Samir S. Ayoub

Fundamentals of Inflammation

The acute inflammatory response is the body’s first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are under way in both academic and industry settings. The purpose of this book is to provide an introduction to the cell types, chemical mediators, and general mechanisms of the host’s first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, postdoctoral Fellows, medical scientists, and researchers from other disciplines who wish to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.

Priming innate immune responses to infection by cyclooxygenase inhibition kills antibiotic-susceptible and -resistant bacteria

Inhibition of cyclooxygenase (COX)-derived prostaglandins (PGs) by nonsteroidal anti-inflammatory drugs (NSAIDs) mediates leukocyte killing of bacteria. However, the relative contribution of COX1 versus COX2 to this process, as well as the mechanisms controlling it in mouse and humans, are unknown. Indeed, the potential of NSAIDs to facilitate leukocyte killing of drug-resistant bacteria warrants investigation. Therefore, we carried out a series of experiments in mice and humans, finding that COX1 is the predominant isoform active in PG synthesis during infection and that its prophylactic or therapeutic inhibition primes leukocytes to kill bacteria by increasing phagocytic uptake and reactive oxygen intermediate-mediated killing in a cyclic adenosine monophosphate (cAMP)-dependent manner. Moreover, NSAIDs enhance bacterial killing in humans, exerting an additive effect when used in combination with antibiotics. Finally, NSAIDs, through the inhibition of COX prime the innate immune system to mediate bacterial clearance of penicillin-resistant Streptococcus pneumoniae serotype 19A, a well-recognized vaccine escape serotype of particular concern given its increasing prevalence and multi-antibiotic resistance. Therefore, these data underline the importance of lipid mediators in host responses to infection and the potential of inhibitors of PG signaling pathways as adjunctive therapies, particularly in the con-text of antibiotic resistance.

Increased susceptibility of annexin‐A1 null mice to nociceptive pain is indicative of a spinal antinociceptive action of annexin‐A1

Annexin‐A1 (ANXA1), a glucocorticoid‐regulated protein, mediates several of the anti‐inflammatory actions of the glucocorticoids. Previous studies demonstrated that ANXA1 is involved in pain modulation. The current study, using ANXA1 knockout mice (ANXA1−/−), is aimed at addressing the site and mechanism of the modulatory action of ANXA1 as well as possible involvement of ANXA1 in mediating the analgesic action of glucocorticoids.

Paracetamol-induced hypothermia is independent of cannabinoids and transient receptor potential vanilloid-1 and is not mediated by AM404

In recent years, there has been increasing interest in hypothermia induced by paracetamol for therapeutic purposes, which, in some instances, has been reported as a side effect. Understanding the mechanism by which paracetamol induces hypothermia is therefore an important question. In this study, we investigated whether the novel metabolite of paracetamol, N-(4-hydroxyphenyl)arachidonylamide (AM404), which activates the cannabinoid (CB) and transient receptor potential vanilloid-1 (TRPV1) systems, mediates the paracetamol-induced hypothermia. The hypothermic response to 300 mg/kg paracetamol in CB1 receptor (CB1R) and TRPV1 knockout mice was compared to wild-type mice. Hypothermia induced by paracetamol was also investigated in animals pretreated with the CB1R or TRPV1 antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperdinyl-1H-pyrazole-3-carboxamide trifluoroacetate salt (AM251) or 4′-chloro-3-methoxycinnamanilide (SB366791), respectively. In CB1R or TRPV1 knockout mice, paracetamol induced hypothermia to the same extent as in wild-type mice. In addition, in C57BL/6 mice pretreated with AM251 or SB366791, paracetamol induced hypothermia to the same extent as in control mice. AM404 failed to induce hypothermia at pharmacological doses. Inhibition of fatty acid amide hydrolase (FAAH), which is involved in the metabolism of paracetamol to AM404, did not prevent the development of hypothermia with paracetamol. Paracetamol also induced hypothermia in FAAH knockout mice to the same extent as wild-type mice. We conclude that paracetamol induces hypothermia independent of cannabinoids and TRPV1 and that AM404 does not mediate this response. In addition, potential therapeutic value of combinational drug-induced hypothermia is supported by experimental evidence.

