ANTI-INFLAMMATORY AND ANALGESIC ACTIVITIES OF CIPROFLOXACIN LINCOMYCIN AND ERYTHROMYCIN IN WISTAR RATS

ABSTRACT

The  effect   of  three  antibiotics   (ciprofloxacin,   lincomycin   and  erythromycin)   on   the inflammatory process was studied both in vitro and in vivo. Agar-induced rat paw oedema was used as a model for acute inflammation, and the antibiotics were administered (10 mg/kg, 20 mg/kg and 40 mg/kg per orally) 1hour before injection of agar. Ciprofloxacin showed a dose- dependent inhibitory effect on the paw oedema though the maximum concentration (40 mg/kg b.w. dose) showed a deviation. The same pattern was observed for erythromycin. Lincomycin showed a dose dependent effect on the paw oedema. At the highest concentrations (40 mg/kg), lincomycin  showed the highest  inhibitory effect on the paw oedema. The only significant (p<0.05) differences between the effects of the antibiotics and the reference drug was observed with ciprofloxacin (20 mg/kg) and lincomycin (40 mg/kg) at the fifth hour post-administration. Thermal-induced pain was used for the analgesic activity test. The hot glass surface on which the rats were placed was able to cause pain on the rat paw. At 15 minutes, of all the doses of the various antibiotics, ciprofloxacin 200 mg/kg evoked the highest reaction latency to thermal- induced pain though it was non-significantly (p>0.05) lower than that of aspirin. Only aspirin and ciprofloxacin prolonged the reaction latency after 60 minutes. At 60 minutes, the reaction latency of the three doses of ciprofloxacin was significantly (p<0.05) higher than the reaction latency of lincomycin  and erythromycin. Effect of the drugs on  agar-induced migration of leukocytes into the peritoneum was also ascertained. All the antibiotics significantly (p<0.05) reduced leucocyte mobilization into the affected tissue. They all had their maximum inhibitory effects at the highest dose (40mg/kg) and these  compared well with that of indomethacin. Erythromycin  40mg/kg  showed  the highest  inhibitory  effect  on leucocyte  migration.  The control  had  the  highest  total  leucocyte  count  (3666.67)  and  the  highest  percentage  of neutrophils that migrated (63.67%). Effects of the drugs on phospholipase A2  activity were tested in vitro. The enzyme activity for the different concentrations of the test antibiotics and prednisolone  were  significantly  (p<0.05)  lower  than  that of the control.  Enzyme  activity increased with increasing concentration in ciprofloxacin and erythromycin while it reduced in lincomycin and prednisolone. Effect of the antibiotics was also tested on platelet aggregation. Ciprofloxacin-treated, erythromycin-treated and the normal control all followed a similar trend. They all had stepwise  increases  in absorbance  from time 0 seconds through to  time 120 seconds. The trend in lincomycin-treated groups differed from that of ciprofloxacin-treated and erythromycin-treated  groups.  There was a sharp decrease in  the absorbance  at around  30 seconds followed by a continuous increase up to 120 seconds and this was similar to what was obtained with indomethacin-treated  groups. The present study showed that these antibiotics had  anti-inflammatory  activity,  which  probably  depended  on their  ability  to prevent  the production of some pro-inflammatory mediators and cytokines, and implies that these agents, might exert anti-inflammatory effects alongside their antibacterial activity.

CHAPTER ONE

1.1 Introduction

Inflammation is an important nonspecific defence reaction to tissue injury, such as that caused by a pathogen or wound. It is derived from the Latin word,   to set on

. It could also be referred to as part of the complex biological response of body tissues to

harmful stimuli, such as pathogens, damaged cells, or irritants (Ferrero-Miliani et al., 2007). Inflammation is a protective response that involves immune cells, blood vessels, and a host of molecular mediators.

Its purpose is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and to initiate tissue repair. When inflammation is too little, it could lead to progressive tissue destruction by the harmful stimulus (e.g. bacteria)  and compromise  the survival of the  organism. On the other hand, chronic     inflammation     may     lead     to     a     host     of     diseases,     such     as     hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer (e.g., gallbladder carcinoma). Therefore, inflammation is normally closely regulated by the body. Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body     to     harmful     stimuli     and     is     achieved     by     the     increased     movement of plasma and leukocytes(especially granulocytes) from the blood into the injured tissues. A series of biochemical events propagates and matures the inflammatory response, involving the local  vascular  system,  the  immune  system,  and  various  cells  within  the  injured  tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type  of  cells  present  at  the  site  of  inflammation  and  is  characterized  by  simultaneous

destruction and healing of the tissue from the inflammatory process. The use of antibiotics for the sole treatment of inflammation is not common but considering the fact that the normal drugs used in its treatment have side effects which could be harmful in the long run, work should be being done to look for alternatives. Antibiotics are mostly antibacterials but they act via a series of mechanisms.  These mechanisms  are being  looked at to know if they could inhibit the formation of mediators responsible for inflammation. Quinolones (e.g. ciprofloxacin) target DNA  synthesis  (Sárközy,  2001).  Macrolides  (e.g.  erythromycin)  and  Lincosamides  (e.g. lincomycin) target protein synthesis (Tenson et al, 2003).

