Inducible cyclooxygenase may have anti-inflammatory properties.

Cyclooxygenase (COX) has two isoforms. Generally, COX 1 is constitutively expressed in most tissues, where it maintains physiological processes; inducible COX 2 is considered a pro-inflammatory enzyme and a chief target for the treatment of inflammatory diseases. Here we present evidence that COX 2 may have anti-inflammatory properties. In carrageenin-induced pleurisy in rats, the predominant cells at 2 hours are polymorphonuclear leucocytes, whereas mononuclear cells dominate from 24 hours until resolution at 48 hours. In this model, COX 2 protein expression peaked initially at 2 hours, associated with maximal prostaglandin E2 synthesis. However, at 48 hours there was a second increase in COX 2 expression, 350% greater than that at 2 hours. Paradoxically, this coincided with inflammatory resolution and was associated with minimal prostaglandin E2 synthesis. In contrast, levels of prostaglandin D2, and 15deoxy delta(12-14)prostaglandin J2 were high at 2 hours, decreased as inflammation increased, but were increased again at 48 hours. The selective COX 2 inhibitor NS-398 and the dual COX 1/COX 2 inhibitor indomethacin inhibited inflammation at 2 hours but significantly exacerbated inflammation at 48 hours. This exacerbation was associated with reduced exudate prostaglandin D2 and 15deoxy delta(12-14)prostaglandin J2 concentrations, and was reversed by replacement of these prostaglandins. Thus, COX 2 may be pro-inflammatory during the early phase of a carrageenin-induced pleurisy, dominated by polymorphonuclear leucocytes, but may aid resolution at the later, mononuclear cell-dominated phase by generating an alternative set of anti-inflammatory prostaglandins.

Pattern recognition receptors and interleukin-8 mediate effects of Gram-positive and Gram-negative bacteria on lung epithelial cell function.

BACKGROUND AND PURPOSE: Lung epithelial cells express pattern recognition receptors, which react to bacteria. We have evaluated the effect of Gram-positive and Gram-negative bacteria on interleukin-8 (CXCL8) release from epithelial cells and the integrity of the epithelial barrier. EXPERIMENTAL APPROACH: Primary cultures of human airway epithelial cells and the epithelial cell line A549 were used, and CXCL8 release was measured after exposure to Gram-negative or Gram-positive bacteria. Epithelial barrier function was assessed in monolayer cultures of A549 cells. RESULTS: Gram-positive bacteria Staphylococcus aureus or Streptococcus pneumoniae, induced release of CXCL8 from human airway epithelial cells. These bacteria also disrupted barrier function in A549 cells, an effect mimicked by CXCL8 and blocked by specific binding antibodies to CXCL8. Gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa induced greater release of CXCL8 than Gram-positive bacteria. However, Gram-negative bacteria did not affect epithelial barrier function directly, but prevented disruption induced by Gram-positive bacteria. These effects of Gram-negative bacteria on barrier function were mimicked by FK565, an agonist of the nucleotide-binding oligomerization domain 1 (NOD1) receptor, but not by the Toll-like receptor (TLR) 4 agonist bacterial lipopolysaccharide. Neither the Gram-negative bacteria nor FK565 blocked CXCL8 release. CONCLUSIONS: These data show differential functional responses induced by Gram-negative and Gram-positive bacteria in human lung epithelial cells. The NOD1 receptors may have a role in preventing disruption of the epithelial barrier in lung, during inflammatory states.

Cigarette smoke inhibits macrophage sensing of Gram-negative bacteria and lipopolysaccharide: relative roles of nicotine and oxidant stress.