Cyclooxygenase expression and prostaglandin levels in central nervous system tissues during the course of chronic relapsing experimental autoimmune encephalomyelitis (EAE)

ObjectiveMultiple sclerosis (MS) and its animal counterpart experimental autoimmune encephalomyelitis (EAE) have a major inflammatory component that drives and orchestrates both diseases. One particular group of mediators are the prostaglandins (PGs), which we have previously shown, through quantitation and pharmacological intervention, to be closely involved in the pathology of MS and EAE. The aim of the current study was to determine the expression of the PG-generating cyclooxygenase (COX) enzymes and the profile of PGE2 and PGD2, in selected central nervous system (CNS) tissues, with the development of the chronic relapsing (CR) form of EAE. In particular, the work investigates the possible relationship between the expression of COX isoenzymes and PG levels during the neurological phases of CR EAE.MethodsCR EAE was induced in Biozzi mice with inoculum containing lyophilised, syngeneic spinal cord emulsified in complete Freund’s adjuvant. The cerebral cortex, cerebellum and spinal cord were dissected from mice during the acute, remission and relapse stages of disease with a minimum of five animals per treatment. The expression of COX-1, COX-1b variant and COX-2, in pooled samples, was determined by Western blotting. PGE2 and PGD2 levels in extracted samples were measured using commercial enzyme immunoassay kits.ResultsCOX-2 expression in spinal cords during acute disease remained unaltered and was in contrast to an enhancement of the enzyme, together with COX-1 and COX-1b, in all other sampled areas. PGE2 and PGD2 levels remained unchanged during the acute phase and the subsequent remission of symptoms. COX-1 and COX-1b expression was elevated in tissues during the relapse stage of CR EAE and concentrations of the prostanoids were markedly increased.ConclusionsThe study examines the implications of COX isoenzyme expression over the course of CR EAE and discusses the reported relationship between PGE2 and PGD2 in the instigation and resolution of CNS inflammation. Consideration is also given to the treatment of CR EAE and suggests that drugs designed to limit the inflammatory effects of the PGs should be administered prior to or during the relapse phase of the disease.

Iloprost-Induced Nociception: Determination of the Site of Anti-nociceptive Action of Cyclooxygenase Inhibitors and the Involvement of Cyclooxygenase Products in Central Mechanisms of Nociception

The writhing response to acute nociception has been used to test the analgesic activity of drugs in rodents. Dilute acetic acid is the most frequently used irritant to induce writhing behaviour. The administration of acetic acid intraperitoneally activates both peripheral and central mechanisms of nociception. It releases nociceptive mediators such as prostaglandins (PG) E(2)and I(2)at the site of noxious stimulation, the peritoneal cavity, and at central sites such as the dorsal horn of the spinal cord and some brain regions. We have used the PGI(2)mimetic, iloprost, an agonist at the IP receptor, to induce the writhing response in mice. Iloprost activates the IP receptors on peripheral nociceptors directly and thus does not release nociceptive prostaglandins into the peritoneal cavity. However, prostaglandins are still involved in nociceptive transmission at the spinal and supraspinal levels. Using this model of nociception, it is possible to identify the site of action of analgesic drugs which reduce prostaglandin release in central tissues through inhibition of cyclooxygenase. Thus, a drug that inhibits the iloprost-induced writhing response and reduces release of prostaglandins in the central nervous system is likely to be a centrally acting analgesic drug. This chapter compares the iloprost- and acetic acid-induced writhing responses in mice and describes a method for measuring central prostaglandin levels. Part of this work has been published previously.

Cyclooxygenase enzymes and their products in the carrageenan-induced pleurisy in rats.

