ELUCIDATION OF SOME IMMUNOLOGICAL AND BIOCHEMICAL NATURE OF THE LEAVES OF SENNA MIMOSOIDES
ABSTRACT
In the present study, the phytochemical composition, immunomodulatory, leukocyte mobilization, haematological and antihepatotoxic effects of the aqueous extract of Senna mimosoides leaves were evaluated. The study also covered the effect of the extract on the activity of lactase and the assessment of the damaging effect of carbon tetrachloride (CCl4) and ameliorative effect of the extract on liver tissue using histopathological technique. This study was aimed at validating the traditional use of S. mimosoides leaves in folklore medicine to treat breast milk toxicity in neonates by elucidating its immunological and biochemical nature. The qualitative and quantitative phytochemical composition showed the presence of 2.67 ± 0.0013 mg of flavonoids; 3.43 ± 0.0028 mg of alkaloids; 1.97 ± 0.0030 mg of saponin; 2.32 ± 0.0032 mg of terpenoids; 0.86 ± 0.0023 mg of steroid; 3.61 ± 0.0025 mg of phenol; 8.31 ± 0.0032 mg of reducing sugar; 4.75 ± 0.0034 m g of tannin; 1.61 ± 0.0031 mg of cyanide; 2.75 ± 0.0029 mg of glycoside and 4.68 ± 0.0033 mg of soluble carbohydrates for every 100 g of the extract. For the animal model experiment, one hundred and thirty (130) albino rats were used. The experimental design was divided into four (4) phases containing five (5) groups of five (5) rats in each group. Rats in group A (control) were administered 0.2 ml of normal saline; rats in groups B, C and D were treated with 50, 100 and 250 mg/kg of the aqueous extract of S. mimosoides leaves respectively; group E rats received levamisole or silymarin (standard drugs) while group F rats were treated with carbon tetrachloride (CCl4) only. Administration of 50, 100 and 250 mg/kg of the extract resulted in a dose-dependent significant (p < 0.05) increase in primary antibody titre with a value of 6, 8, 13, and secondary antibody titre with a value of 11, 26, 34. Delayed type hypersensitivity (DTH) response shows that the extract produced a dose- and time-dependent increase in footpad swelling of the rats. The extract (50, 100 and 250 mg/kg) and levamisole (25 mg/kg) at 24 hr after challenge, significantly (p < 0.05) boosted DTH reactions observed respectively as 1.412, 1.504, 1.816 and 1.827 mm difference in thickness of footpad before challenge and 24 hr after challenge while the control elicited a non-significant (p > 0.05) increase with a difference of 0.614 mm. At 48 hr after challenge, there was an additional increase in footpad swelling observed as 1.908, 1.918, 2.304 and 2.326 mm for the extract and levamisole respectively. The humoural antibody (HA) titre and DTH response compare well with that of levamisole, a standard immunostimulatory drug, at 25 mg/kg. The total leukocyte count of the groups treated with different concentrations of extract increased in a dose-dependent manner while the group treated with indomethacin decreased significantly (p < 0.05) compared with control. The percentage packed cell volume (PCV) for group B, before and after treatment with cyclophosphamide (CP) and later with (50 mg/kg) was 38.8 ± 1.30, 19.4 ± 0.55 and 34.4 ± 0.55 respectively. Groups C, D, and E showed the same trend but in the control group decreased by CP was not reversed. In the control, percentage PCV before and after CP and then extract was 35.8 ± 0.45, 19.4 ± 0.55 and 19.8 ± 1.09 respectively. The same trend was observed in haemoglobin concentration, white blood cell count, red blood cell count and its indices. There was increase in serum alanine aminotransferase (ALT) activity of rats in group F (81.20 ± 0.84 IU/L) after CCl4 administration as compared to the normal control A (53.00 ± 1.00 IU/L). The extract (50, 100, 250 mg/kg) and silymarin (25 mg/kg) caused a significant (p < 0.05) decrease in the activity of ALT (65.00 ± 1.58, 59.20 ± 0.84, 55.20 ± 1.30 and 57.00 ± 1.00 IU/L) respectively. The levels of aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, malondialdehyde, iron, phosphate followed the same trend as ALT compared to control. Administration of CCl4 decreased the level of reduced glutathione in group F (2.21 ± 0.239 mMol/g tissue). However, treatment with different concentrations of the extract and levamisole augmented this decrease (3.08 ± 0.093, 4.17 ± 0.241, 5.16 ± 0.193 and 4.97 ± 0.273 mMol/g tissue) respectively. Activities of glutathione s-transferase, glutathione peroxidase, catalase, superoxide dismutase and concentrations of sodium, magnesium, potassium, calcium, zinc and selenium showed the same trend. Histopathological studies showed that the extract and levamisole ameliorated centrilobular degeneration of the liver tissues induced by CCl4. Moreover, the extract exhibited higher significant (p < 0.05) activity of lactase in a dose-dependent manner when compared to the control. At 10, 20, 30, 40 and 50 µl, the enzyme activity were 17.187, 18.8 22, 20.044, 22.022 and 23.898 IU.The findings of this study show that the vase medicinally important bioactive compounds, present in this extract could be responsible for the immunostimulatory, antihepatotoxic effect, increase in lactase activity and haematological parameters. This justifies the use of this plant in folklore medicine for the treatment of diseases.
TABLE OF CONTENTS
Title Page
Certification
Dedication
Acknowledgement
Abstract
Table of Contents
List of Figures
List of Tables
List of Plates
List of Abbreviation
CHAPTER ONE: INTRODUCTION
1.1 Overview of the Human Immune System
1.2 The Cells of the Immune System
1.2.1 T-Lymphocytes
1.2.2 B-Lymphocytes
1.2.3 Natural Killer (Nk) Cells
1.2.4 Monocyte and Macrophages
1.2.5 Antigen-Presenting Cells (APCs)
1.2.6 Phagocytes
1.2.7 Neutrophils
1.2.8 Basophils and Mast Cells
1.2.9 Eosinophils
1.3 Innate (Nonspecific) Immunity
1.4 Adaptive Immunity
1.5 Humoural Immunity
1.6 Cell-Mediated Immunity (CMI)
1.7 Mediators of the Immune System
1.7.1 Cytokines
1.7.2 Complement System
1.8 Blood
1.9 The Concept of Immunomodulation
1.10 Cyclophosphamide (CP)
1.10.1 Metabolism of Cyclophosphamide
1.10.2 Mechanism of Action of Cyclophosphamide
1.11 Levamisole
1.12 Some Plants with Immunological Potential
1.13 The Liver and Its Function
1.14 The Overview of the Antioxidant Physiology of Human
1.15 Hepatotoxicity
1.16 Liver Histology
1.17 Biotransformation of Hepatotoxicants
1.18 Mechanism of Hepatotoxicity
1.19 Carbon tetrachloride (CCl4)
1.20 Lipid Peroxidation
1.21 Glutathione
1.21.1 Alanine Aminotransferases- the Standard Clinical Biomarker of Hepatotoxicity
1.21.2 Aspartate Aminotransferase (AST)
1.21.3 Alkaline Phosphatase (ALP)
1.21.4 Glutathione S-Transferase
1.22 Silymarin
1.23 Bilirubin
1.24 Serum/ Plasma Proteins
1.25 Phosphate
1.26 Calcium
1.27 Sodium
1.28 Potassium (K)
1.29 Magnesium
1.30 Zinc (Zn)
1.31 Selenium (Se
1.32 Iron
1.33 Breast Milk Toxicity
1.34 Disaccharides
1.35 Botanical Outline of Senna Mimosoides
1.36 Previous Investigation Carried Out on Senna Species
1.37 Aim of the Study
1.40 Specific Research Objectives
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials
2.1.1 Plant Material
2.1.2 Animal Material
2.1.3 Chemicals and Reagents
2.1.3.1Chemicals
2.1.3.2Reagents
2.2 Methods
2.2.1 Aqueous Extraction
2.2.2 Experimental Design
2.2.3 Phytochemical Analysis
2.2.3.1 Qualitative Phytochemical Analysis
2.2.3.1.1 Test for Saponins
2.2.3.1.2 Test for Alkaloids
2.2.3.1.3 Test for Tannins
2.2.3.1.4 Test for Flavonoides
2.2.3.1.5 Test for Terpenoids
2.2.3.1.6 Test for Steroids
2.2.3.1.7 Test for Phenols
2.2.3.1.8 Test for Glycosides
2.2.3.1.9 Test for Reducing Sugar
2.2.3.1.10 Test for Cyanide
2.2.3.1.11 Test for Soluble Carbohydrate (Molisch Test)
2.2.3.2 Quantitative Phytochemical Analysis
2.2.3.2.1 Test for Saponins
2.2.3.2.2 Test for Alkaloids
2.2.3.2.3 Test for Tannins
2.2.3.2.4 Test for Flavonoids
2.2.3.2.5 Test for Terpenoids
2.2.3.2.6 Test for Steroids
2.2.3.2.7 Test for Glycosides
2.2.3.2.8 Test for Reducing Sugar
2.2.3.2.9 Test for Soluble Carbohydrate
2.2.3.2.10 Test for Cyanide
2.2.3.2.11 Test for Phenols
2.2.4 Determination of Biological Activity
2.2.4.1 Acute Toxicity and Lethality
2.2.4.2 Immunomodulatory Activity of Extracts
2.2.4.2.1 Preparation of Antigen
2.2.4.2.2 Delayed Type Hypersensitivity (DTH) Reaction
2.2.4.2.3 Humoural Antibody (HA) Synthesis
2.2.4.3 Cyclophosphamide-Induced Myelosuppression
2.2.4.4 Determination of Haematological Parameter
2.2.4.4.1 Determination of WBC Count
2.2.4.4.2 Determination of PCV Concentration
2.2.4.4.3 Determination of Hb Concentration
2.2.4.4.4 Determination of RBC Count
2.2.4.5 Effect of the Extract on in vivo Leukocyte Mobilization
2.2.4.6 Determination of the Effect of the Extract on CCl4 Induced Hepatotoxicity
2.2.4.6.1 Assay of Serum Alanine Aminotransferase (ALT) Activity
2.2.4.6.2 Assay of Aspartate Aminotransferase (AST) Activity
2.2.4.6.3 Assay of Serum Alkaline Phosphatase (ALP) Activity
2.2.4.6.4 Determination of Serum Bilirubin
2.2.4.6.5 Determination of Catalase Activity
2.2.4.6.6 Determination of Reduced Glutathione Level
2.2.4.6.7 Determination of Superoxide Dismutase (SOD) Activity
2.2.4.6.8 Assay of Glutathione S-Transferase (GST) Activity
2.2.4.6.9 Assay of Glutathione Peroxidase Activity
2.2.4.6.10 Determination of Malondialdehyde (MDA) Concentration
2.2.4.7 Serum Inorganic Ion Determination
2.2.4.7.1 Determination of Serum Iron Concentration
2.2.4.7.2 Determination of Serum Selenium Concentration
2.2.4.7.3 Determination of Serum Zinc Concentration
2.2.4.7.4 Determination of Serum Calcium Concentration
2.2.4.7.5 Determination of Serum Phosphate Concentration
2.2.4.7.6 Determination of Serum Magnesium Concentration
2.2.4.7.7 Determination of Serum Sodium and Potassium
2.2.4.8 Determination of Serum Protein Concentration
2.2.4.9 Histopathological Study
2.2.4.10 Determination of the Effect of the Extract on the Activity of Lactase 65
2.2.5 Statistical Analysis
CHAPTER THREE: RESULTS
3.1 Extract of Senna mimosoides
3.2 Phytochemical Composition of Aqueous Extract of S. mimosoides Leaves
3.2.1 Qualitative Phytochemical Composition of Aqueous Extract of S. mimosoides Leaves
3.2.2 Qualitative Phytochemical Composition of Aqueous Extract of S. mimosoides Leaves
3.3 LD50
3.4 Effect of Aqueous Extract of S. mimosoides Leaves on In Vivo Leukocyte Migration
3.5 Effect of Aqueous Extract of S. mimosoides Leaves on Humoural Immunity
3.6 Effect of Aqueous Extract of S. mimosoides Leaves on Cell Mediated Immunity
3.7 Effect of Aqueous Extract of S. mimosoides Leaves on Serum PCV Level
3.8 Effect of Aqueous Extract of S. mimosoides Leaves on Serum Hb Concentration
3.9 Effect of Aqueous Extract of S. mimosoides Leaves on Serum WBC Level
3.10 Effect of Aqueous Extract of S. mimosoides Leaves on Serum Red Blood Cell Level
3.11 Effect of Aqueous Extract of S. mimosoides Leaves on MCH Concentration Level
3.12 Effect of Aqueous Extract of S. mimosoides Leaves on Mean Cellular Volume
3.13 Effect of Aqueous Extract of S. mimosoides Leaves on Serum MCH Level
3.14 Effect of Aqueous Extract of S. mimosoides on Serum Level of AST
3.15 Effect of Aqueous Extract of S. mimosoides on Serum Level of Alanine Transaminase
3.16 Effect of Aqueous Extract of S. mimosoides on Serum Level of Alkaline Phosphatase
3.17 Effect of Aqueous Extract of S. mimosoides on Serum Level of Bilirubin
3.18 Effect of Aqueous Extract of S. mimosoides on the Activity of Glutathione Peroxidase
3.19 Effect of Aqueous Extract of S. mimosoides on the Activity of Superoxide Dismutase
3.20 Effect of Aqueous Extract of S. mimosoides on the Activity of Catalase
3.21 Effect of Aqueous Extract of S. mimosoides on Lipid Peroxidation
3.22 Effect of Aqueous Extract of S. mimosoides on the Activity of Glutathione S-transferase
3.23 Effect of Aqueous Extract of S. mimosoides on Glutathione Level
3.24 Effect of Aqueous Extract of S. mimosoides on Total Serum Protein
3.25 Effect of Aqueous Extract of S. mimosoides on Serum Level of Sodium
3.26 Effect of Aqueous Extract of S. mimosoides on Serum Level of Magnesium
3.27 Effect of Aqueous Extract of S. mimosoides on Serum Level of Iron
3.28 Effect of Aqueous Extract of S. mimosoides on Serum Level of Potassium
3.29 Effect of Aqueous Extract of S. mimosoides on Serum Level of Phosphate
3.30 Effect of Aqueous Extract of S. mimosoides on Serum Level of Calcium
3.31 Effect of Aqueous Extract of S. mimosoides on Serum Level of Zinc
3.32 Effect of Aqueous Extract of S. mimosoides on Serum Level of Selenium
3.33 Effect of Aqueous Extract of S. mimosoides on the Activity of Lactase
3.34 Histopathological Examination on the Liver Control Group A
3.35 Histopathological Examination of Liver Cells of Rats in Group B
3.36 Histopathological Examination of Liver Cells of Rats in Group C
3.37 Histopathological Examination of Liver Cells of Rats in Group D
3.38 Histopathological Examination of Liver Cells of Rats in Group E
3.39 Histopathological Examination of Liver Cells of Rats in Group F
CHAPTER FOUR: DISCUSSION
4.1 Discussion
4.2 Conclusion
4.3 Suggestions for Further Studies
References
CHAPTER ONE
INTRODUCTION
Plants are known to contain a variety of secondary metabolites. These secondary metabolites or bioactive compounds have definite physiological effects on the human system. According to Yadav and Agarwala (2011), approximately 25 percent of all prescribed medicines today are substances derived from plants. Interestingly, many phytochemicals have been discovered and even isolated from a variety of medicinal plants. However, many more of them are yet to be exploited for clinical use. Phytochemical analysis of plants is important due to the need for alternative drugs of plant origin, made imperative by the high cost of synthetic drugs. These secondary plant metabolites extractable by various solvents exhibit varied biochemical and pharmacological actions in animals when ingested (Nwogu et al., 2008).
The use of Senna mimosoides in folklore medicine, precisely in Ukehe, Nsukka, to treat oedema and breastmilk toxicity in neonates was the rationale behind this work. The anti-inflammatory capacity of the leaf extract of Senna mimosoides and its mechanism of action has been reported by Ekwueme et al. (2011a,b).In Nsukka, immediately after delivery, breast milk is usually dropped on the leaves of cocoyam or on ants to check its toxicity.Toxic breast milk usually burns the leaves of the cocoyam or kills any ants it comes in contact with. The prevalence of industries predisposes mothers to chemicals that might accumulate in breast milk. In this study, the immunomodulatory activity and anti-hepatotoxic effect of the leaf extract of S. mimosoides was investigated because they are the basic mechanism used by the body to prevent or cure diseases. Moreover, the effect of the leaf extract on the activity of lactase,the enzyme that catalyzes the hydrolysis of lactose which is the only carbohydrate present in breast milk was assayed for.
Overview of the Human Immune System
Immunology is the study of the methods by which the body defends itself against infectious agents and other foreign substances in its environment (Wotherspoon, 2012). There are thousands of components to the immune system and it would appear that the immune system is far more complicated than necessary for achieving what is, on the surface, a simple task of eliminating a pathogenic organism or abnormal ‘self’ cells (Parkin and Cohen, 2001). However there are a number of reasons for this complexity, including the desirability of eliminating pathogens without causing damage to the host. Getting rid of a pathogen or dead host cells is theoretically easy, but eliminating these without damaging the host is much more complicated. As a consequence of this dynamic complexity, the immune system is able to generate a tremendous variety of cells and molecules capable of specifically recognising and eliminating an apparently limitless variety of foreign invaders, in addition to the recognition and destruction of abnormal cells (Parkin and Cohen, 2001). Once a foreign protein, microorganism (e.g., bacterium, fungus or virus) or abnormal cell is recognised, the immune system enlists the participation of a variety of cells and molecules to mount an appropriate effector response to eliminate or neutralise them (Parkin and Cohen, 2001). Later exposure to the same foreign organism induces a memory response, characterised by a heightened immune reactivity, which serves to eliminate the microbial pathogen, prevent disease and protect against the development of some tumour cells.
1.2 The Cells of the Immune System
1.2.1 T Lymphocytes
T-lymphocytes do not produce antibody molecules rather they directly attack foreign antigens such as viruses, fungi, or transplanted tissues (Kruisbeek et al., 2004). One T-cell class carries the CD8 molecule which binds to MHC class I while the other carries the CD4 molecule which binds to MHC class II. T-lymphocytes based on their function are grouped into killer or cytotoxic T-lymphocytes, helper T-lymphocytes, and regulatory T-lymphocytes. T cells displaying CD4+ generally function as TH cells, whereas those displaying CD8+ function as TC cells.Killer, or cytotoxic, T-lymphocytes perform the actual destruction of the invading microorganism (Lukashenka et al., 2008). They do this by migrating to the site of an infection or the transplanted tissues, directly binding to their target and killing it by lysing.
The helper T-lymphocyte and “helps” or enhances the function of B-lymphocytes, causing them to produce quickly more antibodies and to switch from the production of IgM to IgG and IgA and and also assist killer T-lymphocytes in their attack on foreign substances (Parkin and Cohen, 2001). Activation of TH cell makes it an effector cell that secretes various cytokines (O’Keefe et al., 2002) that plays an important role in activating B cells, TC cells, macrophages, and various other T cells, and initiate the delayed type hypersensitivity (DTH) response (Parkin and Cohen, 2001).Regulatory T-lymphocytes suppress or turn off other T-lymphocytes. Without regulatory cells, the immune system would keep working even after an infection had been cured and overreact to the infection (Vignali et al., 2008).
B-lymphocytes
B-lymphocytes (sometimes called B-cells) are specialized cells of the immune system whose major function is to produce antibodies (also called immunoglobulins or gamma globulins) (Leen et al., 2013). Antibodies are complex molecules (glycoproteins) that have the property of combining specifically to the antigen that induced its formation. Antibodies are catholic in their recognition; they can recognize free proteins, in solution; proteins displayed on cell walls or membranes; and proteins within higher-order structures, such as viral capsids. When B-lymphocytes are stimulated by antigens, they respond by maturing into plasma cells which are the cells that actually produce the antibodies. These antibodies then find their way into the bloodstream, tissues, respiratory secretions, intestinal secretions, and even tears. The resulting antibodies bind to the invading pathogen, marking it for destruction by killer T-lymphocytes by a process called antibody dependent cell cytotoxicity (ADCC) (Clemenceau, 2008). Antibodies also mark cells for phagocytosis by neutrophils and other phagocytic cells by a process called opsonization. Most of the daughter cells produced by B cell activation die within a few weeks but a proportion of them recirculate in the body for many years as memory cells. If they are reintroduced to the same antigen that elicited an initial response, they rapidly become reactivated and produce antigen-specific antibody (Leen et al., 2013). There are five distinct classes of antibody, based on the type of heavy chain involved; Immunoglobulin G (IgG); Immunoglobulin A (IgA); Immunoglobulin M (IgM); Immunoglobulin E (IgE); Immunoglobulin D (IgD).
The IgG class is the only class of immunoglobulins which crosses the placenta and passes immunity from the mother to the newborn (Walter and Thiel, 2011). Antibodies of the IgA fraction are produced near mucus membranes and find their way into secretions such as tears, bile, saliva, and mucus since it can be transported across, where they protect against infection in the respiratory tract and intestines. Antibodies of the IgM class are the first antibodies formed in response to infection. They are important in protection during the early days of an infection. Antibodies of the IgE class are responsible for allergic reactions. IgE sensitizes specialized ‘mast’ cells, important in protecting against parasitic infections.
1.2.3 Natural killer (NK) Cells
NK cells are large, granular lymphocytes that are capable of lysing or killing infected or tumour cells without overt antigenic stimulation or recognition (recruiting specific immune response) (Parkin and Cohen, 2001). These cells can be considered complementary to cytotoxic T lymphocytes (CTLs). Many viruses attempt to circumvent CTL recognition by preventing the MHC molecule from reaching the cell surface – and here natural killer (NK) cells step into the breach. These cells do not recognize specific foreign antigen, instead being activated by the absence of MHC molecules on a cell’s surface, activated NK cells destroy susceptible target cells by inoculating a protein named perforin into the target cell membrane; perforin molecules assemble in the membrane to form a pore, through which other toxic molecules can flow into the target. NK cells are also prolific producers of the antiviral cytokine interferon g (Kim et al., 2011). At the sites of inflammation, activated macrophages produces IL-12 which stimulate NK cells to produce IFN.
1.2.4 Monocyte and Macrophages
Monocytes which make up 2-8% of the WBCs leave circulation and enter tissue, as macrophages. There are two types of macrophages, one that wander in the tissue spaces and the other that are fixed to vascular endothelium of liver, spleen, lymph node and other tissue (Parkin Cohen, 2001). Macrophages are large leukocytes derived from monocytes that function in phagocytosis, antigen processing and presentation, secretion of cytokines and antibody-dependent cell-mediated cytotoxicity (ADCC). Functions of macrophage include killing of microbes, infected cells, and tumor cells, secretion of immunomodulatory cytokines, antigen processing and presentation to T cells. Macrophages respond to infections as quickly as neutrophils but persist much longer; hence they are dominant effector cells in the later stage of infection.
1.2.5 Antigen-Presenting Cells (APCs)
Specifically, APCs are any cells that can process and present antigenic peptides in association with class II MHC molecules on the surface of antigen-presenting cells or altered self-cells (Accolla and Tosi, 2012).These specialised cells, which include macrophages, B lymphocytes, and dendritic cells, are distinguished by two properties: they express class II MHC molecules on their membrane, and they are able to deliver a co-stimulatory signal that is necessary for TH-cell activation (Kuby, 1997). In the presence of soluble antigen, TH cells primed by dendritic cells can interact with B cells and stimulate antigen-specific antibody production (Girolamo et al., 2008). Dendritic cells are equally important in priming CD8+ or TC cells. Interestingly, dendritic cells can directly induce cytotoxic TC cell proliferation with help from TH cells. Antigen-presenting cells (APC) can also elicit a local rapid reaction or cascade of events that triggers the specific-immune responses.
1.2.6 Phagocytes
Phagocytes are specialized cells of the immune system whose primary function is to ingest and kill microorganisms. There are several different types of phagocytic cells. Polymorphonuclear leukocytes (neutrophils or granulocytes) are found in the bloodstream and can migrate into sites of infection within a matter of minutes. It is this phagocytic cell that increases in number in the bloodstream during infection and is in large part responsible for an elevated white blood cell count during infection. Polymorphs play a major role in controlling many infections, travelling rapidly to the affected site, assisting in the recruitment of other immune responses, and engulfing the microbes and other debris (Wang, et al., 2006). It is also the phagocytic cell that leaves the bloodstream and accumulates in the tissues during the first few hours of infection, and is responsible for the formation of “pus” (Dale et al., 2008). Monocytes, another type of phagocytic cell, are also found circulating in the bloodstream.
Neutrophils
Neutrophils are the most abundant leukocytes in our circulation and become rapidly mobilized to eliminate microbes and necrotic cells in areas of infection or inflammation (Nathan, 2006). Despite having a brief half-life and lacking proliferative potential, neutrophils have the ability to synthesize and release immunoregulatory factors, thereby helping the recruitment of DCs and monocytes that not only complete innate clearance of invading microbes, but also initiate more specific adaptive immune responses (Mantovani et al., 2011). Neutrophils are characterized by the presence of cytoplasmic granules primary (or azurophilic) granules which predominates in early stages of neutrophil maturation and are less capable of exocytosis than secondary (or specific) granules, which are generated in later developmental stages. Primary granules contain myeloperoxidase (MPO), which is important for the digestion of phagocytosed material (Mantovani et al., 2011) while secondary granules contain lactoferrin and gelatinase, which degrade the extracellular matrix, exert antimicrobial activity and initiate inflammation.
In addition to undergoing degranulation, neutrophils generate a respiratory burst by activating an enzymatic complex known as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which generates reactive oxygen species involved in microbial killing (Puga et al., 2012). Moreover, neutrophils can also form neutrophil extracellular traps (NETs), which are cellular projections capable of trapping and killing bacteria. These structures contain decondensed chromatin embedded with cytoplasmic and granular proteins with powerful antimicrobial functions, including serine proteases and antimicrobial peptides such as cathelicidin (Brinkmann et al., 2004).
Basophils and Mast Cells
Mast cells are tissue-resident leukocytes very similar to basophils. There are at least two populations of mast cells, based on the enzymes they contain and their tissue location (Parkin and Cohen, 2001). T mast cells (mucosal mast cells) contain only trypsin, whereas connective tissue mast cells contain both trypsin and chymotrypsin. Mast cells and basophils bear high-affinity receptors for IgE FcRI (CD23) which rapidly absorbs any local IgE (Puga et al., 2012). Crosslinking of these receptors by the binding of antigen to IgE leads to degranulation and release of preformed mediators, such as the vasoactive amines, histamine and serotonin. Membrane derived mediators such as leukotrienes B4, C4, D4 and E4, prostaglandins and platelet activating factor are also produced leading to increased vascular permeability, bronchoconstriction, and induction of an inflammatory response.
Basophils produce histamine and other vasoactive compounds, immunomodulating factors such as platelet-activating factor (PAF), leukotriene C4, granzyme B and retinoic acid as well as antibody-inducing and Th2-differentiating cytokines, including IL-4, IL-6 and IL-13(Karasuyama et al., 2011). