2. Biochemistry of inflammation

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1 Research Signpost 37/661 (2), Fort P.O. Trivandrum Kerala, India Molecular Aspects of Inflammation, 2013: ISBN: Editors: Leonor Pérez-Martínez, Gustavo Pedraza-Alva and Eduardo Ferat Osorio 2. Biochemistry of inflammation Nancy Mora 1, Eileen Uribe-Querol 2 and Carlos Rosales 1 1 Immunology Department, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico; 2 División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, Mexico City, Mexico Abstract. Inflammation is a complex process that involves several cell types and is controlled by many chemical signals. Despite the initial triggering stimuli, inflammation is regulated by several lipid molecules that control important cellular responses. These lipid mediators have important pro-inflammatory activities mediating cell proliferation, apoptosis, cell migration, and even general physiological responses. Cytokines, growth factors, and even nutrients control the activity of key lipid-modifying enzymes: phospholipases, prostaglandin synthase (cyclooxygenase), 5-lipoxygenase, sphingosine kinase, and sphingomyelinase. These enzymes and their downstream targets form a complex signaling network that controls inflammation and metabolism. Imbalances in this network lead to pathogenesis and human disease. This chapter will focus on the major inflammatory lipid mediators, describing their synthesis and metabolism, their receptors and the role individual mediators have in inflammation. Introduction The co-ordination of biochemical events between cells in a tissue or in an intact organism plays a central role in the response of each cell to external Correspondence/Reprint request: Dr. Carlos Rosales, Department of Immunology, Instituto de Investigaciones Biomédicas - UNAM, Apto. Postal 70228, Cd. Universitaria, México D.F , México. carosal@unam.mx

2 16 Nancy Mora et al. stimuli. A large number of molecular substances have been selected to serve the role of cellular mediators. Various mechanisms have also evolved that recognize small molecules as signaling species. Lipids have long since been recognized as signaling molecules that have the capacity to trigger profound physiological responses. Slow-reacting substance of anaphylaxis (SRS-A), now know to belong to the group of cysteinyl leukotrienes, was identified as early as 1930 (1). At about the same time, the vasodilating actions of prostaglandins were described (2). Subsequently arachidonic acid was identified as the source of leukotrienes and prostaglandins, a discovery that has led to our current understanding of eicosanoid signaling (3). The study of the lipid components of cellular membranes (4), and in particular the turnover of phosphoinositides, culminated in the 1980s with the discovery that phosphatidylinositol-4,5-bisphosphate could be hydrolyzed by phospholipase C (PLC) 3 (5) to diacylclycerol (DAG) and inositol-1,4,5- trisphosphate. Both products were identified as second messengers that trigger the activation of protein kinase C (PKC) (6) and the release of Ca 2+ from internal stores, respectively. Ceramides and sphingosines are lipids that have pro-apoptotic and antiproliferative actions (7), when phosphorylated by lipid kinases. However, sphingosine is also converted into sphingosine-1-phosphate (S1P), which promotes cell growth and differentiation through a series of different G protein-coupled receptors (GPCRs) (8). Imbalances of these major lipid-signaling pathways contribute to disease progression in chronic inflammation, autoimmunity, allergy, cancer, atherosclerosis, hypertension, and metabolic syndrome, among others. This chapter will focus on the major inflammatory lipid mediators, describing their synthesis and metabolism, their receptors and the role individual mediators have in inflammation and metabolism. Eicosanoids Eicosanoids are bioactive lipids with a broad range of physiological effects. They are potent mediators of cellular function and participate mainly in regulating the inflammation process and the immune response. Eicosanoids are mainly derived from arachidonic acid, through the action of several lipid-acting enzymes. The first eicosanoid studied was prostaglandin (2). The vaso-dilating properties found in the seminal fluid were attributed to a substance isolated from the prostate gland (9) therefore the name prostaglandin, and characterized to be a polyunsaturated fatty acid. Several prostaglandins are known today and they all have potent cell effects that depend in part on the particular membrane cell receptor that recognizes them (3). Thromboxane A 2 is another eicosanoid, initially characterized as a potent platelet activator (10, 11).

