ABSTRACT
In the last decade intensive research into the mechanisms of inflammation has implicated production of prostaglandins and reactive oxygen species as central to the progression of many inflammatory diseases. This study aimed at investigating/elucidating the biochemical mechanisms of antiinflammatory action and antioxidant activities of the bioactive phytoconstituents of stem bark extract of C. adansonii. The mineral contents, phytochemical analysis and oral acute toxicity (LD50) of the extract were determined. The extract was subjected to solvent-guided fractionation in a silica gel column successively eluted with n-hexane, ethylacetate, dichloromethane, and methanol (100%). The extract and fractions were subjected to biological activity guided studies for antiinflammatory activity using the egg-albumin induced rat hind paw-edema method as activity guide. The ethylacetate fraction was subjected to further separation in silica gel column eluted with graded mixtures of n-hexane and ethylacetate to obtain ethylacetate subfractions (EF1) and (EF2) a white crystalline solid (C.adansonii compound
1; CA1). The antiinflammatory activity of the isolate (CA1) was confirmed using the activity
guide. The extract, bioactive fraction (EF) and isolated compound (CA1) were further subjected to antiinfammatory activity tests (formaldehyde-induced chronic inflammation test in rats, red blood cell membrane stabilization activity test and cyclooxygenase inhibition assays) and antioxidant tests (phosphomolybdate test for total antioxidant capacity, reducing power test, nitric oxide and hydrogen peroxide scavenging activity tests). The molecular structure of CA1 was established using nuclear magnetic resonance (NMR), infra-red, gas and mass spectrophotometers. The n-hexane, ethylacetate and methanol fractions tested positive to glycosides, terpenoids and flavones. Mineral analysis of stem bark showed that it contained selenium, sodium, iron, zinc, calcium, potassium, manganese, and magnesium. Acute toxicity test on the extract gave an oral LD50 > 5 g/kg. Ethylacetate fraction caused the greatest inhibition of rat paw edema. Phytochemical tests on CA1 gave positive reaction to terpenoids. CA1 significantly (P < 0.05) suppressed acute edema. The ethylacetate fraction and CA1 significantly (P < 0.05) inhibited erythrocyte lysis. Analysis of the blood samples from formaldehyde-induced chronic inflammatory rats, indicated that the extract (200 and 500 mg/kg) caused non-significant (P > 0.05) difference in lymphocyte levels, total white blood cells, platelet count and % PCV compared with untreated control group, but exhibited significant decrease in % haemoglobin and % neutrophil levels. In relation to the baseline values, the extract exhibited significant (P < 0.05) increase in lymphocyte level, total white blood cells and platelet count, but significant decrease in % neutrophils level. The results on radical scavenging activities showed that ethylacetate, methanol and n-hexane fractions significantly (P < 0.05) exhibited reducing power. The total antioxidant capacity indicated that n-hexane and ethylactate fractions have significant (P <0.05) inhibition capacity. All the fractions exhibited significant (P < 0.05) percentage inhibition of in vitro hydrogen peroxide and nitric oxide scavenging activities when compared with vitamins E and C. Formalin-induced paw edema formations were significantly inhibited by the extract. The mean decrease in serum zinc was statistically significant in relation to control and the baseline values. Structural elucidation indicated that CA1 is lupeol with M.W. of 426.70 g, molecular formular of C30H50O and melting point of 215°C. Lupeol at the tested concentrations significantly (P < 0.05) and selectively inhibited cyclooxygenase-2 enzymes.
CHARPTER ONE
INTRODUCTION
1.1 Inflammation
The inflammatory response consists of an innate system of cellular and humoral responses following injury (such as after heat or cold exposure, ischemia/reperfusion, blunt trauma) in which the body attempts to restore the tissue to its preinjury state (Peter et al., 2000). In the acute inflammatory response, there is a complex orchestration of events involving leakage of water, salt and proteins from the vascular compartment; activation of endothelial cells; adhesive interactions between leukocytes and the vascular endothelium; recruitment of leucocytes; platelets and their aggregation; activation of the complement clotting and fibrinolytic systems and release of proteases and oxidants from phagocytic cells, all of which may assist in coping with the state of injury. Whether due to physical or chemical causes, infectious organisms, or any number of other reasons that damage tissues, the earliest in vivo hallmark of acute inflammatory response is the adhesion of neutrophils to the vascular endothelium “margination’ (Summera, 2010). The chronic inflammatory response is defined according to the nature of the inflammatory cell appearing in tissues. Resolution of the inflammatory response implies that leukocytes will be removed either via lymphatics or by apoptosis and that the ongoing acute inflammatory response is terminated. During resolution increased vascular permeability is reversed due to closure of the open tight junctions and PMN emigration from the blood compartment ceases (Nottebaum et al., 2008). In both the vascular and extravascular compartments, fibrin deposits are removed by pathways that leads to activation of plasminogen (to plasmin), which degrades fibrin. Cell debris and red blood cells (RBCs) in the extravascular compartment are removed by phagocytosis involving tissue macrophages.
1.2 Types of inflammation based on disease duration
Inflammation is classified based on duration of the lesion and histologic appearances into acute and chronic inflammation.
i. Acute
ii.Chronic Inflammation.
1.2.1 Acute-Inflammatory Response
The acute inflammatory response is defined as a series of tissue responses that can occur within the first few hours following an encounter with a harmful stimulus. As an initial response of the body to injury, there are numerous changes within the vascular compartment that trigger off this response. The initiation of an inflammatory response is dependent on the recognition of invading microbes and/or damaged tissue by resident immune cells. The major sentinel cells involved in this innate immune response are the macrophages and mast cells (Heib et al., 2008; Weissler et al., 2008; Laskin 2009; Dietrich et al., 2010).With microbial invasion, these cells recognize highly conserved components of microbes termed pathogen- associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) (Mogensen,
2009).With sterile tissue injury, the sentinel cells detect substances released by damaged cells
and/or the extracellular matrix (ECM), referred to as damage-associated molecular patterns (DAMPs), such as heat shock proteins, high mobility group protein-B1 (HMGB1), and hyaluronan (Klune et al., 2008; Gauley and Pisetsky, 2009). Both macrophages and mast cells can be activated by PAMPs and DAMPs. Upon activation, both mast cells and macrophages release various inflammatory mediators (e.g., platelet activating factor (PAF)), chemokines (interleukin- 8 (CXCL8)), and cytokines (e.g., tumor necrosis factor (TNF), interleukin-1 (IL-1)) (Theoharides et al., 2007; Szekanecz and Koch, 2007). The major class of membrane receptors for PAMPs and DAMPs are the Toll-like receptors (TLRs) (Mogensen, 2009). There is also evidence that IL-1α released from necrotic cells utilizes the IL-1R on sentinel cells to initiate an inflammatory response (Chen et al., 2007). Activation of TLRs, RAGE, or IL-1R results in the activation/nuclear translocation of transcription factors involved in the inflammatory response (e.g., NF-κB) (Verstrepen et al., 2008; Tapping,
2009). Collectively, most of the available information indicates that TLRs are the major receptors for PAMPs and DAMPs and that there is a convergence of molecular pathways at the level of NF-κB. These numerous changes within the vascular compartment trigger the acute inflammatory response, involving at least six intravascular events including three of the followings;
1. Activation of endothelial cells, during which these cells begin to express on their surfaces adhesion molecules for leukocytes. Activated endothelial cells also engage in the generation and release of proinflammatory cytokines and chemokines which will
chemotactically attract and activate neutrophils as these cells adhere to the endothelium before their transmigration into the extravascular compartment.
2. Reversible opening of endothelial cells tight junctions, which allows for the leakage of protein and fluids from the vascular compartment into the extravascular compartment. When extensive edema develops in closed compartments, this can seriously impair organ function.
3. Adhesive interactions between PMNs and endothelial cells. Ordinarily, PMNs and other leukocytes are carried in the center in the blood stream without contact with endothelial surfaces. PMNs undergo activation responses such as upregulation of CD11b/CD18 on their membranes, while endothelial cells undergo activation most commonly with gene expression, leading to appearance of P-selectin, E-selectin, and ICAM-1.
The five cardinal signs of acute inflammation are;
a. Redness (rubor) which is due to dilation of small blood vessels within damaged tissue. b. Heat (calor) which results from increased blood flow (hyperemia) due to regional
vascular dilation.
c. Swelling (tumor) which is due to accumulation of fluid in the extravascular space which, in turn, is due to increased vascular permeability.
d. Pain (dolor) which results from nociceptor excitation by inflammatory mediators.
e. Loss of function: The inflamed area is inhibited by pain while severe swelling may also physically immobilize the tissue.
1.2.2 Chronic inflammation
Chronic inflammation is a pathological condition characterized by continued active inflammation response and tissue destruction, often called Proliferative Inflammation because it is characterised by a proliferation of cells than exudation of cells and fluids. Many of the immune cells including macrophages, neutrophils and eosinophils are involved directly or by production of inflammatory cytokine, in pathology of chronic inflammation (Khansari et al.,
2009). In chronic inflammation, the proliferating cells may be macrophages, lymphocytes,
plasma cells and fibroblasts. New tissue, made up of fibroblast and capillary loop, is known as granulation tissue. Such tissue fills in the gaps caused by necrosis and removal of debris.
Types of chronic inflammation: unspecific (e.g.: chronic peptic ulcer) and specific (granulomatous). According to the mechanism, granulomatous inflammation may be: immune type (tuberculosis, sarcoidosis) and non-immune type (foreign body reaction). Classification of granulomatous inflammation, according to the etiology (Zumla and James,
1996).
1.3 Pathophysiology of Inflammation
The complex sequence of events that characterized the inflammatory process can be categorized into two – vascular and cellular processes.