BACKGROUND AND PURPOSE: Smoking cigarettes is a major risk factor for the development of cardiovascular and respiratory disease. Moreover, smokers are more prone to infections. This has been associated with a suppression of the immune system by smoke. However, it is not clear how cigarette smoke affects the ability of immune cells to sense pathogens. Cigarette smoke contains a large number of molecules which may mediate responses on immune cells and of these, nicotine and oxidants have both been identified as inhibitory for the sensing of bacterial lipopolysaccharide (LPS). Nitric oxide synthase (NOS) and tumour necrosis factor (TNF)-alpha are both induced in macrophages on stimulation with Gram negative bacteria or LPS. EXPERIMENTAL APPROACH: We used murine macrophages stimulated with whole heat-killed bacteria or LPS. We measured output of NO (as nitrite) and TNFalpha, NOS protein by Western blotting and cellular oxidant stress. KEY RESULTS: Cigarette smoke extract suppressed the ability of murine macrophages to release NO, but not TNFalpha in response to whole bacteria. Cigarette smoke extract also inhibited nitric oxide synthase II protein expression in response to LPS. The effects of cigarette smoke extract on nitrite formation stimulated by LPS were unaffected by inhibition of nicotinic receptors with alpha-bungarotoxin (100 units ml(-1)). However, the effects of cigarette smoke extract on LPS-induced nitrite formation were mimicked by hydrogen peroxide and reversed by the anti-oxidants N-acetyl cysteine and glutathione. CONCLUSIONS AND IMPLICATIONS: We suggest that cigarette smoke exerts its immunosuppressive effects through an oxidant-dependent and not a nicotine-dependent mechanism.

Generation of innovative anti-inflammatory and anti-arthritic glucocorticoid derivatives that release NO: the nitro-steroids.

Addition to the prednisolone structure of a chemical moiety (linker+nitric ester) that releases NO species yielded a novel glucocorticoid (nitro-prednisolone or NCX-1015) with enhanced anti-inflammatory activities. Nitro-prednisolone was much more potent than prednisolone and the derivative devoid of the nitric ester in an acute peritonitis model (higher impact on neutrophil migration and soluble mediator generation) as well as in models of chronic inflammation (air-pouch granuloma and collagen II-induced arthritis). In the collagen II-induced arthritis model, NCX-1015 abrogated the plasma levels of a catabolite of cartilage and bone metabolism, indication of a disease modifying action. In an in vitro assay of bone resorption, NCX-1015 did not activate osteoclast activity, whereas prednisolone did. This lack of effect of NCX-1015 was chiefly due to NO. We propose that NCX-1015 is the prototype of a new class of glucocorticoids, the nitro-steroids, endowed with enhanced anti-inflammatory properties and reduced side effects. These and other experimental observations here reviewed may prompt the assessment of the clinical impact of the nitro-steroids on rheumatoid arthritis and inflammatory bowel disease.

The annexin-1 knockout mouse: what it tells us about the inflammatory response.

The 37kDa protein annexin 1 (Anx-1; lipocortin 1) is a glucocorticoid-regulated protein that has been implicated in the regulation of phagocytosis, cell signalling and proliferation, and postulated to be a mediator of glucocorticoids action in inflammation and in the control of anterior pituitary hormone release. Immuno-neutralisation or antisense strategies support this hypothesis as they can reverse the effect of glucocorticoids in several systems. We recently generated a line of mice lacking the Anx-1 gene noting that some tissues taken from such animals exhibited an increased expression of several proteins including COX-2 and cPLA2. In models of experimental inflammation, Anx-1(-/-) mice exhibit an exaggerated response and a partial or complete resistance to the anti-inflammatory effects of glucocorticoids. Several other anomalies were noted including abnormal leukocyte adhesion molecule expression, an increased spontaneous migratory behaviour of PMN in Anx-1(-/-) mice and a resistance in Anx-1(-/-) macrophages to glucocorticoid inhibition of superoxide generation. This paper reviews these and other data in the light of the development of the 'second messenger' hypothesis of glucocorticoid action.

Pharmacology and therapeutic potential of pattern recognition receptors.

Pharmacologists have used pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) for decades as a stimulus for studying mediators involved in inflammation and for the screening of anti-inflammatory compounds. However, in the view of immunologists, LPS was too non-specific for studying the mechanisms of immune signalling in infection and inflammation, as no receptors had been identified. This changed in the late 1990s with the discovery of the Toll-like receptors. These 'pattern recognition receptors' (PRRs) were able to recognise highly conserved sequences, the so called pathogen associated molecular patterns (PAMPs) present in or on pathogens. This specificity of particular PAMPs and their newly defined receptors provided a common ground between pharmacologists and immunologists for the study of inflammation. PRRs also recognise endogenous agonists, the so called danger-associated molecular patterns (DAMPs), which can result in sterile inflammation. The signalling pathways and ligands of many PRRs have now been characterised and there is no doubt that this rich vein of research will aid the discovery of new therapeutics for infectious conditions and chronic inflammatory disease.