3 Biochemistry of inflammation 17 Prostaglandins and thromboxane A 2 are collectively termed the prostanoids (12). Another group of eicosanoids is the leukotrienes. These lipids got their name because they are produced by leukocytes and have three conjugated double bonds. Leukotrienes induce smooth muscle contraction and are related to respiratory complications in patients with asthma. Arachidonic acid and phospholipase A2 Membrane glycerophospholipids in eukaryotic cells frequently contain an unsaturated fatty acyl moiety esterified at the sn-2 position. Polyunsaturated fatty acids such as arachidonic acid are especially abundant in certain cell types, including those of myeloid origin such as neutrophils and macrophages. The initial step in the synthesis of eicosanoids is the cleavage of this arachidonoyl ester bond through the action of a phospholipase A2 (PLA2) (Fig 1). The hydrolysis reaction yields a lysophospholipid and the 20-carbon unsaturated fatty acid, arachidonic acid. Cellular levels of both arachidonic acid and lysophospholipids are tightly regulated. An important mechanism to maintain low levels of these bioactive lipids is the phospholipid reacylation process known as the Lands cycle (13, 14) (Fig. 2). In this process, fatty acids Figure 1. Action of phospholipases. Several phospholipases have been described with specificity for hydrolysis for one or more of the bonds of phosphoglycerides. The susceptible bonds in phosphatidylcholine are shown by capital letters in the formula above. Enzymes hydrolyzing an acyl group (R or R 1 ) from the phosphoglyceride are phospholipases A. For hydrolysis at bond A, the enzyme is phopholipase A, for that at bond B, phopholipases A2. Phospholipase B hydrolyzes both bonds A and B. Phospholipase C cleaves bond C, generating diacylglycerol and a phosphorylated nitrogenous base. Phospholipase D cleaves the terminal diester bond at D, of glycerophosphatidates containing choline, ethanolamine, inositol, or serine, with the formation of phosphatidic acid.

4 18 Nancy Mora et al. Figure 2. Arachidonic acid cycle (Lands cycle). Arachidonic acid is initially liberated from a glycerophospholipid by cytosolic phopholipase A2 (cpla2). The major lysophopholipid formed is most likely lysoglycerophosphoethanolamine (GPEtn) (according to studies of the liberation of arachidonic acid in the human neutrophil). A fraction of the arachidonic acid is converted to arachidonoyl-coa-ester by long-chain fatty acyl-coa ligase (FACL4). The arachidonoyl-coa-ester can also be religated to a lysoglycerophospho-choline (GPCho) lipid by the action of lysophospholipid acyltransferase (LAT). Transfer of arachidonic acid back to phosphatidylethanolamine phospholipids is possibly carried out by a CoA-independent transacylase. get conjugated to coenzyme A (CoA) through the action of fatty acyl-coa ligases. The, fatty acyl-coa esters are esterified to lysophospholipids in a reaction catalyzed by the enzyme lysophospholipid-acyl-coa acyltransferase (LAT). This reacylation process seems to be central for controlling the availability of free arachidonic acid and the subsequent production of eicosanoids after cell stimulation (15). Inhibition of LAT by the organic mercury compound thimerosal, leads to an increase in leukotriene B 4 released by neutrophils after GM-CSF (granulocyte/macrophage colony-stimulating factor) or fmlp (N-formyl-methionyl-leucyl-phenylalanine) stimulation (15, 16). Several phospholipase A2 (PLA2) enzymes have been described and classified into 15 different groups according to their primary structures, localization, and Ca 2+ ion requirements (17). In general it is practical to separate mammalian PLA2s in five families (14): low-molecular-mass calcium-dependent secretory PLA2 (spla2), which includes groups I, II, III, V, X, and XII; calcium-dependent cytosolic PLA2 (cpla2), which includes group IV; intracellular calcium-independent PLA2 (ipla2), which includes group VI; calcium-independent PAF-acetylhydrolase, which includes groups VII and VIII; and lysosomal PLA2, which includes group XV. Despite various PLA2 have been reported to initiate or amplify arachidonic acid release in

5 Biochemistry of inflammation 19 several experimental models, it is generally recognized that cpla2 is central to the cell-activation events that directly lead to eicosanoid production (18). cpla2 possesses a particular preference for glycerophospholipids containing arachidonic acid esterified at the sn-2 position (19). Mice genetically deficient in this enzyme could not synthesize prostaglandins nor leukotrienes in response to several stimuli (20, 21). cpla2 regulation is complex and not well comprehended. cpla2 is expressed in many tissues, but its expression can be modulated in response to stimuli such as cytokines, growth factors or corticosteroids. Its activity is also regulated by several mechanisms including phosphorylation, interaction with lipids such as ceramide 1-phosphate, interaction with proteins such as annexin, and degradation by caspases (19). Prostanoids Prostanoids, the prostaglandins (PGs) and thromboxane A 2 (TxA 2 ), are a family of lipid mediators formed by the action of the enzyme prostaglandin G/H synthase, or cyclooxygenase (COX) on the 20-carbon (C 20 ) unsaturated fatty acid, arachidonic acid. Prostaglandins are C 20 carboxylic acids containing a cyclopentane ring, and can be considered as derivatives of a hypothetical C 20 acid given the trivial name prostanoic acid (Fig. 3). There are two main groups of prostaglandins (PGs), the E and F series, each having three members, E 1, E 2, and E 3, and F 1, F 2, and F 3. The two series differ from each other in that the PGEs contain a keto oxygen at C-9 and a hydroxyl group at C-11, whereas the PGFs have a hydroxyl group at both positions in the cyclopentane ring (Fig. 3). The subscripts and after the numerical subscripts in the F series indicate the orientation of the hydroxyl group at C-9; that is, means below and means above the plane of projection of the cyclopentane ring. Prostaglandins are formed by the action of the enzyme prostaglandin G/H synthase, or cyclooxygenase (COX) on arachidonic acid. COX is an evolutionary conserved enzyme (22). There are three isoforms of COX. The first, COX-1 is expressed constitutively in most tissues, and synthesizes PGs at low levels, and thus it has been considered to be the source of prostanoids for many housekeeping physiological functions (23). COX-2 is induced by several pro-inflammatory stimuli, cytokines, shear stress, and mitogens. It is considered to be the more important source of prostanoids in inflammation (23). However, both enzymes contribute to the generation of homeostatic prostanoids, and both can also contribute to prostanoid synthesis during inflammation (12, 24). A third recently described isoform, COX-3 is a splice variant of COX-1, is inhibited by acetaminophen and is thus thought to mediate the antipyretic and analgesic effects of this drug (25). However, the