1.3.1 Vascular Response
Inflammatory stimuli alter vascular tone through direct effect on endothelium, which may affect vascular function and remodeling. Endothelium is the active inner monolayer of the blood vessels forming an interface between circulating blood and the vessel wall. It plays a critical role in vascular homeostasis. Endothelial cells regulate vascular tone by releasing various contracting and relaxing factors (inflammatory mediators) such as nitric oxide, arachidonic acid metabolites, reactive oxygen species (ROS) and vasoactive peptides (Luscher and Tanner, 1993). Therefore, the endothelium actively regulates vascular tone and permeability, the balance between coagulation and fibrinolysis, the inflammatory activity as well cell proliferations (Savoia et al., 2011). In inflammatory response, blood vessels undergo a series of changes that are designed to maximize the movement of plasma proteins and circulating cells out of the circulation and into the site of injury or infection. The changes occur in the following order; a transient vasoconstriction, vasodilatation and increased permeability of microvasculature.
1.3.1.1 Vasoconstriction
The initial trauma in inflammatory response, a neurogenic response, results in immediate (momentary) vasoconstriction (within some few seconds) of the blood vessels leading to decrease in blood flow. Vasoconstriction results from the increased concentration of calcium
ions (Ca2+) or inhibition of myosin light-chain phosphatase and soluble guanylate cyclase
(sGC) within vascular smooth muscle cells (Sandoval et al., 2001a; Sandoval et al., 2001b). However, the specific mechanisms for generating an increased intracellular concentration of
calcium depend on the vasoconstrictor. The stimuli responsible for eliciting this neurogenic response are circulating epinephrine and activation of the sympathetic nervous system (through release of norepinephrine) that directly innervates the muscle. These compounds in response to inflammatory stimuli interact with cell surface adrenergic receptors results in a signal transduction cascade that leads to increased intracellular calcium from the sarcoplasmic reticulum through IP3-mediated calcium release, as well as enhanced calcium entry across the sarcolemma through calcium channels. The rise in intracellular calcium complexes with calmodulin, which in turn activates myosin light-chain kinase (Mehta and Malik, 2006). This enzyme is responsible for phosphorylating the light chain of myosin to stimulate cross-bridge cycling. Endothelium-dependent contractions can be enhanced by compromising nitric oxide bioavailability or amplifying ROS bioavailability (Vanhoutte et al., 2005).
1.3.1.2 Vasodilatation
Vasodilatation, which is endothelium smooth muscle cell relaxation and its resulting increased blood flow, is one of the earliest manifestations of inflammation that follows a transient constriction of arterioles, lasting a few seconds. Vasodilatation first involves the arterioles and then results in opening of new capillary beds in the area. Endothelium– dependent vasodilatation has been attributed to the release of prostacyclin (PGI2) and NO, which are referred to as endothelium–derived relaxing factors (EDRFs) (Furchgott and Vanhoutte, 1989). Vasodilatation as one of the cardinal signs of an inflammatory response is produced to a large extent via a NO-dependant process. Various inflammatory mediators, such as bradykinin and histamine, produce vasodilatation through stimulation of endothelial release of NO (Furchgott and Zawadski, 1980; Bryan et al., 2009). NO appears to be the
dominant EDRF. In the EDRF pathway, an increased intracellular Ca2+ activates endothelia
(e)NOs, which generates NO during the conversion of L-arginine to L-citrulline. NO diffuses to the myocytes where it binds to the heme moiety of soluble guanylate cyclase (sGC) and displaces iron from its usual position in the porphyrin ring allowing sGC to catalyze the formation of cGMP. cGMP inhibits myosin light chain kinase thereby preventing the cross- bridge formation between action filaments and myosin head resulting in smooth muscle relaxation (Beckman and Koppenol, 1996). In endothelia smooth muscle, vasodilatation works by lowering intracellular calcium concentration, inhibition of myosin light chain kinase, or dephosphorylation of myosin. Dephosphorylation by myosin light-chain
phosphatase and induction of calcium symporters and antiporters that pump calcium ions out of the intracellular compartment both contribute to smooth muscle cell relaxation and therefore vasodilation.
1.3.1.3 Increased Vascular Permeability
A hallmark of acute inflammation is increased vascular permeability, leading to the escape of a protein-rich fluid (exudates) into the extravascular tissue. The loss of protein from the plasma reduces the intravascular osmotic pressure and fluid. Together with the increased hydrostatic pressure owing to increased blood flow through the dilated vessels, this leads to a marked outflow of fluid and its accumulation in the interstitial tissue. The net increase of extravascular fluid results in edema. The vascular endothelium lining the blood vessels forms a continuous, semi-permeable restrictive barrier enabling the passage of macromolecules, inflammatory cells, and fluid between the blood and interstitial space. There are two different routes of transport across the endothelial barrier, transcellular (across the cell) and paracellular (between the cells) (Mehta and Malik, 2006). The paracellular route is tightly controlled by inter-endothelial junctions (IEJ) and tight junctions (TJ), which provides an unperturbed restrictive barrier to the endothelium. The paracellular permeability is the major route of vascular leakage observed in a variety of inflammatory states.
Among the TJ proteins, occludins are redox-sensitive proteins. Increased oxidative stress has been related to down-regulation of occludin expression, reduced membrane localization, and reduced tightness of the junctional barrier (Krizbai et al., 2005; Maier et al., 2006). Oligomerization of occludin is modulated by the ratio of intracellular GSH/GSSG (Walter et al., 2009a). During inflammation, enhanced oxidative stress drastically alters the GSH/GSSG ratio toward GSSG, which prevents oligomeric assembly of occludin (McCaffrey et al.,
2009). The effects of inflammatory agonists such as thrombin and VEGF on adherent junction proteins are mediated by phosphorylation of VE-cadherin, β-catenin, and p120 catenin, which induce junctional disassembly. Tyrosine phosphorylation of VE-cadherin induces junctional disassembly by preventing interaction with its cytoplasmic binding partners and actin cytoskelton. Phosphorylation of these residues differs according to the inflammatory mediator used. Inhibitors of ROS generations prevent phosphorylation of VE- cadherin (Monaghan-Benson and Burridge, 2009).
The actin cytoskeleton of endothelial cells plays an indispensable role in maintaining endothelial cell morphology, junctional stability, and endothelial motility. Inflammatory agonists and oxidative stress are well-known inducers of actin cytoskeleton reorganization in endothelial cells, leading to junctional opening and gap formation between endothelial cells. Oxidation of actin monomers (G-actin) by H2O2 alters its polymerization ability as well as its interaction with actin regulatory proteins filamin and α-actinin (DalleDonne et al., 1995).
Endothelial barrier integrity is formed by tight cell–cell and cell–matrix adhesions, and it is coupled to cytosolic Ca2+ levels (Mehta and Malik, 2006). Increases in [Ca2+]i induced formation of inter-endothelial cell gaps and vascular hyperpermeability through different signaling pathways (Lum et al., 1989; Sandoval et al., 2001b) Several reports suggest that an
increase in [Ca2+]i leads to activation of Ca2+/calmodulin-dependent myosin light chain
kinase (MLCK), which facilitates reorganization of actin cytoskeleton to induce changes in endothelial cell shape. PKC-α is critical in Ca2+-mediated increase in endothelial permeability (Sandoval et al., 2001a; Sandoval et al., 2001b). In addition, evidence indicates that ROS activates Ca2+ signaling and influences different cellular events which trigger inflammation. An increase in intracellular Ca2+ was associated with enhanced H2O2 generation (Dreher and
Junod, 1995). The converse is also true. H2O2 caused a dose-dependent rise in [Ca2+]i of
endothelial cells (Hecquet and Malik, 2009). ROS-mediated regulation of intracellular free Ca2+ concentration is a major mechanism of increased vascular permeability, the hallmark of inflammation.
1.3.1.3.1 Endothelial Cell Contraction leads to Intercellular Gaps in Venules
Blood vessel walls form a selective barrier to the transport of materials between blood and tissue, and the endothelium contributes significantly to this barrier function. The role of the endothelium is particularly important in thin-walled vessels, such as venules, because during tissue inflammation the endothelial junctions widen in localized areas and gaps form, thus compromising the barrier function (Baldwin and Thurston, 2001). This is the most common form of increased vascular permeability, endothelial cell contraction is a reversible process elicited by histamine, bradykinin, leukotrienes and many other classes of chemical mediators. Cellular contraction occurs rapidly after binding of mediators to specific receptors and is usually short lived (15-30 min). It is known as the immediate transient response. This type of leakage affects venules 20 to 60 µm in diameter, leaving capillaries and arterioles unaffected.
Binding of inflammatory mediators, to their receptors on endothelial cells activates intracellular signaling pathways that lead to phosphorylation of contractile and cytoskeletal proteins. These proteins contract, leading to contraction of the endothelial cells and separation of intercellular junctions (Wang et al., 2014).
1.3.1.3.2 Endothelial Cell Retraction
Retraction of the endothelium due to cytoskeletal and junctional reorganization leading to widened interendothelial junctions. Cell migration and retraction are interrelated activities that are crucial for a range of physiological processes such as wound healing and vascular permeability. A specific protein-phosphatase-1 inhibitor protein (PHI-1) participates in regulatory events at the trailing edge of migrating cells and modulates retraction of endothelial and epithelial cells (Tountas and Brautigan, 2004). Cytokines such as interleukin-
1 (IL-1), tumor necrosis factor (TNF), and interferon-γ (IFN-γ) also increase vascular permeability by inducing a structural reorganization of the cytoskeleton such that the endothelial cells retract from one another. In contrast to immediate transient response by histamine, the cytokine-induced response is somewhat delayed (4 to 6 hours) and long-lived (24 hours or more).