1.2      Acute inflammation

Acute inflammation is a short-term process, usually appearing within a few minutes or hours and begins to cease upon the removal of the injurious stimulus.It is  characterized  by five cardinal signs (Parakrama and Clive.,2005). These signs, in Latin, are Dolor, Calor, Rubor, Tumour (Vogel and Berke, 2009) and Functio laesa (Porth, 2007). Rubor (redness) and Calor (heat) are as a result of increased blood flow at body core temperature to the inflamed site. Tumour (swelling) is as a result of fluid accumulation from the blood vessels. Dolor (pain) is as a result of the release of chemicals such as bradykinin and histamine that stimulate nerve endings, inducing pain and itching. Functio laesa (altered function) results from multiple causes but is probably the result of a neurological reflex in response to pain (Rather, 1971).

The process  of acute  inflammation  is initiated  by occupant  immune  cells  already present in the involved tissue. On the surfaces of these cells are certain receptors named pattern recognition receptors (PRRs) (Dardick and Ronald, 2006), which recognise generic molecules that are broadly shared by pathogens but distinguishable from host  molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs).At the onset of an infection, burn, or other injuries, these cells undergo activation (one of their PRRs recognize a PAMP) and  release  inflammatory  mediators  responsible  for  the  clinical  signs  of  inflammation discussed earlier. These chemical  signals (chemokines)  activate the inner lining of nearby capillaries. Within these capillaries, a group of cell adhesion molecules called selectins are displayed on the  activated endothelial  cells. These selectins attract and temporarily attach circulating neutrophils in the blood to the endothelial cells. They do this by forming weak bonds  that bind and break. This slows the neutrophils  and causes them to roll along the endothelial  wall until they come across the inflammatory  chemicals that act as  activating signals. These signals would activate adhesion receptors on the neutrophils called integrins.

These integrins then bind tightly to the selectins. This makes the neutrophils to adhere to the endothelium and stop rolling. This is called margination. These neutrophils then experience histrionic  changes  in shape and squeeze  through  the endothelial  wall in a  process  called diapedesis.  After  passing  through  the  wall,  they  then  migrate  to  the  scene  of  injury (extravasation) and attack the pathogen or any other cause of the injury. Chemotactic factors called chemotaxins attract neutrophils and other leukocytes to the site of injury. The acute inflammatory response requires constant stimulation to be sustained. Inflammatory mediators are short-lived and are quickly degraded in the tissue. Hence, acute inflammation begins to cease once the stimulus has been removed.

1.3.     Components of Inflammation

Acute inflammation is an immune-vascular response to an inflammatory stimulus. It can be broadly divided into a vascular phase that occurs first, followed by a cellular phase involving immune cells (more specifically myeloid granulocytes in the acute setting).

1.3.1    Vascular component

The vascular  component  of acute  inflammation  involves  the  movement  of  plasma  fluid, containing important proteins such as fibrin and immunoglobulins (antibodies), into inflamed tissue.       Upon       contact       with       pathogen       associated       molecular       patterns, tissue macrophages and mastocytes release vasoactive amines such as histamine and serotonin, as well as eicosanoids  such as prostaglandin  E2  and  leukotriene  B4  to remodel  the local vasculature, macrophages and endothelial cells release nitric oxide. These mediators vasodilate and permeabilize the blood vessels, which results in the net distribution of blood plasma from the vessel into the tissue space. The increased collection of fluid into the tissue causes it to swell (oedema). The exuded  tissue fluid contain various antimicrobial  mediators from the plasma such as complement, lysozyme, antibodies, which can immediately cause damage to microbes, and opsonise the microbes in preparation for the cellular phase. If the inflammatory stimulus is a lacerating wound, exuded platelets,coagulants,  plasmin and kinins can clot the wounded area and provide haemostasis in the first instance. These clotting  mediators also provide  a  structural  staging  framework  at  the  inflammatory  tissue  site  in  the  form  of a fibrin lattice for the purpose of aiding phagocytic debridement and wound repair later on. Some of the exuded tissue fluid is also funnelled by lymphatics to the regional lymph nodes, flushing bacteria along to start the recognition and attack phase of the adaptive immune system.

Acute inflammation  is characterized  by marked  vascular  changes,  including  vasodilation, increased permeability and increased blood flow, which are induced by the actions of various inflammatory  mediators.  Vasodilation  occurs  first  at  the  arteriole  level,  progressing  to the capillary level, and brings about a net increase in the amount of blood present, causing the redness  and  heat  of  inflammation.  Increased  permeability  of  the  vessels  results  in  the movement  of  plasma  into  the  tissues,  with  resultant  stasis  due  to  the  increase  in  the concentration of the cells within blood – a condition characterized by enlarged vessels packed with cells. Stasis allows leukocytes to  marginate (move) along the endothelium, a process critical to their recruitment into the tissues. Normal flowing blood prevents this, as the shearing force along the periphery of the vessels moves cells in the blood into the middle of the vessel.

1.3.1.1 Vascular Systems

The  complement  system,  when  activated,  creates  a  cascade  of  chemical  reactions  that promote  opsonization,  chemotaxis,  and agglutination,  and produces  the  membrane  attack complex (MAC).The kinin system generates proteins capable of sustaining vasodilation and other physical inflammatory effects.The coagulation system or clotting cascade, which forms a protective protein mesh over sites of injury.The fibrinolysis system, which acts in opposition to the coagulation system, to counterbalance clotting and generate several other inflammatory mediators.

1.3.1.2 Plasma-derived mediators

These are mediators derived from the plasma and they aid the inflammatory process. Examples include bradykinin, components C3 and C5, plasmin, thrombin etc.