6 20 Nancy Mora et al. functional relevance of acetominophen-sensitive COX-3 in humans is not completely clear (26). COX-1 and COX-2 function as homodimers located at the endoplasmic reticular membrane, to convert arachidonic acid into the unstable cyclic endoperoxides PGG 2 and PGH 2 (Fig. 4). These endoperoxides in turn are substrates for various downstream synthases and isomerases responsible for the generation of thromboxane A 2 (TxA 2 ), and the series D, E, F, and I of PGs (Fig. 4). Both COX-1 and COX-2 present similar structural properties (27), including a hydrophobic tunnel that allows arachidonic acid access to the active site of the enzyme. Isoform specific preference for downstream enzymes has been reported in heterologous expression systems; though the biological significance of this preference is unknown. COX-1 cooperates mainly with Figure 3. Prostaglandins. Prostaglandins (PGs) are considered to be derivatives of the hypothetical 20-carbon prostanoic acid shown with the numeration of the carbon atoms. The PGEs contain a keto oxygen at C-9 and a hydroxyl group at C-11, whereas PGFs have a hydroxyl group at both positions. The structure of the three PGEs is shown to indicate the number of double bonds they present.

7 Biochemistry of inflammation 21 Figure 4. Biosynthesis of prostanoids. Following the catalytic release of arachidonic acid (AA) from membrane phospholipids by phospholipase A2, cyclooxygenase (COX) converts AA into the cyclic endoperoxides PGG 2 and PGH 2. The later is then metabolized by the indicated specific enzymes to yield the various prostanoids: prostaglandin D 2 (PGD 2 ), prostaglandin E 2 (PGE 2 ), prostaglandin F 2 (PGF 2 ), prostaglandin I 2, also known as prostacyclin (PGI 2 ), and thromboxane A 2 (TxA 2 ). thromboxane synthase, prostaglandin F synthase, and cytosolic prostaglandin E synthase isozymes (12). COX-2 cooperates mainly with prostaglandin I synthase, and microsomal prostaglandin E synthase isozymes, both of which are induced by cytokines and tumor promoters (12). No steroidal anti-inflammatory drugs (NSAIDs) No steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, and naproxen (Fig. 5) are compounds traditionally used for the treatment of

8 22 Nancy Mora et al. inflammatory joint diseases. NSAIDs, especially aspirin, inhibit COX and thus reduce the synthesis of prostanoids, underscoring the importance of these arachidonic acid metabolites as pro-inflammatory mediators. The use of traditional NSAIDs is however associated with renal and gastrointestinal toxicity, due to inhibition of COX-1 (28). NSAIDs were generally shown to inhibit either COX-1 more selectively or both COX isoforms coincidentally (29). Therefore, research focused on developing new selective COX-2 inhibitors (30). The development of selective COX-2 inhibitors and the growing evidence that placed COX-2 at many sites of inflammation reinforced the idea of a more relevant pro-inflammatory role for this inducible enzyme (31). However, in COX-1-deficient mice, arachidonic acid induces much less edema than in wild-type mice, suggesting a more prominent role for COX-1 than for COX-2 in this model of inflammation (32). Clearly, then depending on the context both COX isoforms contribute to inflammation. Prostanoid receptors Prostanoids induce important and potent cellular responses through activation of membrane receptors. Specific G protein-coupled receptors Figure 5. No steroidal anti-inflammatory drugs (NSAIDs). A) Aspirin acetylates a serine residue in the active site of the enzyme cyclooxygenase (COX), inactivating it to convert arachidonic acid into PGG2. B) The structure of the other common NSAIDs, Ibuprofen and Naproxen, is shown.