1.3.1.3.3 Direct Endothelial Injury, resulting in Endothelial Cell Necrosis and detachment
This effect is usually encountered in necrotizing injuries and is due to direct damage to the endothelium by the injurious stimulus (Severe burns or lytic bacterial infections) (Wang et al., 2014), but the adherence of neutrophils (PMN) to endothelium is a crucial step in neutrophil-mediated vascular injury. However, vascular injury is not a necessary event in inflammatory states, which suggests that endogenous mechanisms may protect endothelial cells from neutrophil-mediated injury (Render and Rounds, 1988). In most instances, leakage starts immediately after injury and is sustained at a high level for several hours until the damaged vessels are thrombosed or repaired. This reaction is known as the immediate sustained response and all levels of the microcirculation are affected, including venules, capillaries, and arterioles. Endothelial cell detachment is often associated with platelet adhesion and thrombosis.
1.3.1.3.4 Delayed Prolonged Leakage
This is curious but relatively common type of increased permeability that begins after a delay of 2 to 12 hours, lasts for several hours or even days, and involves venules as well as capillaries. Such leakage is caused, by mild to moderate thermal injury, x-radiation, or ultraviolet radiation, and certain bacterial toxins (Clydesdale et al., 2001). Late-appearing sundown is a good example of a delayed reaction. This may result from the direct effect of the injurious agent, leading to delayed endothelial cell damage or the effect of cytokines causing endothelial retraction.
1.3.1.3.5 Leukocyte-Mediated Endothelial Injury
Leukocytes adhere to endothelium relatively early in inflammation. Circulating leukocytes other than neutrophils may also contribute to vascular pathology. Mediators released by these cell types during the process of adherence and emigration may also provoked vascular endothelial injury endothelial or detachment, resulting in increased permeability. Joussen et al., (2001) demonstrated that adherent leukocytes are temporary and spatially associated with retinal endothelial cell injury and death within 1 week of streptozotocin-induced experimental diabetes in rats. Leukocytes adherence to endothelium appears to be necessary for leukocyte- mediated vascular injury to occur. Close approximation of the leukocytes to the endothelium allows short-lived mediators such as oxidants to act. Adherence also produces a protected microenvironment at the cell-cell interface that is inaccessible to plans inhibitors (Shasby et al., 1983). In acute inflammation, this form of injury is largely restricted to vascular sites, such as vanules and pulmonary and glomerular capillaries, where leukocytes adhere for prolonged period to the endothelium.
1.3.1.3.6 Increased Transcytosis across the Endothelial Cytoplasm
Transcytosis occurs across channels consisting of clusters of interconnected, uncoated vesicles and vacuoles called the vasiculovascuolar organelle, many of which are located close to intercellular junctions. Certain factors, for example, vascular endothelial growth factor (VEGF), appear to cause vascular leakage by increasing the number and perhaps the size of these channels.
1.3.1.3.7 Leakage from new Blood Vessels
During repair, endothelial cells proliferate and form new blood vessels, a process called angiogenesis. New vessel sprouts remain leaky until the endothelial cell mature and form intercellular junctions. In addition, certain factors that cause angiogenesis (e.g VEGF) also increased vascular permeability and endothelial cells in foci of angiogenesis have increased density of receptors for vasoactive mediators, including histamine, substance P and VEGF (Hoeben et al., 2004). Three patterns in the time course of vascular permeability occurring at different times after injury have been recognized. These include;
Fluid loss from vessels with increased permeability occurs in distinct phases.
i. An immediate transient response lasting for 30 minutes or less, mediated mainly by the actions of histamine and leukotrienes on endothelium
ii. A delayed response starting at about 3 hours and lasting for 8 hours, mediated by bradykinins, complement products, and other factors released from dead neutrophils in exudates
iii. A prolonged response that is most noticeable after direct endothelial injury, for example after burns.
1.3.2 Cellular Changes (Events)
Leukocyte emigration is essential in inflammation and lymphocyte homing, as a central part of immune surveillance. The emigration of leukocytes requires the interplay of adhesion molecules of the selectin and integrin families and chemokines. Selectin-dependent cell-cell interaction is essential in localizing leukocytes within tissues by promoting the rolling of leukocytes along the endothelial cell surface before development of tight adhesion and subsequent transendothelial migration (Crockett-Toradi, 1995). Adhesion molecules are therefore particularly implicated in a wide variety of pathogenic disorders that involve inflammation (Barreiro and Sanchez-Madrid, 2009).
1.3.2.1 Margination (Tethering, Rolling and Capture of Leukocytes)
To initiate the inflammatory response, circulating leukocytes in the bloodstream have to establish contact (tethering) with the vascular wall and adhere to it, while withstanding the shear forces (Barreiro and Sanchez-Madrid, 2009). Tethering and rolling of the leukocytes over the activated endothelium are the first steps in the sequential process of extravasation. Hematopoietic cells use a multistep process in which they initially tether to and roll along the
vessel wall, then decelerate and arrest, and finally aggregate or emigrate into the underlying tissues. For cells to tether, interactions between adhesion molecules must form rapidly. For cells to roll, these interactions must break rapidly (McEver and Cheng, 2010). The initial contact or tethering is largely mediated by selectins and their ligands and blood flow must be present for it to be efficient (Alon and Ley, 2008). Although selectins and their ligands tend to interact with a variable affinity; the much frequency of association – dissociation of interactions allows them to mediate labile and transient dethers between leukocytes and the endothelium (Mehta et al., 1998). Tethering slows the speed of travel of the leukocytes and allows them to roll over the endothelial surface, favouring subsequent interactions mediated by integrins and their ligands and increasing leukocyte adherence.
The selectins (P, E and L) are type-1 transmenbrance glycoproteins that bind to fucosylated and sialylated hydrocarbons present in their ligands in a Ca2+-dependent fashion. L-selectin is expressed on endothelial cells activated by proinflammatory stimuli (Barreiro and Sanchez- Madoid, 2009). In addition to the interaction of leukocyte selectin (L-selectin) with endothelial selectin (P- and E- selectin), the P-selectin glycoprotein ligand – 1 (PSGL-1) protein is a major ligand of these 3 selectins. The binding of PSGL-1 to P- and E-selectin promotes the interaction of leukocytes with the endothelium, whereas the binding of PSGL-1 to L-selectin enables leukocyte-leukocyte interactions, whereby the adhered leukocytes facilitate the capture of other circulating leukocytes at sites where the endothelium is inflamed, regardless of whether these cells express ligands for endothelial selectins in a process denoted besides the selectins and their ligands, the α4β1- and α4β7- integrins – through their interaction with vascular cell adhesion molecules (VCAM)-1 and mucosal addressin cell adhesion molecule (MAdCAM)-1, respectively–can independently mediate this initial tethering (Alon et al., 1995). The interaction between lymphocyte function associated antigen (LFA)-1 and cell-cell adhesion molecule (ICAM)-1 collaborates with the function of L-selectin, thereby stabilizing the transient contact phase and reducing the rolling velocity. It has been shown that selectins activate multiple signaling pathways that are linked to processes such as actin cytoskeletal reorganization, like the MAPK, Ras, or Rac2 cascade (Eriksson et al., 2001). In addition to PSGL-1, selectins can also bind to other glucoproteins such as CD44 or E-selectin ligand-1 (ESL-1) in the case of E-selectin. Each particular ligand seems to have a distinct function during the process of neutrophil capture. PGSL-1 is primarily implicated in initial leukocyte tethering, whereas ESL-1 is necessary to convert the transient initial tethers into a slower and more stable rolling. Finally, CD44 controls the
rolling velocity and intervenes in the polarization of PPSGL-1 and L-selectin, probably to allow secondary recruitment (Hidalgo et al., 2007). Platelets can also aids as secondary recruiters of leukocytes because of their capacity to interact with both the circulating leukocytes and the endothelium at the same time. In addition, they can release chemokines that are immobilized on the luminal endothelial surfaces, therefore favouring the adhesion process.
1.3.2.2 Activation
Rolling leukocytes are in intimate contact with the endothelium for prolonged periods of time, allowing them to effectively sample the endothelial surface. Activated endothelial cells produce chemoattractants which may be secreted or remain surface bound. These include interleukin-8 (IL-8) and platelet-activating factor (PAF). Stimulation of chemoattractant receptors causes conversion of G-actin to F-actin, making the cell more rigid and enabling it to migrate (Ley, 1996). L-selection is known to be proteolygically lost from the leukocyte surface upon activation (Kishimoto et al., 1989). As tethering to the vascular endothelium occurs, the rolling velocity of the leukocytes slows and they are activated on encountering immobilized chemokines and integrin ligands exposed on the apical endothelial surface (Barreiro and Sanchez-Madrid, 2009). This activation step enables the arrest of leukocytes and their subsequent firm adhesion to the endothelium under physiological flow conditions (Alon et al., 2003). Leukocyte activation implies a marked morphological change: the rounded circulating cell is transformed into a promigratory cell with polarized morphology in which at least 2 regions can be identified, the cell front and the cellular uropod. The polarization of the leukocytes allows the cell to coordinate the intracellular forces to produce the necessary cell crawling during the extravasations process (Geiger and Bershadsky, 2002). The chemokines bound to the glycosaminoglycans of the apical endothelial membrane act by signaling via the G-protein coupled receptors (GPCR) located on the microvilli of the leukocyte, thereby inducing a wide range of “outside in” signals in a fraction of a second. Chemokine signaling through G-protein-coupled receptors on leukocytes activates α1β2 to an extended, high-affinity state that curses firm adhesion to endothelial cells (McEver and Zhu,
2010). IL-8 is a cytokine with powerful chemotactic and activating effects on phagocytes, which has been implicated as a key mediator of many inflammatory disorders (Baggiolini and Clark-Lewis, 1992). The major role of IL-8 is to induce, specifically, the migration of neutrophils to the site of inflammation with the subsequent activation of neutrophils leading
to the generation of O2- and degranulation; processes directly associated with the inflammatory responses (Elahe Crochett-Torabi, 1998). Integrins are fundamental molecules in cell migration. They control the cell-cell and cell-extracellular matrix interactions during recirculation and inflammation (Barreiro and Sanchez-Madrid, 2009). One of their most
important characteristics lies in their ability to alter their adherent activity, regardless of how extensively expressed they are on the membrane. Thus, circulating leukocytes in blood maintain their integrins in an inactive conformation to avoid nonspecific contact with uninflamed vascular walls, but when they arrive at the inflammatory focus, a rapid in situ activation of the integrins occurs (Campbell et al., 1998). Cellular activation via extracellular chemokines causes pre-formed β2 integrins to be released from cellular stores. Integrin molecules migrate to the cell surface and congregate in high-avidity patches. Intracellular integrin domains associated with the leukocyte cytoskeleton, via mediation with cytosolic factors such as talin, α-actinin and vinculin. This association causes a conformational shift in the integrin’s tertiary structure, allowing ligand access to the binding site. Divalent cations
(e.g Mg2+) are also required for integrin ligand binding.