1.3.1.2.1          Bradykinin

Produced by the kinin system, bradykinin is an inflammatory mediator. It is a peptide that causes blood vessels to dilate (enlarge), and therefore causes blood pressure to fall.Bradykinin dilates blood vessels via the release of prostacyclin, nitric oxide, and Endothelium-Derived Hyperpolarizing Factor. Bradykinin is a physiologically and pharmacologically active peptide of the kinin group of proteins, consisting of nine amino acids. The amino acid sequence of bradykinin  is:  Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg. Its empirical formula is therefore C50H73N15O11.The kinin-kallikrein  system makes bradykinin by proteolytic cleavage of  its kininogen  precursor,  high-molecular-weight  kininogen  (HMWK  or  HK),  by the  enzyme kallikrein.In humans, bradykinin is broken down by three kininases: angiotensin-converting

enzyme (ACE), aminopeptidase P (APP), and carboxypeptidase N (CPN), which cleave the 7-

8,  1-2,  and  8-9  positions,  respectively  (Dendorferet  al.,  2001),  Bradykinin  is  a  potent endothelium-dependent  vasodilator,  leading  to  a  drop  in  blood  pressure.  It  also  causes contraction  of  non-vascular  smooth  muscle  in  the  bronchus  and  gut,  increases  vascular permeability and is also involved in the mechanism of pain (Mutschleret al., 1997). Bradykinin also causes natriuresis, contributing to the drop in blood pressure. Bradykinin raises internal calcium levels in neocortical astrocytes causing them to release glutamate (Parpuraet al., 1994). Bradykinin is also thought to be the cause of the dry cough in some patients on angiotensin- converting enzyme (ACE) inhibitor drugs. It is thought that bradykinin is converted to inactive metabolites by ACE, therefore inhibition of this enzyme leads to increased levels of bradykinin, which causes a dry cough via bronchoconstriction. This refractory cough is a common cause for stopping ACE inhibitor therapy, in which case angiotensin II receptor antagonists (ARBs) are the next line of treatment.Over-activation of bradykinin is thought to play a role in a rare disease called hereditary angioedema, formerly known as hereditary angio-neurotic oedema (Bas et al., 2007). Initial secretion of bradykinin post-natally causes constriction and eventual atrophy of the ductus arteriosus, forming the ligamentum arteriosum between the pulmonary trunk and aortic arch.

1.3.1.2.2          Component C3

Complement component 3, often simply called C3, is a protein of the immune system. It plays a central role in the complement system and contributes to innate immunity (Sahu and Lambris,

2001). In humans it is encoded on chromosome 19 by a gene called C3. One form of C3-

convertase, also known as C4b2a, is formed by a heterodimer of activated forms of C4 and C2. It catalyses the proteolytic cleavage of C3 into C3a and C3b, generated during  activation through the classical pathway as well as the lectin pathway. C3a, an anaphylotoxin, stimulates histamine release by mast cells, thereby producing vasodilation. C3b is able to bind to bacterial cell walls and act as an opsonin, which marks the invader as a target for phagocytosis. Several crystallographic structures of C3 have been determined and reveal that this protein contains 13 domains (Jannsen et al., 2005).

1.3.1.2.3          Component C5a

This is produced by the complement system. C5a is a protein fragment released from cleavage complement component C5 by protease C5-convertase into C5a and C5b fragments. It acts as

a highly inflammatory peptide. The origin of C5 is in the hepatocyte but its synthesis can also be found in macrophages  that may cause local increase of C5a. C5a has  chemotactic  and anaphylatoxic properties. It is essential in the innate immunity but it is also linked with the adaptive  immunity.  The  increase  production  of  C5a  is   connected  with  a  number  of inflammatory diseases (Manthey et al., 2009).Human polypeptide C5a contains 74 amino acids (Andreas et al., 2013). C5a is an  anaphylatoxin,  causing increased expression of adhesion molecules on endothelium, contraction of smooth muscle, and increased vascular permeability. C5a des-Arg is a much less potent anaphylatoxin. Both C5a and C5a des-Arg can trigger mast cell degranulation, releasing pro-inflammatory molecules histamine and TNF- effective chemoattractant, initiating accumulation of complement and phagocytic cells at sites of infection or recruitment of antigen-presenting cells to lymph nodes. C5a plays a key role in increasing migration and adherence of neutrophils and monocytes to vessel walls. White blood cells  are  activated  by  up-regulation  of  integrin  avidity,  the  lipoxygenase  pathway  and arachidonic  acid  metabolism.  C5a  also  modulates  the  balance  between  activating  versus inhibitory    IgG   Fc   receptors    on   leukocytes,    thereby   enhancing    the    autoimmune response(Manthey et al., 2009). C5a is a powerful inflammatory mediator, and seems to be a key factor in the development  of pathology of many inflammatory  diseases involving the complement system such as sepsis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythemotosis, and psoriasis. The inhibitor of C5a that can block its effects would be helpful in medical applications (Ward, 2004).

1.3.1.2.4          Factor XII (Hageman factor)

This is produced  by the liver. It is a protein  that circulates  inactively,  until activated  by collagen,  platelets,  or  exposed  basement  membranes  via  conformational  change.  When activated, it in turn is able to activate three plasma systems involved in inflammation: the kinin system, fibrinolysis system, and coagulation system.