9 Biochemistry of inflammation 23 (GPCRs) have been cloned for all the prostanoids (33).The I prostanoid (IP) receptor recognizes prostacyclin, the F prostanoid (FP) receptor binds PGF 2, and the T prostanoid (TP) receptor binds TxA 2. There are four receptors for PGE 2, EP 1 to EP 4, and two PDG 2 receptors, DP 1 and DP 2 (Fig. 6). Two additional isoforms of the human FP and TP and eight EP 3 variants are generated through differential splicing. IP, DP 1, EP 2, and EP 4 receptors activate adenylyl cyclase via Gs, increasing intracellular camp. FP, TP, and EP 1 can activate PLC via Gq and initiate phosphatidylinositol metabolism leading to the formation of inositol triphosphate (IP 3 ), which induces release of Ca 2+ from intracellular stores. TP can activate multiple G proteins, including G 12/13 and G 16 to stimulate small GTPase signaling pathways, and may activate or inhibit adenylyl cyclase. EP 3 isoforms can induce via Gi or G 12 elevation of intracellular Ca 2+, inhibition of camp production, and activation of the small GTPase Rho (12, 33). Figure 6. Prostanoid receptors. Receptors for the various prostanoids are listed together with the main tissues where they are expressed. The I prostanoid (IP) receptor recognizes prostacyclin (PGI 2 ), the F prostanoid (FP) receptor binds PGF 2, the T prostanoid (TP) receptor binds TxA 2, the EP 1 to EP 4 receptors bind PGE 2, and the DP 1 and DP 2 receptors bind PGD 2. There are two additional isoforms of the human FP and TP receptors, and eight EP 3 variants generated through differential splicing.

10 24 Nancy Mora et al. Prostanoid functions Prostanoids have several functions important in inflammation and in maintenance of homeostasis. Table 1 summarizes the main site of action and function of prostanoids. Activated platelets produce TxA2, amplifying further platelet activation (34). Mature platelets express only COX-1, although megakaryocytes and immature platelets also express COX-2 (35). In platelets, the TP receptor couples mainly to Gq activating protein kinase C-dependent pathways, which promote platelet aggregation. Prostacyclin (PGI 2 ) is the major platelet inhibitory prostanoid. It is produced mainly by COX-2 in vascular endothelial and smooth muscle cells (36). The IP receptor activates Gs and PKA via camp. PKA in turn deactivates myosin light chain kinase, to reduce myosin phophorylation and decrease platelet aggregation (37). The effects of PGE2 on platelets in vivo remain unclear. High concentrations of PGE 2 (>10 mm) inhibit platelet function, whereas low concentrations of PGE 2, via the EP 3 receptor, seem to increase platelet stimulation (38). During inflammation prostanoid biosynthesis is considerably increased, mainly by COX-2 activity in inflammatory cells and tissues. However, COX-1 also contributes to the production of pro-inflammatory prostanoids. In both, COX-1- and COX-2-deficient mice reduced inflammatory responses are produced underlining the importance of both isozymes in inducing inflammation. Human data suggest that COX-1-derived products participate in the initial phase of an acute inflammation, while COX-2 upregulation is more important at several hours after initiation of an inflammatory reaction (12). PGE 2 and PGI 2 are the main pro-inflammatory prostanoids. They increase edema formation and leukocyte infiltration by promoting blood flow. They also increase vascular permeability (33). PGD 2 is produced by mast cells and contributes to inflammation in allergic responses, particularly in the lung. It also increases blood flow, vascular permeability, and promotes T-cell polarization to the Th2 phenotype. Thus, PGD 2 promotes inflammation by activation of Th2 cells and eosinophils (39). PGE 2 and PGI 2 also reduce the threshold of nocireceptor sensory neurons to stimulation. Thus, they potentiate the pain-inducing activity of bradykinin and other autacoids. There is some basal expression of COX-2 in neurons and glia. The prostanoids produced by this enzyme contribute to central sensitization, a process with an increase in spinal dorsal horn neuron excitability that augments pain intensity, mainly in the early phase of peripheral inflammation (40). After few hours, COX-2 is upregulated throughout the spinal cord, leading to prolonged central sensitization. EP1 and IP receptors seem to participate more during the early phase of inflammation-

11 Biochemistry of inflammation 25 induced pain, while EP2 receptors seem more relevant at later times of inflammation (41). Several prostaglandins have important functions in the kidney. Both COX are expressed in different parts of renal tissue. Prostanoids derived from the cortex and from the medulla seem to have contrasting effects on blood pressure and liquid retention (42). This complexity is in part due to the regulated expression of prostanoid receptors in different parts of the kidney (43). In addition, prostanoids are also involved in maintaining cardiovascular homeostasis. Selective inhibition of COX-2 increase the risk of myocardial infarction (36). Table 1. Location and function of the main prostanoids. Prostanoid a Principal Location Function TxA 2 Platelets Monocytes Platelet aggregation, broncoand vaso-constriction, cellular proliferation PGI 2 Vascular endothelium Inhibition of platelet aggregation, bronco- and vasodilation, vascular leakage PGD 2 PGE 2 Mast cells Some parts of the brain Renal medulla Gastric mucosa Platelets Microvascular endothelium Bronchospasm and allergic asthma, inhibition of platelet aggregation, sleep Inhibition of sodium reabsorption, bronco- and vasodilation, uterine contraction, lymphocyte function, presynaptic adrenergic modulation PGF 2 Brain Uterus Bronchial and uterine contraction, uterine vasoconstriction a TxA2, thromboxane A2; PGI 2, prostacyclin; PGD 2, PGE 2, PGF 2, prostaglandins of the series D, E, and F. Leukotrienes Leukotrienes are metabolites of arachidonic acid formed by the action of the enzyme 5-lipoxygenase (5-LO). They have potent biological activities including that of contracting smooth muscle and inducing PMN chemotaxis (44). Leukotrienes were characterized as the main arachidonic acid metabolites of PMN (45) as well as being the main component of the slow-reacting substance of anaphylaxis (SRS-A) (46). The name leukotriene was established