Aspirin and the in vitro linear relationship between thromboxane A2-mediated platelet aggregation and platelet production of thromboxane A2.

BACKGROUND: Currently, 'aspirin resistance', the anti-platelet effects of non-steroid anti-inflammatory drugs (NSAIDs) and NSAID-aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A(2) formation is non-linear and TXA(2) generation must be inhibited by at least 95% to inhibit TXA(2)-dependent aggregation. This relationship, however, has never been rigorously tested. OBJECTIVES: To characterize, in vitro and ex vivo, the concentration-dependent relationships between TXA(2) generation and platelet activity. METHOD: Platelet aggregation, thrombi adhesion and TXA(2) production in response to arachidonic acid (0.03-1 mmol L(-1)), collagen (0.1-30 microg mL(-1)), epinephrine (0.001-100 micromol L(-1)), ADP, TRAP-6 amide and U46619 (all 0.1-30 micromol L(-1)), in the presence of aspirin or vehicle, were determined in 96-well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. RESULTS: Platelet aggregation, adhesion and TXA(2) production induced by either arachidonic acid or collagen were inhibited in concentration-dependent manners by aspirin, with logIC(50) values that did not differ. A linear relationship existed between aggregation and TXA(2) production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R(2) 0.92; n = 224). The same relationships were seen in combinations of aspirin-treated and naïve platelets, and in blood from individuals taking an anti-thrombotic dose of aspirin. CONCLUSIONS: These studies demonstrate a linear relationship between inhibition of platelet TXA(2) generation and TXA(2)-mediated aggregation. This finding is important for our understanding of the anti-platelet effects of aspirin and NSAIDs, NSAID-aspirin interactions and 'aspirin resistance'.

Modulation of heme oxygenase activity in rat brain and spleen by inhibitors and donors of nitric oxide.

The capacity of nitric oxide to activate or inhibit metalloprotein-containing enzymes underlies many of its biological actions. Heme oxygenase, involved in a variety of biological processes, does not contain heme but utilises it as a substrate. The substrate for nitric oxide, L-arginine (0.1-10mM), but not D-arginine, decreased heme oxygenase activity in rat brain homogenates. The arginine analogue L-NAME (0.1-10mM) increased activity in the same tissue. In spleen homogenates where endogenous nitric oxide activity is lower than in brain, these compounds had no effect. The nitric oxide donor sodium nitroprusside (0.001mM-10mM) reduced heme oxygenase activity in both brain and spleen. These results suggest that biological effects attributed to modulation of nitric oxide synthase may act via heme oxygenase.

Nitric oxide synthase inhibitors have opposite effects on acute inflammation depending on their route of administration.

The bulk of published data has shown that NO is proinflammatory. However, there also exists the conflicting notion that NO may be protective during an inflammatory insult. In an attempt to resolve this issue, we have compared the effects on inflammation of a range of NO synthase (NOS) inhibitors given either directly to the site of the inflammatory lesion or systemically. It was found that in the carrageenin-induced pleurisy, a single intrapleural injection of the selective inducible NO inhibitors S-(2-aminoethyl) isothiourea (AE-ITU; 3 and 10 mg/kg) and N-(3-(aminomethyl)-benzyl) acetamidine (1400W; 10 mg/kg) or the selective endothelial cell NOS inhibitor L-N(5)(1-iminoethyl)-ornithine (10 mg/kg) not only exacerbated inflammation at the very early stages of the lesion (1-6 h), but also prevented inflammatory resolution. By contrast, administering NOS inhibitors systemically ameliorated the severity of inflammation throughout the reaction. To elucidate the mechanisms by which inhibition of NO synthesis locally worsened inflammation, we found an increase in histamine, cytokine-induced neutrophil chemoattractant, superoxide, and leukotriene B(4) levels at the inflammatory site. In conclusion, this work shows that the local production of NO is protective by virtue of its ability to regulate the release of typical proinflammatory mediators and, importantly, that NOS inhibitors have differential anti-inflammatory effects depending on their route of administration.

Neutrophil interaction with inflamed postcapillary venule endothelium alters annexin 1 expression.

Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.