1.3.1.2.5          Membrane Attack Complex

This is produced  by the complement  system. The membrane  attack complex (MAC)  is  a structure  typically  formed  on the surface  of pathogenic  bacterial  cells  as a result  of the

activation  of  the  host’s  alternative  pathway,  classical  pathway,  or lectin  pathway  of  the complement  system,  and  it  is  one  of  the  effector  proteins  of  the  immune  system.  The membrane-attack complex (MAC) forms trans-membrane channels. These channels disrupt the phospholipid bilayer of target cells, leading to cell lysis and death (Peitsch and Tschopp, 1991). A number of proteins participate in the assembly of the MAC. Freshly activated C5b binds to C6 to form a C5b-6 complex, then to C7 forming the C5b-6-7 complex. The C5b-6-7 complex binds to C8, which is composed of three chains (alpha, beta, and gamma), thus forming the C5b-6-7-8 complex. C5b-6-7-8 subsequently binds to C9 (Stanley et al., 1988) and acts as a catalyst in the polymerization of C9. It is composed of a complex of four complement proteins (C5b, C6, C7, and C8) that bind to the outer surface of the plasma membrane, and many copies of a fifth protein (C9) that hook up to one another, forming a ring in the membrane.A complex of the complement proteins C5b, C6, C7, C8, and multiple units of C9.Active MAC has a subunit composition of C5b-C6-C7-C8-C9{n}. The combination and activation of this range of complement  proteins forms the membrane  attack complex, which is able to insert into bacterial cell walls and causes cell lysis with ensuing death.

1.3.1.2.6     Plasmin

Plasmin is an important enzyme present in blood that degrades many blood plasma proteins including fibrin clots. The degradation of fibrin is termed fibrinolysis. In humans, the plasmin protein is encoded by the PLG gene. Plasmin is a serine protease that acts to dissolve fibrin blood clots. Apart from fibrinolysis, plasmin proteolyses proteins in various other systems: It activates collagenases, some mediators of the complement system and weakens the wall of the Graafian follicle (leading to ovulation). It cleaves fibrin, fibronectin, thrombospondin, laminin, and  von  Willebrand   factor.  Plasmin,   like   trypsin,   belongs   to  the  family   of  serine proteases.Plasmin is released as a zymogen called plasminogen (PLG) from the liver into the factor IX systemic circulation and placed into the MD5+ that leads into the lungs. Two major glycoforms  of  plasminogen  are  present  in  humans  –  type  I  plasminogen  contains  two glycosylation moieties (N-linked to N289 and O-linked to T346), whereas type II plasminogen contains only a single O-linked sugar (O-linked to T346). Type II plasminogen is preferentially recruited to the cell surface over the type I glycoform. Conversely, type I plasminogen appears more readily recruited to blood clots.In circulation, plasminogen adopts a closed, activation resistant conformation. Upon binding to clots, or to the cell surface, plasminogen adopts an open form that can be converted into active plasmin by a variety of enzymes, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and factor XII

(Hageman  factor).  Fibrin  is a cofactor  for plasminogen  activation  by tissue  plasminogen activator. Urokinase plasminogen  activator receptor (uPAR) is a cofactor  for plasminogen activation by urokinase plasminogen  activator. The conversion of  plasminogen  to plasmin involves the cleavage of the peptide bond between Arg-561 and Val-562 (Miyata et al., 1982; Forsgrenet al., 1987; Law et al., 2012). Deficiency in plasmin may lead to thrombosis, as clots are not degraded adequately. Plasminogen deficiency in mice leads to defective liver repair (Bezerra et al., 1999), defective wound healing, reproductive abnormalities. Plasmin cleavage produces angiostatin.

1.3.1.2.7     Thrombin

Thrombin is a serine protease that in humans is encoded by the F2 gene (Royle et al., 1987; Degen and Davie, 1987). Prothrombin (coagulation factor II) is proteolytically cleaved to form thrombin in the coagulation cascade, which ultimately results in the reduction of blood loss. Thrombin in turn acts as a serine protease that converts soluble fibrinogen into insoluble strands of  fibrin,  as  well  as  catalysing  many  other  coagulation-related  reactions.  In  the  blood coagulation  pathway, thrombin acts to  convert factor XI to XIa, VIII to VIIIa, V to Va, fibrinogen to fibrin, and XIII to XIIIa.Factor XIIIa is a transglutaminase that catalyzes the formation of covalent bonds between lysine and glutamine residues in fibrin. The covalent bonds increase the stability of the fibrin clot. Thrombin interacts with thrombomodulin (Bajzar et al., 1996; Jakubowski and Owen, 1989). As part of its activity in the coagulation cascade, thrombin also promotes platelet activation and aggregation via activation of protease-activated receptors  on  the  cell  membrane  of  the  platelet.  Activation  of  prothrombin  is  crucial  in physiological and pathological coagulation. Various rare diseases involving prothrombin have been  described  (e.g.,  hypoprothrombinemia).  Anti-prothrombin  antibodies  in autoimmune disease  may  be  a  factor  in  the  formation  of  the  lupus  anticoagulant  also  known  as (antiphospholipid syndrome). Thrombin, a potent vasoconstrictor and mitogen, is implicated as a major factor in vasospasm following subarachnoid hemorrhage. Blood from a ruptured cerebral aneurysm clots around a cerebral artery, releasing thrombin. This can induce an acute and prolonged narrowing of the blood vessel, potentially resulting in cerebral ischemia and infarction (stroke).Beyond its key role in the dynamic process of thrombus formation, thrombin has a pronounced pro-inflammatory character, which may influence the onset and progression of  atherosclerosis.  Acting  via  its  specific  cell  membrane  receptors  (protease  activated receptors: PAR-1, PAR-3 and PAR-4), which are abundantly expressed in all arterial vessel wall  constituents,  thrombin  has  the  potential  to  exert  pro-atherogenic  actions  such  as

inflammation, leukocyte recruitment into the atherosclerotic plaque, enhanced oxidative stress, migration  and proliferation  of vascular  smooth  muscle  cells,  apoptosis  and  angiogenesis (Borissoff et al., 2009; Borissoff et al., 2010; Borissoff et al., 2011). Thrombin is implicated in the physiology of blood clots