12 26 Nancy Mora et al. to indicate that these compounds present three conjugated double bonds within the structure of arachidonic acid and that were produced from leukocytes such as PMN or activated mast cells. The enzyme 5-LO is expressed in cells of myeloid origin including PMN, eosinophils, mast cells, macrophages, basophils, and monocytes. Leukotriene biosynthesis begins with the specific oxidation of arachidonic acid by 5-LO. First oxygen is added to the C-7 of arachidonic acid to form 5-hydroperoxyeicosatetraenoic acid (5-HpETE) (Fig. 7). Then in a second step also catalyzed by 5-LO a hydrogen atom is removed from C-10 to produce the conjugated triene epoxide leukotriene A 4 (LTA 4 ) (Fig. 7). LTA 4 becomes then the substrate of either LTA 4 -H (LTA 4 hydrolase) or LTC 4 -S [LTC 4 (leukotriene C 4 ) synthase]. LTA 4 -H stereospecificlly adds water to C-12 to produce leukotriene B 4 (LTB 4 ); whereas LTC 4 -S opens the reactive Figure 7. Biosynthesis of leukotrienes. Following the catalytic release of arachidonic acid (AA) from membrane phospholipids by phospholipase A2, 5-lipoxygenase (5-LO) converts AA into 5-hydroperoxyeicosatetraenoic acid (5-HpETE), which is, in a second step also catalyzed by 5-LO, converted into the conjugated triene epoxide leukotriene A 4 (LTA 4 ). Then, LTA 4 becomes the substrate of either LTA 4 -H (LTA 4 hydrolase) or LTC 4 -S [LTC 4 (leukotriene C 4 ) synthase] to produce leukotriene B 4 (LTB 4 ) and LTC 4, respectively.

13 Biochemistry of inflammation 27 epoxide at C-6 with the thiol anion of glutathione to produce LTC 4 (Fig. 7). LTC 4 is the first of the so-called cysteinyl leukotrienes, which also include leukotriene D 4 (LTD 4 ) and leukotriene E 4 (LTE 4 ). LTD 4 is one of the constituents of SRS-A and is produced by the metabolism of LTC 4 by the enzyme -glutamyl transpeptidase (Fig. 8). LTE 4 is also one of the constituents of SRS-A and is produced from LTD 4 by the action of a dipeptidase, which leaves only the cysteinyl group still attached to the fatty acid backbone (Fig. 8). Figure 8. Cysteinyl leukotrienes. Leukotriene C 4 (LTC 4 ) is the first of the so-called cysteinyl leukotrienes, which also include leukotriene D 4 (LTD 4 ) and leukotriene E 4 (LTE 4 ). LTD 4 is produced by the metabolism of LTC 4 by the enzyme -glutamyl transpeptidase, and LTE 4 is produced from LTD 4 by the action of a dipeptidase.

14 28 Nancy Mora et al. Leukotrienes are all biologically active lipid mediators. LTB 4 induces chemotaxis and aggregation of PMN. It also promotes activation of various leukocyte functions such as enhancement of lysosomal enzyme release and superoxide anion production (47). LTC 4 is produced by neutrophils, macrophages, mast cells, and by transcellular metabolism in platelets. It exhibits potent smooth muscle contracting activity leading to bronchoconstriction and also enhanced vascular permeability. These effects contribute to the pathogenesis of asthma and acute allergic hypersensitivity (14). Just like LTC 4, LTD 4 also induces bronchoconstriction and vascular permeability. LTD 4 is equipotent to LTC 4 in its biological activities, except that it is 100-fold more potent in contracting peripheral airway smooth muscle (48). LTE 4 is less active (around 10-fold) than other cysteinyl leukotrienes, and unlike other leukotrienes, it is excreted in urine as a major metabolite, accounting for about 4 % of total LTC 4 produced in humans (49). This is a sufficiently abundant metabolite that sensitive and specific assays have been developed to measure biosynthesis of LTC 4 in vivo, particularly in asthmatic patients (50). Leukotriene receptors Leukotrienes induce important and potent cellular responses through activation of specific membrane G protein-coupled receptors. Two LTB 4 receptors are known and are named BLT-1 and BLT-2 (51-53), and two receptors for the cysteinyl leukotrienes are named CysLT 1 and CysLT 2 (54, 55). Recently a novel receptor that binds uracil diphosphate (UDP) and also LTC 4 has been described (56). The two LTB 4 receptors differ in their affinity and specificity for this leukotriene: BLT-1 is a high-affinity receptor specific for LTB 4, whereas BLT-2 is a low-affinity receptor that also binds other eicosanoids. The two receptors also differ in their pattern of expression with BLT-1 being expressed primarily in leukocytes, whereas BLT-2 is expressed more ubiquitously. Cysteinyl-leukotrienes induce a range of pro-inflammatory effects, such as constriction of airways and vascular smooth muscle, increase of endothelial cell permeability leading to plasma exudation and edema, and enhanced mucus secretion. Receptors for these leukotrienes (57) were initially classified into subtypes based on binding and functional data, obtained using the natural agonists and a wide range of antagonists (58). These receptors have proved remarkably resistant to cloning. However, in 1999 and 2000, the CysLT 1 and CysLT 2 were successfully cloned and both shown to be members of the GPCRs superfamily. Molecular cloning has confirmed most of the previous pharmacological characterization and identified distinct expression patterns

