and at the lower dose (10 mg/kg) from day 14. Nimesulide (5 mg/kg) however, significantly increased levels of PGE2 at days 5 and 21, but at 0.5 mg/kg was without effect. Aspirin (200 mg/kg) significantly reduced granuloma dry weight at day 14 but had no effect on granuloma vascularity at day 7. In contrast, nimesulide (5 mg/kg) significantly increased granuloma vascularity at day 7 and granuloma dry weight at day 14. NS-398 at all doses had no effect on granuloma dry weight, vascularity or COX activity 7 days after the injection of the inflammatory stimulus.¶Conclusion: In this model of chronic inflammation, aspirin, more selective for the inhibition of COX-1 is more effective than the selective COX-2 inhibitors nimesulide and NS-398 at inhibiting granuloma dry weight, vascularity and COX activity.

Growth factors in angiogenesis: current interest and therapeutic potential

Angiogenesis, the process of new blood vessel development, is an essential component of the bodys physiology and contributes to the pathogenesis of a variety of diseases such as benign and malignant neoplasia and rheumatoid arthritis. Failure of this physiological response is also important in abnormalities of wound healing in diseases such as duodenal ulceration and diabetes. Angiogenesis is controlled by a variety of factors that initiate, control and terminate this complex, multi-stage process. This review covers those factors that are exciting much interest currently and have potential for incorporation into clinical medicine.