1.3.2    Cellular Component

The cellular component involves leukocytes, which normally reside in blood and must move into the inflamed tissue via extravasation to aid in inflammation. Some act as  phagocytes, ingesting bacteria, viruses, and cellular debris. Others release enzymatic granules that destroy pathogenic  invaders.  Leukocytes  also  release  inflammatory  mediators  that  develop  and maintain   the   inflammatory   response.   In   general,   acute   inflammation   is   mediated by  granulocytes,  whereas  chronic  inflammation  is  mediated  by  mononuclear  cells  such as monocytes and lymphocytes.

1.3.2.1 Macrophages

Macrophages are white blood cells that engulf and digest cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the types of proteins specific to the surface of healthy body cells on its surface in a process called phagocytosis. They are found in essentially  all  tissues  (Ovchinnikov,  2008)  where  they  patrol  for  potential  pathogens  by amoeboid movement. They play a critical role in non-specific defence (innate immunity), and also help initiate specific defence mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. In humans, dysfunctional macrophages cause severe diseases such as  chronic  granulomatous  disease  that  result  in  frequent  infections.Beyond   increasing inflammation and stimulating the immune system, macrophages also play an important anti- inflammatory  role  and  can  decrease  immune  reactions  through  the  release  of cytokines. Macrophages that encourage inflammation are called M1 macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages (Mills, 2012). This difference is reflected in their metabolism, M1 macrophages have the unique ability to metabolize arginine to the “killer” molecule nitric oxide, whereas M2 macrophages have the unique ability to metabolize arginine to the “repair” molecule ornithine.Human macrophages are about 21 micrometres (0.00083 in) in diameter (Krombachet al., 1997) and are produced by the differentiation of monocytes in tissues. Macrophages are essential for wound healing. They replace Polymorphonuclear neutrophils as the predominant cells in the wound by two days after injury. Attracted to the wound site by growth factors released by platelets and other

cells, monocytes from the bloodstream enter the area through blood vessel walls. Numbers of monocytes in the wound peak one to one and a half days after the injury occurs. Once they are in the wound site, monocytes mature into macrophages. The spleen contains half the body’s monocytes  in reserve  ready to be deployed  to injured  tissue (Jia and Pamer, 2009). The macrophage’s main role is to phagocytize bacteria and damaged tissue, and they also debride damaged tissue by releasing proteases (Deodhar and Rana, 1997). Macrophages also secrete a number of factors such as growth factors and other cytokines, especially during the third and fourth post-wounding days. These factors attract cells involved in the proliferation stage of healing to the area. Macrophages may also restrain the contraction phase (Newton et al., 2004). Macrophages are stimulated by the low oxygen content of their surroundings to produce factors that induce and speed angiogenesis (Greenhalgh, 1998) and they also stimulate cells that re- epithelialize the wound, create granulation tissue, and lay down a new extracellular matrix (Stashaket al., 2004) By secreting these factors, macrophages contribute to pushing the wound healing process into the next phase.

1.3.2.2   Monocytes

Monocytes are a type of white blood cells (leukocytes). They are the largest of all leukocytes. They are part of the innate immune system of vertebrates including all  mammals (humans included), birds, reptiles, and fish. They are amoeboid in shape, having agranulated cytoplasm. Monocytes  have  unilobar  nuclei,  which  makes  them  one  of  the  types  of  mononuclear leukocytes (containing azurophil granules). The archetypal idea of the nucleus is that it is bean- shaped or kidney-shaped,  although  the most  important  distinction  is that it is not deeply furcated into lobes, as occurs in polymorphonuclear leukocytes. Monocytes constitute 2% to

10% of all leukocytes in the human body. They play multiple roles in immune function. Such

roles  include  replenishing  resident  macrophages  under  normal  states;  and in response  to inflammation signals, monocytes can move quickly (approx. 8  12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. Half of them are stored in the spleen (Swirski et al., 2009). Monocytes are usually identified in stained smears by their large kidney shaped or notched nucleus. These change into macrophages after entering into the tissue spaces, and in endothelium can transform into foam cells. Monocytes are produced by the bone marrow from precursors called monoblasts, bipotent cells that differentiated from hematopoietic stem cells. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. They constitute between three to eight percent of the leukocytes in the blood. Half of them are stored as a reserve in the spleen in clusters in the red pulp’s Cords of Billroth (Swirski et al., 2009). In the tissues, monocytes mature into different types of macrophages at different anatomical locations. Monocytes are the largest corpuscles in the blood.Monocytes which migrate from the bloodstream to other tissues will then  differentiate into tissue resident macrophages or dendritic cells. Macrophages are responsible for protecting tissues from foreign substances, but are also suspected to be important in the formation of important organs like the heart and brain. They are cells that possess a large smooth nucleus, a large area of cytoplasm, and many internal vesicles for processing foreign material.Monocytes  and their macrophage and dendritic-cell progeny serve three main functions in the immune system. These are phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake of microbes and particles  followed  by digestion  and destruction  of this  material.  Monocytes  can  perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen,  as well as by binding  to the microbe  directly  via  pattern-recognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibody-dependent cell-mediated cytotoxicity. Vacuolization may be present in a cell that has recently phagocytized foreign matter.Microbial fragments that remain after such digestion can serve as antigens.  The  fragments  can be incorporated  into MHC molecules  and then trafficked to the cell surface of monocytes (and macrophages and dendritic cells). This process is called antigen presentation and it leads to activation of T lymphocytes, which then mount a specific immune response against the antigen.Other microbial products can directly activate monocytes and this leads to production of pro-inflammatory and, with some delay, of anti- inflammatory cytokines. Typical cytokines produced by monocytes are TNF, IL-1, and IL-12.