15 Biochemistry of inflammation 29 only partially overlapping. Recombinant cysteinyl-leukotriene receptors couple to the Gq/11 pathway that modulates inositol phospholipids hydrolysis and calcium mobilization, whereas in native systems, they often activate a pertussis toxin-insensitive Gi/o-protein, or are coupled promiscuously to both G-proteins. The CysLT1 receptor is mainly expressed in spleen, peripheral blood leukocytes, and lung smooth muscle cells and interstitial lung macrophages. The CysLT2 receptor is most highly expressed in the heart, adrenal medulla, placenta and peripheral blood leukocytes. Sphingolipids Sphingolipids are produced by the metabolism of sphingomyelin, an important lipid of the plasma membrane. Various enzymatic pathways lead to the formation of different lipid mediators with important roles in many cellular functions including proliferation, apoptosis, and migration. Sphingolipid metabolites, including ceramide, ceramide 1-phosphate, and sphingosine 1-phosphate (S1P) are now recognized as important inflammation mediators (59). They can promote inflammation by activating pro-inflammation transcription factors, such as the Nuclear Factor B (NF- B) in various cell types, or by inducing COX-2 with the consequent production of prostaglandins. The mode of action of each sphingolipid is different. Ceramide can accumulate in certain areas of the plasma membrane, where it may promote the assembly of signaling complexes, whereas S1P binds to specific S1P receptors that belong to the family of GPCRs (7). The metabolism of sphingomyelin is controlled by the activity of sphingomyelinase (SMase) enzymes (60). SMases are activated by a variety of stimuli, including inflammatory cytokines, growth factors, GPCRs, and cell stress (7). Different isoforms of SMases are found in mammalian cells: acid SMase, with an optimum activity around ph = 5; neutral SMase, which is Mg 2+ -dependent; and secretory SMase (61). Acid SMase is present mainly in lysosomes (where there is an acid environment), neutral SMase is a membranebound protein that seems to be expressed in all types of cells, and secretory SMase is targeted to the Golgi secretory pathway, and could potentially restrict the effects of sphingolipid mediators produced in certain intracellular locations. The action of SMases on sphingomyelin leads to the formation of ceramide (Fig. 9), which can be either degraded by ceramidase to yield sphingosine or phosphorylated by ceramide kinase to form ceramide 1-phosphate (C1P). Sphingosine can also be phosphorylated by sphingosine kinases to produce sphingosine 1-phosphate (S1P) (Fig. 9). There are two isoforms of sphingosine kinase, SK1 and SK2, in mammalian cells (62, 63). S1P in turn, can be dephosphorylated by sphingosine phosphatase or degraded

16 30 Nancy Mora et al. by S1P lyase (64). Another sphingolipid closely related to S1P, sphingosylphosphorylcholine (SPC) is probably also derived from sphingomyelin but via the action of another enzyme, sphingomyelin deacylase (7, 65). Because these sphingolipids are interconnected in their synthesis and metabolism. The increase in one lipid is associated with the decrease in another, and this can be an important regulator of cell function. Indeed, the so-called sphingolipid rheostat regulates pro- and anti-apoptotic signals (66). An increase in ceramide with the associated decrease in S1P leads to activation of cell death, while a decrease in ceramide and the corresponding increase in S1P leads to stimulation of anti-apoptotic signals (7). Figure 9. The sphingomyelin cycle. The structure of main sphingolipids and their metabolic cycle is shown. Enzymes responsible for each step are indicated over reaction arrows.

