BIOCHEMICAL CHARACTERIZATION OF THE ANTIINFLAMMATORY CONSTITUENT AND ANTIOXIDANT ACTIVITY STUDIES ON STEM BARK EXTRACT OF CRATAEVA ADANSONII

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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