1.3.2.3            Mast cells

A mast cell is a cell that is derived from the myeloid stem cell. It contains many granules rich in histamine and heparin. They are known to be involved in wound healing, angiogenesis, defence against pathogens and blood-brain barrier function (da Silva et al, 2014; Polyzoidis et al, 2015). They are also known to play roles in allergy and anaphylaxis. Mast cells look very much like basophils but are different cells as they develop from different hematopoietic stem cells. Unlike basophils, which leave the bone marrow in the mature form, mast cells circulate in an immature form and only mature when they get to their target tissue site (Prussin and

Metcalfe, 2003). They are seen in tissues surrounding blood vessels and nerves and also in the skin, mucosa of the lungs, digestive tract, conjunctiva, nose and mouth (Prussin and Metcalfe,

2003). Mast cells play important roles in the inflammatory process. They release histamine which  dilates  post-capillary  venules,  activates  the  endothelium,  increases  blood  vessel permeability leading to local oedema and also depolarizes the nerve endings leading to pain or itching.

1.3.2.4             Neutrophils

Neutrophil granulocytes (also known as neutrophils) are the most abundant (40% to 75%) type of white blood cells in most mammals and form an essential part of the innate immune system. Functionality varies in different animals (Ermert et al., 2013). They are formed from stem cells in the bone marrow. They are short-lived and highly motile. Neutrophils may be subdivided into  segmented  neutrophils  and  banded  neutrophils  (or  bands).  They  form  part  of  the polymorphonuclear  cell  family  (PMNs)  together  with  basophils  and  eosinophils  (Witko- Sarsatet al., 2000; Nathan, 2006).The name neutrophil derives from staining characteristics on hematoxylin and eosin (H&E)  histological or cytological preparations. Whereas basophilic white blood cells stain dark blue and eosinophilic white blood cells stain bright red, neutrophils stain a neutral pink. Normally, neutrophils contain a nucleus divided into 2  5 lobes.Neutrophils are a type of  phagocyte and are normally found in the bloodstream. During the beginning (acute) phase of inflammation, particularly as a result of bacterial infection, environmental exposure (Jacobs et al., 2010) and some cancers (Waugh and Wilson, 2008; De Larco et al.,

2004), neutrophils are one of the first-responders of inflammatory cells to migrate towards the

site of inflammation. They migrate through the blood vessels, then through interstitial tissue, following chemical signals such as Interleukin-8 (IL-8), C5a, fMLP and Leukotriene B4 in a process  called  chemotaxis.  They  are  the  predominant  cells  in  pus,  accounting  for  its whitish/yellowish  appearance.Neutrophils  are recruited to the  site of injury within minutes following   trauma,   and  are  the  hallmark   of  acute   inflammation   (Cohen   and  Burns,

2002).However,   due  to  some  pathogens  being  indigestible,   they  can  be  less   useful alone.Neutrophil  granulocytes  have  an  average  diameter  of  12-15  micrometers  (µm)  in peripheral  blood  smears.  When  analyzing  neutrophils  in  suspension,  neutrophils  have anaverage diameter of 8.85 µm. With the eosinophil and the basophil, they form the class of polymorphonuclear  cells,  named  for  the  nucleus’  multilobulated  shape  (as  compared  to lymphocytes and monocytes, the other types of white cells). The nucleus has a characteristic lobed appearance, the separate lobes connected by chromatin. The nucleolus disappears as the

neutrophil matures, which is something that happens in only a few other types of nucleated cells (Zucker-Franklinet  al., 1988). Neutrophils are the most abundant white blood cells in humans (approximately 1011 are produced daily); they account for approximately 50-70% of all white blood cells (leukocytes). The stated normal range  for human blood counts varies between laboratories, but a neutrophil count of 2.5  7.5 x 109/L is a standard normal range. People of African and Middle Eastern descent may have lower counts, which are still normal. The average lifespan of (non-activated human) neutrophils in the circulation has been reported by different approaches to be between 5 and 90 hours (Tak et al., 2013). Upon activation, they marginate  (position  themselves  adjacent  to  the  blood  vessel  endothelium),  and  undergo selectin-dependent capture followed by integrin-dependent adhesion in most cases, after which they migrate  into  tissues,  where  they  survive  for 1  2 days.  Neutrophils  are much  more numerous  than  the  longer-lived  monocyte/macrophage  phagocytes.  A  pathogen  (disease- causing  microorganism  or  virus)  is  likely  to  first  encounter  a neutrophil.  Some  experts hypothesize  that the short lifetime of neutrophils is an evolutionary  adaptation. The  short lifetime of neutrophils minimizes propagation of those pathogens that parasitize phagocytes because the more time such parasites spend outside a host cell, the more likely they will be destroyed by some component of the body’s defenses. Also, because neutrophil antimicrobial products  can also  damage  host tissues,  their  short life limits  damage  to the host during inflammation. Neutrophils undergo a process called chemotaxis, which allows them to migrate toward sites of infection or inflammation. Cell surface receptors allow neutrophils to detect chemical gradients of molecules such as interleukin-8 (IL-8), interferon gamma (IFN-gamma), C3a, C5a, and Leukotriene B4, which these cells use to direct the path of their migration.Being highly motile, neutrophils quickly congregate at a focus of infection, attracted by cytokines expressed by activated endothelium, mast cells, and macrophages. Neutrophils express (Ear and McDonald, 2008) and release cytokines, which in turn amplify inflammatory reactions by several other cell types.