17 Biochemistry of inflammation 31 Sphingolipids in inflammation The role of sphingolipids as mediators of inflammation is now becoming evident (7, 67). Several sphingolipids can in some cell types induce particular responses that regulate the inflammatory process. This regulation takes place by various mechanisms that we describe briefly next. Tumor necrosis factor alpha (TNF), a potent pro-inflammatory cytokine can activate acid SMase leading to ceramide production and subsequent activation of the pro-inflammatory transcription factor NF- B (68). NF- B is responsible for induction of many inflammatory genes that code for cytokines and chemokines such as interleukin (IL)-1, IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1), and also enzymes such as COX-2 (69). Figure 10. Intracellular signals relevant to inflammation where sphingolipids participate. TNFR, receptor for tumor necrosis factor alpha; SMase, sphingomyelinase; C1P, ceramide 1-phosphate; c/ebp, CCAAT/enhancer binding proteins; NF- B, nuclear factor B; S1P, sphingosine 1-phosphate; S1PR, receptor for S1P; cpla2, cytosolic phospholipase A2; COX-2, cyclooxygenase-2.

18 32 Nancy Mora et al. COX-2 in turn leads to the production of pro-inflammatory prostaglandins (Fig. 10). Ceramide can also activate another family of transcription factors related to inflammation, the CCAAT/enhancer binding protein (c/ebp), which induce gene expression of pro-inflammatory proteins such as TNF, IL-6, IL-8, and IL-1 (70). TNF can also activate neutral SMase and this leads via ceramide, or possibly C1P, to activation of cpla2 (71) (Fig. 10). These effects of ceramide on inflammation have been observed in several cell lines, but it is not clear ceramide can regulate the inflammatory process in all cell types. Ceramide kinase is responsible for production of C1P, but not much is known about its regulation (72). Therefore, it is not clear whether some of the effects attributed to ceramide are in fact mediated by activation of ceramide kinase and production of C1P (73), but recent reports suggest that the effects on inflammation are mediated by activation of cpla2 (71). The effects of S1P on inflammation are more clearly established, in part due to the cloning and characterization of high-affinity specific receptors. These receptors belong to the GPCR family and are classified in five subtypes, named S1P 1 to S1P 5 (74, 75). The receptors can associate with multiple G proteins (with the exception of S1P 1 that joins only to G i) and therefore may activate multiple signaling pathways (76). S1P 2 and S1P 3 associate preferentially to G q (activating PLC) and G 12/13 (activating the GTPase Rho) (77). Because of the expression of the various receptors, S1P effects vary depending on the cell type. In mast cells, SK activation and production of S1P take place after engagement of the high-affinity receptor for IgE (78). Mast cells express S1P 1 and S1P 2 receptors, and they mediate different effects on these cells. S1P 1 participates in cell migration, while S1P 2 plays a role in degranulation (79). In other cell types, such as fibroblasts, lung epithelial cells, and macrophages, S1P is involved in up-regulation of COX-2 (80); and in lung epithelial cells it has also been reported to activate cpla2 (81) (Fig. 10). Resolution of inflammation Inflammation in response to an injurious stimulus is a beneficial event that leads to removal of the offending agent and restoration of the tissue structure and function. Elimination of the offending agent is achieved through the well-known inflammatory response that involves vascular changes, production of chemokines, upregulation of cell adhesion molecules, and accumulation of leukocytes at the site of tissue damage. After the injurious stimulus has been neutralized inflammation can resolve. Resolution of inflammation was thought to slowly terminate after the pro-inflammatory stimuli were not longer present. Today, it is recognized that resolution of inflammation is an active process mediated by an increased number of soluble mediators. These mediators

19 Biochemistry of inflammation 33 activate mechanisms that lead to the switching off of inflammation and the restoration of the tissue normal physiology (82). Thus, failure of acute inflammation to resolve may influence the development of chronic inflammation and auto-immunity (24). Among the signals required for a successful resolution of inflammation, the COX and LOX pathways are essential (82, 83). This novel idea is in contrast to the role of COX established through the initial literature on NSAIDs, according to which COX-derived arachidonic acid metabolites are mainly pro-inflammatory. It is clear now that there are novel fatty acid metabolites and other signaling molecules that actively participate in the resolution of inflammation. Reports on COX-deficient mice (84) indicated that both COX-1 and COX-2 contributed to prostanoid production at sites of inflammation, but also that COX-2-derived prostanoids participate in the resolution phase as well as in the early phase of inflammation. COX-2-deficient mice developed suppurative peritonitis with and other abdominal lesions, consistent with chronic inflammation (85). In a different study, COX-2 was found to be involved in resolution of inflammation in a carrageenan-induced pleurisy model (86). Two phases of COX-2 expression were reported in the time course of this inflammatory response. The fist peak (at about two hours) was associated with initiation of inflammation and presented polymorphonuclear leukocyte infiltration, PGE 2 production, and COX activity (86). A second peak (at about 48 hours) was associated with resolution of inflammation and late influx of mononuclear cells. This second peak of COX-2 expression was associated not with PGE 2 production, but with PGD 2 instead. In addition COX-2 inhibitors given after 24 hours inhibited the resolution of inflammation (86). Moreover, mice deficient in the enzyme hematopoietic PGD 2 synthase (hpgd 2 S), presented a more aggressive inflammatory response that failed to resolve, whereas mice overexpressing hpgd 2 S showed little inflammation (87). These reports suggest that both COX-2 and PGD 2 S are checkpoint controllers in the progression from acute to resolving inflammation (24). Similarly to the COX pathway of arachidonic metabolism, the 5-LO pathway is not completely pro-inflammatory either. For example, LTB 4 is pro-inflammatory due to its chemoattractant action on polymorphonuclear leukocytes (PMN) (88). However, the 5-LO-derived arachidonic products may be further metabolized by three different biochemical pathways to produce new anti-inflammatory and pro-resolving eicosanoids named lipoxins (Fig. 11). In one pathway, a 15-lipoxygenase present in eosinophils, monocytes, or epithelial cells, adds oxygen to C-15 of arachidonic acid to produce 15(s)hydroperoxyeicosatetraenoic acid (15S-H(p)ETE). This compound is released from the cells and taken up by PMN cells or monocytes, where 5-LO