The rolling of leukocytes (Neutrophils) facilitates their contact with chemokines-decorated endothelium to induce activation. Full activation may be a two-step process initiated by specific priming by pro-inflammatory cytokines, such as tumour-necrosis factor-α (TNF-α) and IL-Iβ or by contact with activated endothelial cells followed by an exposure to pathogen- associated molecular patterns (PAMPs), chemoattractants or growth factors (Summera,
2010). Chemokines, which are positively charged molecules, are immobilized on endothelium by binding to negatively charged heparan sulphates, which serve as anchors to prevent the shear forces from washing these molecules away (Massena, 2010). This allows the formation of intravascular chemotactic gradients. Eventually, apically sequestered chemokines are removed by endothelial endocytosis. The activation of G-protein-coupled chemokine receptors on neutrophils induces changes in the conformation of cell surface expressed integrins (inside-out-signalling), which subsequently show higher affinity for their ligands, including cell adhesion molecules (CAMs).
1.3.2.3 Pavementing (Arrest and Firm Adhesion)
After chemokine-induced activation, the conformation of the integrins changes reversibly from the inactive (bent) form to the extended form with intermediate affinity. This event prepares the integrin for binding for its endothelial ligand. The integrins that contain an I-
domain inserted into their α-subunits undergo a subsequent conformational change after binding to the ligand, culminating in the complete activation of the integrin and leukocyte arrest (Cabanas and Hogg, 1993). Therefore, the high affinity conformational state for immediate arrest of the leukocyte on the endothelium requires immobilized chemokines and the integrin ligands. However, the α4 integrins, which contain in I-like domain on the β- chains, can spontaneously interact with their endothelial ligands without a prior chemotactic trigger (Alon et al., 2003). The signaling induced by the binding to the ligand leads to the separation of the cytoplasmic regions of the subunits of the integrin, thereby favouring its association with the cortical actin cytoskeleton. The α4 integrins are basically linked through paxillin while β2 integrins are linked through talin, filamin, and other structural molecules (Barreiro and Sanchez-Madrid, 2009). In addition, the binding to the ligand increases the recruitment of additional integrins to increase the firm adhesion of the leukocyte in conditions of flow stress. This clustering of integrins depends on the release from their tether to the cytoskeleton–a process mediated by protein kinase C (PKC) and calpain–to increase their lateral mobility on the membrane (Stewart et al., 1998). Flow stress also regulates the integrins, reinforcing their bonds and even increasing their affinity (Marschel and Schmid- Schonbein, 2002). The integration of signaling derived from chemokines and the external forces to favour transmigration has been defined as the phenomenon of chemorheotaxis (Cinamon et al., 2001). Firm adhesion of neutrophils (“sticking”) is largely CD18-(β2) dependent (Arfors et al., 1987), although additional mechanisms appear to exist. Neutrophils use both Mac-1 (CD11b/CD18) and LFA-1 (CD11a/CD18) for adhesion (Ley, 1996).
1.3.2.4 Endothelial Transmigration
Leukocyte transendothelial migration is a multistep process coordinated by chemokine receptors, integrins and cell adhesion molecules. The interaction between leukocytes and endothelial cells is accompanied by bidirectional signaling in both cell types, which is initiated following formation of specialized, “docking” structures. This signaling induces a focal and transient loss of endothelial cell-cell adhesion, which is dependent on vascular- endothelial cadherin. GTPases, reactive oxygen species, changes in the actin cytoskeleton and protein tyrosine phosphorylation are implicated in the control of VE-cadherin and associated proteins (Hordijk, 2003). ICAM-1 and VCAM-1 signaling, cytosolic free calcium flux, RhoA and Rac1 activation, VE-cadherin removal from the junction and MLCK activation have all been shown to be necessary for efficient transmigration (Muller, 2009). During
endothelial transmigration, the endothelial junctions are partially dismantled to avoid damage to the monolayer or substantial changes in permeability. Thus, the leukocyte membranes and the endothelium remain in close contact during diapedesis and, afterwards, the endothelial membranes reseal their links. Once the leukocytes have reached an appropriate site for transmigration (preferably the intercellular junctions), they deploy exploratory pseudopods between two adjacent endothelial cells. The pseudopods then transform into a lamella that moves across the open space on the monolayer.
The clustering of ICAM-1 and VCAM-1 on the endothelial cell has been observed as the leukocyte approaches the endothelial cell border (Barreiro et al., 2002; Carman and Springer,
2004). The initial leukocyte-facilitated clustering of ICAM-1 and VCAM-1 requires Src- dependent phosphorylation of the actin-binding protein cortactin, which leads to actin polymerization and recruitment of more ICAM-1 to the site of leukocyte adhesion, which in turn induces more cortactin phosphorylation. Clustering of ICAM-1 and VCAM-1 stimulates signaling in the endothelial cells that promote diapedesis. Clustering of ICAM-1 and VCAM-
1 may occur in three dimensions. So-called docking structures or transmigratory cups are finger-like projections of endothelial apical surface membrane reported to surround the lower portion of adherent leukocytes. The membrane is enriched in ICAM-1 and VCAM-1 and overlies cytoplasm enriched in F-actin and actin-binding proteins.
Evidence shows that loosening the endothelial cell junctions is important for efficient transmigration. Clustering of ICAM-1 and VCAM-1 on endothelial cells transmits a number of signals into the endothelial cell. Cross-linking VCAM-1 and ICAM-1 (van Buul et al.,
2007) on the endothelial cell stimulates an increase in cytosolic free calcium ions, which has long been known to be a requirement for diapedesis (Huang et al., 1993). The increase in cytosolic free calcium ion activates myosin light-chain kinase (MLCK), which leads to actin– myosin fiber contraction. Stimulation of ICAM-1 leads to phosphorylation of VE-cadherin, which is a prerequisite for adherens junction disassembly (Turowski et al., 2008). Cross- linking VCAM-1 also activates Rac1 and stimulates an increase in reactive oxygen species in endothelial cells (Cook-Mills et al., 2004) that leads to loosening of adherens junctions. Under resting conditions, the vascular endothelial protein tyrosine phosphatase (VE-PTP) associates with VE-cadherin via plakoglobin (γ-catenin) and maintains VE-cadherin in a hypophosphorylated state at the junction. Interaction between leukocytes and cytokine- activated endothelial cells triggers rapid dissociation of VE-PTP from VE-cadherin, which
allows it to be phosphorylated on tyrosine, thereby increasing junctional permeability and facilitating TEM (Nottebaum et al., 2008).
1.3.2.5 Leukocyte interstitial migration (Chemotaxis)
After penetration through the endothelial barrier, leukocytes move in the interstitium to the site of injury. Leukocyte interstitial migration is suggested to be of a critical importance not only for tissue injury, but also for reparative events (Ley, 2003). Emigrated immune cells moving within the interstitium are suggested to accomplish leukocyte chemotaxis – directed migration along gradients of chemotactic agents toward their destinations (Snyderman and Goetzl, 1981; Sixt et al., 2006), Leukocyte chemotaxis is dependent on the ability of migrating cells to sense gradients of chemoattractants, serving as a “driving force” for immune cells (Middleton et al., 2002; Moser et al., 2004; Colditz et al., 2007). Several signaling pathways have been proposed to be involved in this gradient-amplification process, the most predominant being the phosphatidylinositol-3 kinase (PI3K) pathway (Heit et al.,
2008). Leukocytes sense the direction of chemotactic gradients, and undergo polarization characterized by the formation of lamellipodia at the leading edge of the cell and a uropod at the trailing edge (Vicente-Manzanares and Sanchez-Madrid, 2000; Franca-Koh and Devreotes, 2004). The intercellular communication in the extracellular matrix (ECM) is mediated by glycosaminoglycans which interact with chemokines using low affinity interaction and, therefore, control the site and duration of soluble chemokine gradient (Patel et al., 2001). In addition, glycosaminoglycans are suggested to be involved in transport, clearance, and degradation of chemokines (Colditz et al., 2007). The effect of chemokines on leukocyte migration is accomplished by triggering GPCRs on the leukocyte surface. The combinatorial effects of multiple chemokines and cytokines affect interstitially migrating leukocytes in a step-by-step manner, whereby cells react to chemotactic signals in their immediate vicinity by directional movement and remodeling of the ECM. Leukocytes moving within the interstitial tissue receive signals from neighboring cells as well as from the ECM, activate intracellular processes, release inflammatory mediators, and upregulate adhesion molecules and enzymes (Ridley, 2001; Friedl et al., 2001). Leukocyte-released proteolytic enzymes (proteases) such as heparase, elastase, and matrix metalloproteinases (MMP-2 and MMP-9) seem to play a critical role for leukocyte interstitial migration. They are required for the degradation of the components of the basement membrane as well as of the ECM and therefore allow leukocyte moving within interstitial microenvironment (Lindner
et al., 2000). The next important issue is the cytoskeleton reorganization during leukocyte interstitial migration. In response to chemoattractants, leukocytes rapidly polarize in the direction of the signal, forming a pseudopod on the side of highest chemotactic concentration and an uropod or posterior domain on the opposite side of the cell; these structures become the leading and trailing edges of the chemotaxing cell, respectively (Charest and Firtel, 2006). This process is mediated by leukocyte cytoskeletal dynamics. Leukocyte interstitial migration is suggested to be of critical importance for the development of an inflammatory response and tissue repair. Chemotaxing cells have an amazing ability to detect small changes in the concentration of a chemoattractant. Several signaling pathways have been proposed to be involved in this gradient-amplification process, the most predominant being the phosphatidylinositol-3 kinase (PI3K) pathway (Li et al., 2000; Sasaki et al., 2000; Stephens et al., 2002). Upon detecting a chemotactic stimulus, cells will activate PI3K in such a way that PI3K is active along the region of the cell facing the chemoattractant. This result in the accumulation of the product of PI3K, phosphatidylinositol triphosphate (PIP3), along the leading edge of the cell (Funamoto et al., 2002; Huang et al., 2003; Merlot and Firtel, 2003; Sasaki et al., 2004; Zhelev et al., 2004). This is followed by the accumulation of proteins containing PIP3 –binding domains, thus recruiting the proteins required to form the leading edge of the migrating cell (Sasaki et al., 2004; Sossey-Alaoui et al., 2005; Van Keymeulen et al., 2006). At the same time, enzymes that mediate the breakdown of PIP3 will be active on the sides and back of the cell, thus limiting PI3K activity to the front of the cell (Funamoto et al., 2002; Wain et al., 2005). This process is believed to be a major mechanism of gradient amplification in migrating cells and has been proposed to be indispensable for chemotaxis to all stimuli.