1.3.2.5             Lymphocytes

A lymphocyte is one of the subtypes of white blood cell in a vertebrate’s immune system. They include  natural  killer  cells  (NK  cells)  (which  function  in cell-mediated,  cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). They are the main type of cell found in lymph, which prompted the name lymphocyte. The three major types of lymphocyte are T cells, B cells and natural killer (NK) cells. Lymphocytes can be identified by their large nucleus.

1.3.2.5.1      T-cells and B-cells

T cells (thymus cells) and B cells (bone marrow- or bursa-derived cells) are the major cellular components of the adaptive immune response. T cells are involved in cell-mediated immunity, whereas B cells are primarily responsible for humoral immunity (relating to antibodies). The -ocess known as antigen presentation. Once they have identified an invader, the cells generate specific responses that are tailored to maximally eliminate specific pathogens or pathogen-infected cells. B cells respond to pathogens by producing large quantities of antibodies which then neutralize foreign objects like bacteria and viruses. In response to pathogens some T cells, called T helper cells, produce cytokines that direct the immune response, while other T cells, called cytotoxic T cells, produce toxic granules that contain powerful enzymes which induce the death of pathogen-infected cells. Following activation, B cells and T cells leave a lasting legacy of the antigens they have encountered, in the form of memory cells. Throughout the lifetime of an an and are able to mount a strong and rapid response if the pathogen is detected again.

1.3.2.5.2      Natural Killer cells

NK cells are a part of the innate immune system and play a major role in defending the host from both tumours and virally infected cells. NK cells distinguish infected cells and tumors from normal and uninfected cells by recognizing changes of a surface molecule called MHC (major histocompatibility complex) class I. NK cells are activated in response to a family of cytokines called interferons. Activated NK cells release cytotoxic (cell-killing) granules which then destroy the altered cells (Janeway et al., 2001). They were named “natural killer cells” because of the initial notion that they do not require prior activation in order to kill cells which are missing MHC class I.

1.3.2.6             Basophils

Basophil granulocytes are the least common of the granulocytes, representing about 0.01% to

0.3% of circulating white blood cells. Standard Range is 0.0 – 2.0 % via differential blood count.Basophils contain large cytoplasmic granules which obscure the cell nucleus under the microscope when stained. However, when unstained, the nucleus is visible and it usually has two lobes. Basophils appear in many specific kinds of inflammatory reactions, particularly those that cause allergic symptoms. Basophils contain anticoagulant heparin, which prevents blood from clotting too quickly. They also contain the vasodilator histamine, which promotes

blood flow to tissues. They can be found in unusually high numbers at sites of ectoparasite infection, e.g., ticks. Like eosinophils, basophils play a role in both parasitic infections and allergies (Voehringer, 2009). They are found in tissues where allergic reactions are occurring and probably contribute to the severity of these reactions. Basophils have protein receptors on their cell surface that bind IgE, an immunoglobulin involved in macroparasite defence and allergy.  It is the bound  IgE  antibody  that  confers  a selective  response  of  these  cells  to environmental substances, for example, pollen proteins or helminth antigens. Recent studies in mice suggest that basophils  may  also  regulate  the behaviour  of T cells and mediate  the magnitude of the secondary immune response (Nakanishi, 2010). Basophils arise and mature in bone marrow. When activated, basophils degranulate to release histamine, proteoglycans (e.g. heparin and chondroitin), and proteolytic enzymes (e.g. elastase and lysophospholipase). They also secrete lipid mediators like leukotrienes (LTD-4), and several cytokines. Histamine and proteoglycans are pre-stored in the cell’s granules while the other secreted substances are newly generated.  Each  of these  substances  contributes  to inflammation.  Recent  evidence suggests that basophils are an important source of the cytokine, interleukin-4, perhaps more important  than  T  cells.  Interleukin-4  is  considered  one  of  the  critical  cytokines  in  the development of allergies and the production of IgE antibody by the immune system. There are other substances that can activate basophils to secrete which suggests that these cells have other roles  in  inflammation  (Janeway  et  al.,  2001).  The  degranulation  of  basophils  can  be investigated in vitro by using flow cytometry and the so-called basophil-activation-test (BAT). Especially, in the diagnosis of allergies including of drug reactions (e.g. induced by contrast medium), the BAT is of great impact (Böhmet al., 2011). Basopenia (a low basophil count) is difficult  to demonstrate  as the  normal  basophil  count  is so low; it has been  reported  in association with autoimmune urticarial which is a chronic itching condition (Grattan et al., 2003).

1.3.2.7             Eosinophils

Eosinophil granulocytes are white blood cells and one of the immune system  components responsible for combating multicellular parasites and certain infections in vertebrates. Along with mast cells, they also control mechanisms associated with allergy and asthma. They are granulocytes that develop during hematopoiesis  in the bone  marrow before migrating into blood.These cells are eosinophilic or ‘acid-loving’ as shown by their affinity to coal tar dyes: Normally transparent, it is this affinity that causes them to appear brick-red after staining with eosin, a red dye, using the Romanowsky method. The staining is concentrated in small granules

within the cellular cytoplasm, which contain many chemical mediators, such as histamines and proteins such as eosinophil peroxidase, ribonuclease (RNase), deoxyribonucleases (DNase), lipase, plasminogen, and major basic protein. These mediators are released by a process called degranulation following activation of the eosinophil, and are toxic to both parasite and host tissues.In normal individuals, eosinophils make up about 1-6% of white blood cells, and are about 12-17 micrometres in size (Young et al., 2006). They are found in the medulla and the junction between the cortex and medulla of the thymus, and, in the lower gastrointestinal tract, ovary, uterus, spleen, and lymph nodes, but not in the lung, skin, oesophagus, or some other internal organs under normal conditions. The presence of eosinophils in these latter organs is associated with disease. Eosinophils persist in the circulation for 8  12 hours, and can survive in tissue for an additional 8  12 days in the absence of stimulation (Young et al., 2006).

These leukocytes are critically involved in the initiation and maintenance of inflammation. These cells must be able to get to the site of injury from their usual location in the blood, therefore  mechanisms  exist to recruit and direct leukocytes  to  the  appropriate  place. The process of leukocyte movement from the blood to the  tissues through the blood vessels is known as extravasation, and can be divided up into a number of broad steps.

1.3.3    Cell derived mediators

These are mediators that are derived directly from the cells. They include lysosome granules, histamine, interfe implicated in the inflammatory process.

1.3.3.1            Lysosome granules

These are enzymes derived from granulocytes. These cells contain a large variety of enzymes that     perform     a     number     of     functions.     Granules     can     be      classified     as either specific or azurophilic depending upon the contents, and are able to break down a number of substances, some of which may be plasma-derived proteins that allow these enzymes to act as inflammatory mediators.

1.3.3.2             Histamine

Histamine is an organic nitrogenous compound involved in local immune responses as well as regulating physiological function in the gut and acting as a neurotransmitter (Marieb, 2001). Histamine is involved in the inflammatory response and has a central role as a mediator of pruritus (Andersen et al., 2015). As part of an immune response to foreign pathogens, histamine

is produced by basophils and by mast cells found in nearby connective tissues.  Histamine increases the permeability of the capillaries to white blood cells and some proteins, to allow them to engage pathogens in the infected tissues (Di Guiseppe et al.,  2003). Histamine is derived from the decarboxylation  of the amino acid histidine, a  reaction catalysed by the enzyme  L-histidine  decarboxylase.  It  is  a  hydrophilic  vasoactive  amine.  Once  formed, histamine is either stored or rapidly inactivated by its primary degradative enzymes, histamine- N-methyltransferase or diamine oxidase. In the central nervous system, histamine released into the synapses is primarily broken down by histamine-N-methyltransferase, while in other tissues both enzymes may play a role. Several other enzymes, including MAO-B and ALDH2, further process the immediate metabolites of histamine for excretion or recycling.Most histamine in the body is generated in granules in mast cells and in white blood cells called basophils and eosinophils. Mast cells are especially numerous at sites of potential injury      the nose, mouth, and feet, internal body surfaces, and blood vessels. Non-mast cell histamine is found in several tissues, including the brain, where it functions as a neurotransmitter. Another important site of histamine storage and release is the enterochromaffin-like (ECL) cell of the stomach. The most important  pathophysiologic  mechanism  of  mast  cell  and  basophil  histamine  release  is immunologic.  These  cells,  if sensitized  by  IgE  antibodies  attached  to  their  membranes, degranulate when exposed to the appropriate antigen. Certain amines and alkaloids, including such drugs as morphine, and curare alkaloids, can displace histamine in granules and cause its release. Antibiotics like polymyxin are also found to stimulate histamine release. Histamine release  occurs  when  allergens  bind to mast-cell-bound  IgE antibodies.  Reduction  of  IgE overproduction may lower the likelihood of allergens finding sufficient free IgE to trigger a mast-cell-release of histamine.In humans, histamine exerts its effects primarily by binding to G protein-coupled  histamine  receptors,  designated  H1 through  H4 (Panula  et al., 2015). Although  histamine  is small compared  to other biological  molecules  (containing  only 17 atoms), it plays an important role in the body. It is known to be  involved in 23 different physiological functions. Histamine is known to be involved in many physiological functions because of its chemical properties that allow it to be versatile in binding. It is Coulombic (able to carry a charge), conformational, and flexible. This allows it to interact and bind more easily (Noszal et al., 2004). When injected intravenously, histamine causes most blood vessels to dilate, and hence causes a fall in the blood pressure Dale and Laidlaw,1910). This is a key mechanism in anaphylaxis, and is thought to be caused when histamine releases nitric oxide, endothelium-derived  hyperpolarizing  factors  and other  compounds  from the endotholelial cells.

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