20 34 Nancy Mora et al. Figure 11. Biosynthetic pathways of lipoxins. I) 15-lipoxygenase (15-LO) converts arachidonic acid (AA) into 15(S)hydroperoxyeicosatetraenoic acid (15S-HpETE), which serves as a substrate for polymorphonuclear leukocyte (PMN) 5-LO to produce 5(6)epoxytetraene. The latter is unstable and is transformed into lipoxin A 4 (LXA 4 ) and lipoxin B 4 (LXB 4 ) by lipoxin hydrolases. II) 5-LO in PMN converts AA into leukotriene A 4 (LTA 4 ), which is taken up by platelets and transformed by platelet 12-lipoxygenase (12-LO) into LXA 4. III) Acetylation of the active site of cyclooxygenase-2 (COX-2) by aspirin results in the transformation of AA into15(r)hydroxyeicosatetraenoic acid (15R-HETE), which in turn after released by endothelial and epithelial cells can be converted by leukocyte 5-LO into 15-epi-lipoxin A 4 (15-epi-LXA 4 ) and 15-epi-lipoxin B 4 (15-epi-LXB 4 ). converts it into a 5(6) epoxytetraene, which is then hydrolyzed by lipoxin (LX) A 4 hydrolase or LXB 4 hydrolase to produce the corresponding bioactive lipoxins (89, 90). The second pathway in PMN uses 5-LO to produce LTA 4, which is then released and taken up by platelets where a 12-LO leads also to

21 Biochemistry of inflammation 35 lipoxin LXA 4 (91). The third pathway results from the action of aspirin on COX-2, leading to the production of epi-lipoxins (92). Aspirin acetylates the active site of COX-2 in endothelial and epithelial cells, resulting not in inhibition of the COX-2 activity, as has always been assumed, but in a modified enzyme. Acetylated-COX-2 converts arachidonic acid into 15R-hydroxyeicosatetranoic acid (15R-HETE) (92), which is rapidly transformed by leukocyte 5-LO in 15-epi-LxA4 or 15-epi-LxB4 (Fig. 11). Lipoxins are recognized by an intracellular Ah (aryl hydrocarbon) receptor (93) or a GPCR named ALX-R expressed on PMN, monocytes, epithelial cells, and activated T cells (94). Lipoxins function as anti-inflammatory mediators due to their capacity to block PMN chemotaxis and diapedesis. However, LXA4 also stimulates chemotaxis and adherence of monocytes at sites of inflammation. Thus lipoxins help in the resolution of inflammation by attracting monocytes and inhibiting more PMN recruitment (95). There are also other anti-inflammatory mediators derived from the action of COX and LO pathways. Resolvins and docosatrienes are fatty acid metabolites produced from omega-3 fatty acids docosahexanoic acid and eicosapentaenoic acid are used instead of arachidonic acid as substrates of these enzymes (96, 97). Resolvins and docosatrienes were identified in inflammatory exudates during the resolving phase of acute inflammation and shown to be potent modulators of inflammation. They strongly inhibit PMN recruitment, and leukocyte accumulation in peritonitis (96, 98). There is a lot of interest in deciphering how resolvins and other lipid mediators control inflammation, particularly after a publication suggesting that lipoxins are orally active in models of acute inflammation (99). Acknowledgements Research work at the authors laboratories was supported by grants IN (to CR), IACOD IB200811, and IA (to EUQ) from Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México, and by grant (to CR) from Consejo Nacional de Ciencia y Tecnología, Mexico. References 1. Feldberg W, Holden HF, & Kellaway CH (1938) The formation of lysocithin and of a muscle-stimulating substance by snake venoms. J. Physiol. 94: von Euler US (1936) On the specific vaso-dilating and plain muscle stimulating substances from accesory genital glands in man and certain animals (protaglandin and vesiglandin). J. Physiol. 88:

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