1.3.2.6 Phagocytosis and intracellular degradation
In the course of inflammation blood phagocytes, neutrophils, eosinophils, and monocytes, have a main function in common: they attack and kill invading microbes and parasites (Baggiolini, 1984). The prey is usually phagocytosed by these cells, i.e. fully enclosed in a vacuole where killing and digestion take place without harm to the surrounding tissues. On interaction with the microorganisms and other particles, the phagocytes suddenly increase their oxygen consumption. This phenomenon is called respiratory burst. Phagocytosis involves three distinct but interrelated steps; (i). Recognition and attachement of bacteria,
(ii).Engulfment, with subsequent formation of a phagocytic vacuole, (iii). Intracellular killing and degradation.
i Recognition and attachment
Phagocytosis of microbes and dead cells is initiated by recognition of the particles by receptors expressed on the leukocyte surface. Mannose receptors and scavenger receptors are two important receptors that function to bind and ingest microbes (Emst, 1998). The mannose receptor is a macrophage lectin that binds terminal mannose and fucose residues of glycoproteins and glycolipids. These sugars are typically part of molecules found on microbial cell walls, whereas mammalian glycoproteins and glycolipids contain terminal sialic acid or N-acetyl-galactosamine. The macrophage mannose receptor recognizes microbes and not host cell. Macrophage scavenger receptors bind a variety of microbes in addition to modified LDL particles (oxidized or acetylated low-density lipoprotein) (Eugene et al., 2000). The efficiency of phagocytosis is greatly enhanced when microbes are opsonized by specific proteins (opsonins) for which the phagocytes express high-affinity receptors. The major opsonins are IgG antibodies, the C3b breakdown product of complement, and plasma lectins.
ii Engulfment
Binding of a particle to phagocytic leukocyte receptors initiates the process of active phagocytosis of the particle. During engulfment, extensions of the cytoplasm (pseudopods) flow around the particle to be engulfed, eventually resulting in complete enclosure of the particle within a phagosome created by the plasma membrane of the cell. The limiting membrane of this phagocytic vacuole then fuses with the limiting membrane of a lysosomal granule, resulting in discharge of the granule’s contents into the phagolysosome. Phagocytosis is dependent on polymerization of actin filaments (Robin and Laura, 1990).
iii Killing and Degradation
The ultimate step of bacterial phagocytosis in the elimination of infectious agents and necrotic cells is their killing and degradation within neutrophils and macrophages, which occur most efficiently after activation of the phagocytes. Microbial killing is accomplished largely by oxygen-dependent mechanisms. Phagocytosis stimulates a burst in oxygen consumption, glycogenolysis, increased glucose oxidation via the hexose-monophosphate shunt, and production of reactive oxygen intermediates (ROS, also called reactive oxygen species) (Baggiolini, 1984). Bacteria phagocytosed by leukocytes are killed and degraded by
toxic oxygen metabolites produced in the phagosome via an NADPH oxidase (Baggiolini,
1984). NADPH oxidase activity is regulated by small GTP-binding proteins in response to phagocytic stimuli.
1.3.2.7 Regulation of antioxidant defense systems in inflammation
To prevent the damaging effects of oxidants, vertebrate cells have evolved an array of antioxidant defense systems that functions to remove free radicals generated during phagocytosis. The antioxidant enzymes SOD (dismutates O2•− to H2O2), catalase, glutathione
peroxidase (GPx) converts H2O2 to H2O (Handy and Loscalzo, 2012). Thus, cells experience
an oxidative stress when the capacity of antioxidant enzymes is overcome by enhanced oxidant production. There are three different isoforms of SOD expressed in mammalian cells: SOD1, also known as CuZn SOD expressed in the cytosol and nucleus; SOD2 or MnSOD expressed in mitochondrial matrix and extracellular (EC-SOD); and a plasma membrane- associated enzyme which has four Cu atoms per molecule. The EC-SOD isoform, although
plasma-membrane associated, is a secreted glycoprotein that scavenges extracellular O2•−
(Handy and Loscalzo, 2012). Catalase a cytoplasmic protein is an important intracellular antioxidant enzyme that detoxifies H2O2 to oxygen and water. The expression of catalase has been reported in alveolar type II cells and macrophages, and highest expression has been reported in the liver and erythrocytes (Rahman et al., 2006). Targeting of catalase directly to the mitochondria in lung epithelial cells has been shown to protect them from H2O2-induced apoptosis (Arita et al., 2006). Moreover, the LPS treatment decreased the expression and activity of catalase in mouse lung, a response preceding NF-κB activation (Clerch et al.,
1996). The congenital deficiency of catalase known as acatalasia is benign. The family of GPx enzymes serves the similar function of detoxifying H2O2 as catalase. There are four selenium-dependent GPx enzymes (GPx1–4) in mammalian tissue with a wide tissue distribution (Margis et al., 2008). GPx enzymes are tetrameric protein and carry four atoms of selenium bound in their catalytic core. These enzymes detoxify H2O2 by oxidizing monomeric glutathione (GSH) into dimeric glutathione disulfide (GSSG). Oxidized GSSG is converted back to its monomeric GSH form by glutathione reductase. The expression and activity of GPx is induced by hyperoxia in endothelial cells (Jornot and Junod, 1992). Peroxiredoxins are a group of related antioxidant enzymes that catalyze the degradation of H2O2 to water. There are six different (Prx1–6) identified so far with molecular weights
ranging between 17 and 31 kD (Ishii et al., 2012). All subtypes of Peroxiredoxins are expressed in lung tissue (Ishii et al., 2012).
1.4 Mediators of Inflammation
Soluble mediators of inflammatory cells
During both acute and chronic inflammation processes, a variety of soluble factors are involved in leukocytes recruitment through increased expression of cellular adhesion molecules and chemoattraction. Many of these soluble mediators regulate the activation of the resident cells (such as fibroblast, endothelial cells, tissue macrophages and mast cells) and the newly recruited inflammatory cells (such as monocytes, lymphocytes, neutrophils, basophils and eosinophils) and some of these inflammation mediators result in the systemic responses to the inflammatory process (e.g. fever, hemodynamic effects, leukocytosis). The soluble factors that mediate these inflammatory responses fall into four main categories;
1. Inflammatory lipid metabolites such as platelet activating factor (PAF) and the numerous derivatives of arachidonic acid (Prostaglandins, Lukotrienes, Lipoxins) which are generated from cellular phospholipids (Amin, 2012).
2. Four Cascades of soluble proteases/substrates (clotting, complement and kinins, Immunoglobin) which generate numerous pro-inflammatory peptides.
3. Nitric Oxide, a potent endogenous vasodilator.
4. A group of cell-derived polypeptides known as cytokines, which to a larger extent orchestrate the inflammatory response (Feghali and Wright, 1997).
1.4.1 Eicosanoids
As major constituents of biological membranes, phospholipids play a key role in all living cells. The two principal groups of phospholipids are the glycerophospholipids (glycerophosphatides) which contain the alcohol glycerol and sphingophospholipids which contain the alcohol sphingosine. Arachidonic acid and other polyenoic acids are present in relatively small amounts (e.g, ~1% of total plasma fatty acids), but they are concentrated in the sn-2 position of phospholipids. Chemical, toxins, thermal, physical, hormonal, radiation and neural stimuli activate Phospholipase A2 to cleave arachidonic acid from phospholipid of the membrane lipid to give free arachidonic acid and lysophospholipid. This activation occurs when a variety of stimuli (agonists), such as histamine and cytokines, interact with a specific
plasma membrane receptor on the target cell surface. Prostanoids are biological active lipids (products) that are derived from 20-carbon polyunsaturated essential fatty acids (Eicosanoids). Prostaglandins contain a cyclopentane ring and two side chains named α and ω attached to the ring. According to the modifications of this cyclopentane ring, they are classified into types A to I, in which types A, B, and C, are believed not to occur naturally but are produced only artificially during extraction procedures. Prostaglandins G and H share the same ring structure but differs at C-15, having a hydroperoxy and hydroxyl group, respectively. Another cyclooxygenase product, thromboxane A2, has an oxane ring instead of the cyclopentane ring. Prostanoids are among the most potent regulators of cellular function in nature and are produced by almost every cell in the body (Tilley et al., 2001). As paracrine hormones they exert effects only within/at the point of synthesis or close adjacent cells and are easily deactivated.
Eicosanoids are classified into three different series: eicosatrinoic acid (α-linolenic acid) 1 series wih three double bonds, eicosatetranoic acid (arachidonic acid) 2 series with four double bonds and eicosapentanoic acid (Omega 3 fatty acid) 3 series with five double bonds. Prostaglandins and thromboxane A2, collectively termed prostanoids, play a key role in the generation of the inflammatory response. Their biosynthesis is significantly increased in inflamed tissue, and they contribute to the development of the cardinal signs of acute inflammation (Tilley et al., 2001). The most typical actions are the relaxation and contraction of various types of smooth muscles. They also modulate neuronal activity by either inhibiting or stimulating neurotransmitter release, sensitizing sensory fibers to noxious stimuli or inducing central actions such as fever generation and sleep induction (Kluger, 1991). Prostaglandins also regulate secretion and motility in the gastroinstestinal tract as well as transport of ions and water in the kidney. Prostanglandin production depends on the activity of PGG/H synthases, known as cyclooxygenases (COXs), bifunctional enzymes that contain both COX and peroxidase activity and that exist as distinct isoforms referred to as COX-1, COX-2 and COX-3 (Smith et al., 2000; Chandrasekharan et al., 2002). Cyclooxygenase enzyme was found to exist as three distinct isoforms, designated COX-1, COX-2 and COX-3. COX-1 is regarded as a constitutive form of the enzyme, widely expressed in almost all tissues, and involved in the production of prostaglandins and thromboxanes for “normal” physiologic functions. COX-2 is an inducible form of the enzyme regulated by a variety of cytokines and growth factors (Smith et al., 2000; Chandrasekharan et al., 2002). COX-2 mRNA and protein level are usually low in most healthy tissue, but are expressed at high
levels in inflamed tissue. COX-1, expressed constitutively in most cells, is the dominant source of prostanoids that subserve these functions, such as gastric epithelial cytoprotection and homeostasis (Dubois et al., 1998). COX -2, induced by inflammatory stimuli, hormones, and growth factors, is the more important source of prostanoid formation in inflammation and in proliferative diseases, such as cancer (Dubois et al., 1998). In addition to serving as a substrate for the cyclooxygenase pathway, arachidonic acid also acts as a substrate for the lipoxygenase pathway. The lipoxygenase enzymes catalyse the incorporation of an oxygen molecule onto a carbon of one of several double bonds of arachidonic acid, forming a hydroperoxy (-OOH) group at these positions (Funk, 2006). With this oxygenation, the double bond isomerizes to a position one carbon removed from the hydroperoxy group and is transformed from the cis to the trans configuration. The unstable hydroperoxy group is then converted to the more stable hydroxyl group. Soy bean lipoxygenase is a member of a family of related lipoxygenases that are found in all eukaryotes. All appear to have similar iron- or manganese-containing active sites. The synthesis of the leukotrienes begins with the formation of 5-Hydroperoxy- 6, 8, 11, 14 -eicosatetraenoic acid [5-HPETE] (Funk, 2006). Some of these peroxides have physiological effects of their own, but they are largely transformed by peroxidases to more stable compounds such as the corresponding alcohols [Hydroxy-eicosatetraenoic acids or HETE’s]. Leukotrienes are formed from 5-HPETE. Dehydration of HPETE produces the unstable epoxide leukotriene A4, which can be hydrolyzed enzymatically by leukocytes to the diol leukotriene B4 (Back, 2009). The second metabolic pathway involves the addition of reduced glutathione to carbon 6 to form LTC4, a reaction catalyzed by glutathione S-transferase. Glutamate is removed from the glutathione moiety of LTC4 through the action of γ-glutamyl transpeptidase to form LTD4. A dipeptidase cleaves the glycine residue from LTD4 to form LTE4 (whereas removal of the glycine is hydrolytic).
1.4.2 Platelet Activating Factor
Platelet-activating factor is a family of structurally related phospholipids mediators (PAF, 1- O-alkyl-2-acetyl-sn-glycero-3-phosphoryl-choline) which possesses a wide spectrum of potent pro-inflammatory actions and significant mediator of the immune system (McManus and Pinckard, 1985). PAF is unique in its role as a phospholipid that functions as an intercellular mediator, and it may also function as an intracellular messager (Venable et al., 1993). These unique acetylated phospholipids are frequently synthesized in concert with pro-
inflammatory lipid mediators derived from arachidonic acid. Platelet-activating-factor (PAF) is one of the most potent and versatile mediator found in mammals. Numerous cell types and tissues have been shown to produce PAF upon appropriate stimulation (Braquet et al., 1989). In particular, PAF is produced by a variety of cells that may participate in the development of inflammatory reaction such as monocytes/macrophages, ploymorphonuclear neutrophils (PMN), eosinophils, basophils and platelets (Bratton and Henson, 1989). In addition, human endothelial cells were found to produce PAF after stimulation by several inflammatory mediators including thrombin, angiotensin, vasopressin, leukotrienes C4 and D4, histamine, bradykinin, elastase, cathepsin, hydrogen peroxide, plasmin, interleukin-8, Il-1α, or TNF-α (Camussi et al., 1983). Most of the cells that produce PAF also possess PAF receptors and are target for PAF action. In vitro, PAF promotes the aggregation, chemotaxis, granule secretion and oxygen radical generation from leukocytes and the adherence of leukocytes to the endothelium (Camussi et al., 1981). PAF increases the permeability of endothelial cell monolayer (Bussolino et al., 1987), stimulates the contraction of smooth muscle (Kester et al., 1984). PAF can be synthesized by two distinct routes: (i). The remodeling and (ii). The de novo pathways, by a variety of cell types, including macrophages, endothelial cells, neutrophils, basophils, eosinophils, lymphocytes, mast cells, platelet and fibroblasts (Prescott et al., 2000), many of which are central to the inflammatory and homeostatic systems. That is, enzymes involved in the PAF biosynthetic pathway are common to diverse cell types which initiate or sustain acute and chronic inflammation. The actions of PAF are abolished by hydrolysis of the acetyl residue, a reaction catalysed by PAF acetylhydrolase (an anti- inflammatory enzyme). There are at least two forms of this enzyme-one intracellular and another that circulates in plasma and is likely to regulate inflammation.
1.4.3 Nitric oxide
Nitric oxide (NO) plays an important role in mediating many aspects of inflammatory responses. NO is an effector molecule of cellular injury, and can act as an antioxidant. It can modulate the release of various inflammatory mediators from a wide range of cells participating in inflammatory responses (e.g., leukocytes, macrophages, mast cells, endothelial cells, and platelets). It can modulate blood flow, adhesion of leukocytes to the vascular endothelium and the activity of numerous enzymes, all of which can have an impact on inflammatory responses. Vasodilation is one of the cardinal signs of an inflammatory response, and it is produced to a large extent via a NO-dependent process. Various
inflammatory mediators, such as bradykinin and histamine, produce vasodilation through stimulation of endothelial release of NO. NO can diffuse out of both the luminal and abluminal sides of the endothelial cell. The NO that diffuses to the vascular smooth muscle activates soluble guanylate cyclase, leading to increased intracellular cGMP levels and, in turn, to relaxation of the smooth muscle (Katsuki et al., 1977; Furchgott and Zawadski, 1980; Ignarro et al., 1987; Palmer et al., 1987). The NO that diffuses into the blood vessel is rapidly inactivated, primarily through an interaction with oxyhemoglobin (Moncada et al., 1991). The half-life of NO in the blood is usually only a few seconds, but can be extended significantly by superoxide dismutase, indicating that NO is also inactivated through an interaction with superoxide anion (Gryglewski et al., 1986). The actions of NO on vascular permeability appear to be predominantly anti-inflammatory; that is, NO diminishes endothelial permeability. NO donors have been found to reduce edema formation in various experimental models, while inhibitors of NO synthesis can exacerbate edema formation (Hinder et al., 1999; Mundy and Dorrington, 2000; Persson et al., 2003). NO appears to play an important role in regulation of P-selectin ex-pression. NO reduces P-selectin expression, while inhibitors of NO synthase elicit an increase in P-selectin expression, and a corresponding increase in leukocyte adherence to the endothelium (Kubes et al., 1991; Armstead et al., 1997). Down-regulation of P-selectin expression by NO is mediated via soluble guanylate cyclase/cGMP (Ahluwalia et al., 2004). Infiltration of leukocytes to a site of injury or infection is a hallmark feature of inflammation. NO has been shown to inhibit the expression of the adhesion molecules on neutrophils (Banick et al., 1997). Inhibition of NO synthesis results in a marked increase in leukocyte adherence to the endothelium (Kubes et al., 1991), while adherence of leukocytes to the vascular endothelium in response to stimulation with a chemotactic factor can be markedly suppressed by NO donors (Wallace et al., 1994, 1997, 1999, 2002; Davies et al., 1997). NO can also down-regulate neutrophil aggregation and secretion (May et al., 1991), and may protect the neutrophil from damage induced by the potent reactive oxygen metabolites that it is capable of producing (Rubanyi, et al., 1991). When activated by exposure to antigens, bacterial products, or a variety of other factors, mast cells release numerous chemical signals including histamine, serotonin, platelet- activating factor (PAF), leukotrienes, tumour necrosis factor (TNF), heparin, and prostaglandins. Thus, mast cells can play a very important role in coordinating an inflammatory response. Mast cell reactivity appears to be carefully regulated by NO, either coming from another cellular source, or from the mast cell itself. The rate of release of NO
from mast cells can be rapidly up-regulated by stimulation with interleukin-1 (Hogaboam, et al., 1993). Interestingly, NO produced by the mast cell appears to down-regulate the release of a number of other inflammatory mediators from these cells, including histamine, PAF, and TNF (Salvemini et al., 1990; Bissonnette et al., 1991; Masini et al., 1991; Hogaboam et al., 1993; Van Overveld et al., 1993). The ability of platelets to adhere to the vascular endothelium and to aggregate is under the control of many soluble mediators, including NO. Thus, NO acts to down-regulate platelet aggregation and adherence, and therefore play an important role in down-regulating inflammatory processes. NO has been reported to be a free radical scavenger (Kanner et al., 1991). For example, the ability of NO to scavenge
superoxide anion (O2-) has been documented both in vitro (Rubanyi et al., 1991) and in vivo
(Gaboury et al., 1993). The antioxidant capacity of plasma was found to be doubled by the administration of NO donors. NO can inhibit transcriptional events by inhibiting the transcription factor NF-kB (Katsuyama et al., 1998). This has been suggested to be an important mechanism underlying the antiinflammatory actions of some NO-releasing drugs (Fiorucci et al., 2002). Likewise, interaction of NO with the glucocorticoid receptor appears to contribute to enhanced antiinflammatory effects of some NO donating drugs (Paul-Clark et al., 2003).