Rodent models of inflammation have helped in our understanding of the inflammatory process and also for the screening of compounds with anti-inflammatory potential. Although they do not represent a particular inflammatory disease in humans, cavity models of inflammation in rodents are easy to induce and to quantify the inflammatory reaction as well as to harvest the inflammatory exudates for cytological, biochemical and molecular biological analysis. Of these models, the carrageenan-induced pleurisy model has been extensively used to study the role of the cyclooxygenase (COX) enzymes and the prostaglandins in acute inflammation and also for the screening of COX-inhibiting anti-inflammatory drugs.

Inhibition of the diclofenac‐induced cyclooxygenase‐2 activity by paracetamol in cultured macrophages is not related to the intracellular lipid hydroperoxide tone

Paracetamol, a weak inhibitor of cyclooxygenase COX‐1 and COX‐2 activities, has been reported to inhibit the activity of COX‐2 induced by diclofenac in J774.2 macrophage cell line. The lack of inhibition of COX‐2 by paracetamol in inflamed tissues and thereby the lack of anti‐inflammatory activity has been attributed to high lipid hydroperoxide (LHP) tone. In this study, we demonstrate that the inhibition of the diclofenac‐induced COX‐2 activity in J774.2 cells by paracetamol is not related to the intracellular LHP tone as paracetamol inhibited this activity in the absence and presence of T‐butyl hydroperoxide, which is an LHP donor, to the same extents. In fact, treatment of the cells with diclofenac resulted in an increase in the LHP tone. Stimulation of the cells with lipopolysaccharide (LPS) results in the induction of a COX‐2 activity, which was not inhibited by paracetamol. This represents the classical induction pathway for COX‐2. LPS stimulation did not alter the LHP tone. These results suggest that the enzymatic activity of the diclofenac‐induced COX‐2 protein does not depend on the supply of hydroperoxides to its peroxidase active site.

N-Methyl-D-aspartate (NMDA) receptor involvement in central nervous system prostaglandin production during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE).

Our previous studies have established that major changes in central nervous system (CNS) prostaglandin (PG) levels occur during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE), an animal model of the human demyelinating disease multiple sclerosis. PG production is controlled through a series of enzymic pathways that, in EAE, are influenced by neuroantigen‐driven autoimmune events. In non‐immune‐based models of CNS disease, endogenous glucocorticoids have been proposed as instigators of PG synthesis via activation of the N‐methyl‐D‐aspartate (NMDA) receptor. Glucocorticoids have an important regulatory role in the pathogenesis EAE and the NMDA receptor is intimately involved in many of the characteristic neuroinflammatory processes that govern the disease. Therefore, the alterations in prostanoid concentrations during the relapse stage of CR EAE may ultimately be governed by glucocorticoid‐induced NMDA receptor activation. The current investigation has examined the proposed glucocorticoid–NMDA receptor link by determining the effects of the receptor antagonist, (+) MK‐801, on CNS PGE 2 and PGD 2 levels in Biozzi mice with relapse symptoms of CR EAE. Prostanoid concentrations in the cerebral cortex were not altered by drug administration, and in cerebellar tissues, a vehicle effect negated any drug‐induced changes. However, the level of PGD 2 in spinal cords from (+) MK‐801‐dosed mice was significantly lower, compared to controls, but PGE 2 concentrations remained unchanged. The results suggest that glucocorticoid–NMDA receptor‐linked events are not primarily responsible for PG generation in the brain but may influence prostanoid production in discrete areas of the CNS.

In Vitro Cyclooxygenase Activity Assay in Tissue Homogenates

The use of in vitro cyclooxygenase (COX) activity assays has been particularly useful for comparing the effect of different drugs on COX activity in different tissues. In addition, this assay is relatively quick, cheap, and a large number of samples can be tested at the same time. However, one limitation of this assay is the fact that it does not discriminate between the activities of different COX isoforms.