1.4.4 Histamine -(4-imidazolyl)-ethylamine]
Histamine [2-(4-imidazolyl)-ethylamine] is an endogenous short-acting biogenic amine synthesized from the basic amino acid histidine through the catalytic activity of the rate- limiting enzyme histidine decarboxylase and widely distributed throughout the body (Dy and Schneider, 2004). One of the first described functions was its ability to mimic anaphylaxis and has since been demonstrated to play a major role in inflammatory processes. The classical source of histamine is the pluripotent heterogeneous mast cell (MC) (Riley and West, 1952), where it is stored in cytosolic granules and released by exocytosis to exert its immunomodulatory role in response to various immunological and non-immunological stimuli, including allergens, drugs, mechanical stimulation, cold, ultra violet rays and endogenous polypeptides such as substance P and bradykinin (Kakavas et al., 2006; Krishnaswamy et al., 2006). Non-mast cell histamine is derived from numerous sources, indicative examples being gastric enterochromaffin-like cells (Prinz et al., 2003), various types of blood cells, such as basophils (Falcone et al., 2006), macrophages, lymphocytes and platelets, neurons (Arrang et al., 1983; Haas et al., 2008), chondrocytes (Maslinska et al.,
2004) and tumours (Falus et al., 2001). In the gastric mucosa, enterochromaffin-like cell- derived histamine acts as a paracrine stimulant to control acid secretion in response to hormonal and neural stimuli (Prinz et al., 2003; Grandi et al., 2008). In the brain, histamine is synthesized exclusively in histaminergic neurons of the tuberomamillary nucleus of the posterior hypothalamus that project all over the central nervous system (Haas and Panula,
2003). In a mutual interaction network with other transmitter systems, brain histamine is implicated in basic homeostatic and higher brain functions, including sleep–wake regulation, circadian and feeding rhythms, immunity, learning and memory (Haas et al., 2008). Thus, in addition to the predominately H1 receptor-mediated actions on smooth muscle, vascular permeability and modulation of allergic responses, the main functions of histamine include gastric acid secretion basically via H2 receptors (Black et al., 1972), neurotransmission in the central nervous system largely via H3 receptor signalling (Arrang et al., 1983; Haas et al.,
2008) and modulation of immune system processes through the H1 receptor and the recently
identified H4 receptor (Oda et al., 2000; Liu et al., 2001a). H4 receptor activation mediates chemotaxis and intracellular Ca2+ mobilization in murine MCs, without affecting degranulation, thus providing a mechanism for the selective recruitment of these effector cells into the tissues and the amplification of the histamine-mediated reaction eventually leading to chronic allergic inflammation (Hofstra et al., 2003).
1.4.5 Neuropeptides
Successful repair of injured tissues requires diverse interactions between cells, biochemical mediators, and the cellular microenvironment (Barbul, 1990). Neurogenic stimuli profoundly affect cellular events that are involved in inflammation, proliferation, and matrix, as well as cytokine and growth factor synthesis. Immune cells regulated by neuropeptides include lymphocyte subsets, macrophages, and mast cells. In addition, neuropeptides may affect the proliferative and synthetic activity of epithelial, vascular, and connective tissue cells. Furthermore, a close interaction between the nervous and the immune systems has become obvious (Payan, 1989). The peripheral nervous system (PNS), acting through neuropeptides, not only relays sensory information to the central nervous system (CNS) but also plays an effector role in the inflammatory, proliferative, and reparative processes after injury. These effects range from growth factor and cytokine responses to control of local blood flow. Neuropeptides mediate many of the actions important in tissue–nervous system
communication. It has been recognized that stimulation of afferent nerve fibers is associated with peripheral inflammatory responses such as vasodilation and plasma extravasation. This observation has led to the notion that afferent neurons not only serve a sensory role but also take part in local effector systems that are involved in inflammatory responses to tissue irritation and injury (Holzer, 1988). The hypothesis that neuropeptides act as a link between the immune and nervous systems has been supported by the demonstration of (1) a direct peptidergic innervation of primary and secondary lymphoid organs, (2) a close proximity between sensory nerve endings and immune cells, and (3) specific neuropeptide receptors on immune effector cells (Felten et al., 1985; Payan, 1989). It has become clear that neuropeptides are capable of interacting with virtually all components of the immune system. A host inflammatory response is necessary to orchestrate tissue repair following injury (Guirao and Lowry, 1996). There is increasing evidence that neuropeptides participate in many of the inflammatory processes that are crucial for normal wound healing. Like other secretory proteins, neuropeptides or their precursors are processed in the endoplasmatic reticulum and then move to the Golgi apparatus to be processed further. They leave the Golgi apparatus within secretory granules and are transferred to terminals by fast axonal transport (Jessell et al., 1991). In the PNS, neuropeptides occur in the perivascular terminals of noradrenergic (sympathetic) and cholinergic nerve fibers, as well as in the free nerve endings of primary afferent neurons (Holzer, 1988). Numerous neuropeptides are localized in nociceptive afferent nerve fibers, including thinly myelinated Aδ pain fibers and unmyelinated C fibers (Hokfelt et al., 1994). Antidromic stimulation of these fibers induces the release of the stored neuropeptides, resulting in vasodilation, increased vascular permeability, and edema (neurogenic inflammation) (Kenins, 1981; Holzer, 1988). These effects are not only restricted to the point of the initial stimulus but also can be observed in the surrounding area, indicating that the nerve impulses travel not only centrally but at the collateral branches they also pass antidromically to unstimulated nerve endings to cause release of neuropeptides (axon reflex) (Holzer, 1988).
1.4.6 Serotonin or 5-Hydroxytryptamine (5-HT)
Serotonin or 5-Hydroxytryptamine (5-HT) is a monoamine neurotransmitter. Biochemically derived from tryptophan, serotonin is primarily found in the gastrointestinal (GI) tract, platelets, and in the central nervous system (CNS) of humans and animals. Approximately 80
percent of the human body’s total serotonin is located in the enterochromaffin cells in the gut, where it is used to regulate intestinal movements (Harvey 2003). The remainder is synthesized in serotonergic neurons in the CNS where it has various functions. These include the regulation of mood, appetite, sleep, as well as muscle contraction. Serotonin plays a very significant role in cognitive functions, including learning and memory (Harvey 2003; Williams et al. 2002) and in the deficits in attention and associative processes seen in schizophrenia (Meltzer 1999). Modulation of serotonin at synapses is thought to be a major action of several classes of pharmacological antidepressants. Serotonin secreted from the enterochromaffin cells eventually finds its way out of tissues into the blood, where it is actively taken up by blood platelets, which store it. When the platelets bind to a clot, they disgorge serotonin, where it serves as a vasoconstrictor and helps to regulate hemostasis and blood clotting. Serotonin also is a growth factor for some types of cells, which may give it a role in wound healing. Serotonin is mainly metabolized to 5-HIAA, chiefly by the liver. Metabolism involves first oxidation by monoamine oxidase (MAO) to the corresponding aldehyde. This is followed by oxidation by aldehyde dehydrogenase to 5-HIAA, the indole acetic acid derivative. The latter is then excreted by the kidneys. In addition to animals, serotonin is also found in fungi and plants. Serotonin’s presence in insect venoms and plant spines serves to cause pain, which is a side effect of serotonin injection.
1.4.7 Substance P
Substance P (SP), an 11–amino acid peptide, is a member of a family of structurally related peptides called tachykinins, which are characterized by a conserved carboxyl terminal sequence of Phe-X-Gly-Leu-Met-NH2 (in the mammalian forms of these peptides, X represents Phe or Val) (Maggi et al., 1993). Substance P is present in many areas of the CNS and PNS. In the periphery, SP is located especially in areas of immunologic importance, such as the skin, gastrointestinal tract, and respiratory tract (Pernow, 1983). Substance P is synthesized in the dorsal root ganglia, from which it migrates centrally to the dorsal horn of the spinal cord and peripherally to nerve terminals of sensory neurons (Barber et al., 1979). The tachykinins bring about their actions mainly by activating 3 primary types of receptors: NK1, NK2, and NK3. All 3 receptors are members of the superfamily of receptors coupled to G-regulatory proteins. Receptor stimulation leads to the activation of phospholipase C and
thus to the generation of inositol triphosphate and diacylglycerol and to the release of Ca2+ from internal stores (Krause et al., 1992; MacDonald et al., 1996). Substance P and other tachykinins are able to cause vasodilation because of direct actions on vascular smooth muscle and enhanced production of nitric oxide by the endothelium (Hokfelt et al., 1975; Bolton and Clapp, 1986). In addition, SP can initiate increased vascular permeability and protein extravasation after tissue injury (Lundberg et al., 1983; Devillier et al., 1986). Many of the inflammatory actions of SP, such as plasma leakage, are mediated by NK1 receptors, which are rapidly desensitized after exposure to agonists and then gradually become resensitized (Baluk et al., 1994). The receptors are internalized after ligand binding, which may be a limiting factor in the inflammatory response.
1.4.8 Calcitonin Gene-Related Peptide
Calcitonin gene-related peptide (CGRP), a 37–amino acid peptide, is known to exist in 2 forms, α and β. In humans, they differ from each other by 3 amino acid residues. Binding sites for CGRP with properties consistent with those of receptors are present in central and peripheral tissue. Stimulation of CGRP receptors in various cells and tissue has been shown to increase intracellular cyclic adenosine monophosphate concentration and to activate adenylate cyclase (van Valen et al., 1990). Pharmacologically, a division into CGRP1 and CGRP2 receptor subtypes has been proposed (Poyner, 1995; Hall et al., 1996). Peripheral secretion of CGRP causes prolonged increases in blood flow (Brain et al., 1985). Unlike SP, CGRP is not capable of enhancing vascular permeability on its own but potentiates the protein extravasation induced by tachykinins (Gamse and Saria, 1985; Louis et al., 1989).
1.4.9 Sphingosine 1-phosphate (S1P)
Sphingosine 1-phosphate (S1P) is a lipid mediator produced from sphingomyelin by the sequential enzymatic actions of sphingomyelinase, ceramidase, and sphingosine kinase. S1P is enriched in blood and lymph but is present at much lower concentrations in interstitial fluids of tissues. This vascular S1P gradient is important for the regulation of trafficking of various immune cells. S1P also plays critical roles in the vascular barrier integrity, thereby regulating inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Among all sphingolipids, S1P is the most potent intercellular signaling molecules. S1P is enriched in blood and lymph in the submicromolar range, whereas it is much lower in interstitial fluids of tissues, creating a steep S1P gradient (Hla et al., 2008). This vascular S1P gradient is utilized to regulate trafficking of immune cells such as lymphocytes, hematopoietic progenitor cells, and dendritic cells. Since half-life of S1P when bound to albumin is <15 min in plasma (Venkataraman et al., 2008), it is likely that S1P is continuously produced to maintain the high concentration in blood and lymph. Platelets store S1P abundantly and have been regarded as a main source of plasma S1P (Yatomi et al., 1997). Recent studies have shown that erythrocytes play important roles in the maintenance of S1P concentration in plasma (Pappu et al., 2007; Hanel et al., 2007). The vascular endothelial cells also contribute to plasma S1P levels and that S1P production is stimulated by fluid shear stress in endothelial cells (Venkataraman et al., 2008). The main pathway for S1P production is from sphingomyelin hydrolysis. Sphingomyelin is sequentially converted to ceramide, sphingosine, and S1P by the action of sphingomyelinase, ceramidase, and sphingosine kinase, respectively. Sphingosine kinases (SphK) phosphorylation of sphingosine to produce S1P, Importantly, enzymatic activity of SphK1 and SphK2 are regulated by extracellular stimulus. SphK1 is also activated by tumor necrosis factor-α (TNF-α) stimulation in a mechanism dependent on TNF receptor-associated factor 2 (TRAF2) (Xia et al., 2002), and knockdown studies have implicated that SphK1 mediates the effects of TNF-α on induction of cyclooxygenase-2 (COX-2) and adhesion molecules, production of prostaglandins, and activation of endothelial nitric oxide synthase (eNOS) (Pettus et al.,2003; De Palma et al., 2003). It seems that many extracellular stimulus including growth factors and pro- inflammatory mediators converge on SphK1 as an intracellular target and exert their effects via subsequent S1P production. S1P is dephosphorylated to sphingosine either by a family of lipid phosphate phosphatases, LPP1-3, at the cell surface, or by S1P specific phosphatases, SPP1 and SPP2, at the endoplasmic reticulum. This is thought to be the primary mechanism by which extracellular S1P signaling is attenuated (Pyne et al., 2005). An alternative pathway to regulate S1P levels is irreversible degradation of S1P to phosphoethanol amine and hexadecenal by S1P lyase (SPL), which serves as the final degradation of sphingolipids species (Ikeda et al., 2004). As mentioned above, S1P is maintained rich in blood and lymph and low in tissues, creating a vascular S1P gradient (Hla et al., 2008). This marks a sharp contrast with most of pro-inflammatory mediators such as eicosanoids and cytokines that are acutely produced upon stimulation. However, disturbances of the vascular S1P gradient by local exaggerated production of S1P or by ectopic S1P increase in tissues make S1P exert both pro- and anti-inflammatory actions in various types of cell, including endothelial cells, smooth muscle cells, fibroblasts, monocyte/macrophages, mast cells, lymphocytes, and so on. Platelets contain high concentrations of S1P because platelets have constitutive SphK activity, while they lack S1P lyase activity (Yatomi et al., 1995; Yatomi et al., 1997; Ikeda et
al., 2004). In fact, several reports showed that platelets secreted S1P upon stimulation, such as thrombin, collagen, and phorbol ester (Yatomi et al., 1995; Yatomi et al., 1997). Very high concentration of S1P (>10 μM) was shown to induce platelet activation such as intracellular calcium increase, shape change, and aggregation (Yatomi et al., 1995), but physiological range of S1P does not activate platelets. Important events to maintain vascular integrity are rearrangements of cytoskeleton and assembly of adherens junctions in endothelial cells. The role of S1P3 in endothelial barrier integrity is complex and may be context dependent. Recent studies showed that silencing of S1P3 receptor attenuated increases of vascular permeability by edemagenic stimulus (Singleton et al., 2006; Singleton et al., 2007). Activation of S1P3 leads to Rho activation through coupling with G12/13 dominant activation of Rho leads to stress fiber formation and disruption of adherens junction (Spindler et al., 2010). Similarly, activation of S1P2 promotes Rho-dependent stress fiber formation and disruption of adherens junction that increases vascular permeability (Sanchez et al., 2007). Mast cells are tissue- resident cells that are important for both innate and acquired immunity. Mast cells play a critical role especially in allergy and anaphylaxis. Antigen engagement of the high-affinity Fc receptor for IgE (FcεRI) on mast cells results in activation of SphK1 and SphK2 and subsequent production of S1P. SphK2 is predominantly responsible for S1P production in mast cells dependent on activation of non-receptor tyrosine kinase.
1.4.10 Endocannabinoids
Endocannabinoids (eCBs) are lipid mediators that exert most of their functions by binding and activating two G protein-coupled receptors, cannabinoid receptor 1 (CB1) and 2 (CB2). The two main eCBs are arachidonic acid derivatives, the N-acylethanolamine N- arachidonoylethanolamine (anandamide) and the monoacylglycerol 2-arachidonoylglycerol (2-AG). Their production from cell membrane lipid precursors is activity-dependent, and their actions are terminated via hydrolysis by specific lipases such as FAAH, MAGL, or ABHD6. CB2 receptor-dependent anti-inflammatory therapeutic effects have also been observed in other conditions such as sepsis (Tschop et al., 2009). More recently, the CB1 receptor antagonist rimonabant (SR141716) has been shown to provide anti-inflammatory protection against atherosclerosis (Dol-Gleizes et al., 2009). The endocannabinoid system may modulate the inflammatory activity of mononuclear immune cells such as monocytes and macrophages (Han et al. 2009). These cells are major players in the development and progression of atherosclerosis (Glass and Witztum, 2001), and Han et al. (2009), show that cannabinoid receptor expression on these cells is regulated in a species-specific manner. Importantly, the differential expression of CB1 and CB2 receptors on mononuclear cells is functionally significant. The ratio of CB1:CB2 receptor expression regulates the inflammatory activity of these cells and their ability to produce reactive oxygen species. Inhibition of human mononuclear cells activity of CB1 receptors together with a selective up- regulation of CB2 receptor activity markedly attenuates the inflammatory response. This combined modulation of CB1 and CB2 receptors in macrophages may therefore be an important novel therapeutic approach to treat inflammatory disease. With respect to the role of endocannabinoid signalling in macrophages for the development and progression of atherosclerosis, it is noteworthy that selective blockade of the CB1 receptor has also been shown to be beneficial via reducing the accumulation of oxidized low-density lipoproteins in these cells (Jiang et al., 2009). Historically, due to its psychotropic effects, the endocannabinoid system has been considered to be primarily involved in signal transduction of the central nervous system. Within the past decade, however, it has been appreciated that this system also plays a major physiological regulatory role in other peripheral tissues and organs (DiMarzo and Petrosino, 2007). Endocannabinoids are endogenous lipid mediators that are released immediately at the site of their biosynthesis. Under physiological conditions, they are rapidly inactivated via degradation by a number of specific metabolic enzymes (Alexander and Kendall, 2007). The high complexity of the endocannabinoid system has been further revealed by the identification of endocannabinoid-like molecules that have effects partially overlapping with those of endocannabinoids. In addition, a number of atypical cannabinoid receptors have been characterized whose significance still needs to be elucidated in further detail (Alexander and Kendall, 2007). An important aspect of conflicting regulatory pathways of the endocannabinoid system under pathological conditions is emerging from recent studies in experimental disease models. Observations from these studies appear to be highly relevant for future clinical applications, because independent groups have demonstrated that pharmacological approaches that modulate the same specific targets of the endocannabinoid system can have both positive and negative effects in comparable disease models (DiMarzo, 2008). As an example, it has been shown in inflammatory disease models that the up- or down-regulation of a particular pathway of this signalling system may be pro- or anti-inflammatory under comparable conditions (DiMarzo,
2008). These contradictory findings, which may be explained by a significantly altered endocannabinoid metabolism in a pathophysiological environment, add another level of
complexity to this system and give a preview of future challenges in potential therapeutic applications. Despite these conflicting findings, a number of promising pharmacological compounds have been characterized in recent years that function both as agonists and antagonists of CB1 and CB2 receptors (Pacher et al., 2006). Difficulties that may be encountered when applying such compounds in a clinical setting have been demonstrated for the CB1 receptor antagonist rimonabant. Clinical trials in which rimonabant has been applied for the treatment of patients with atherosclerosis have recently been stopped because of serious adverse psychiatric effects.
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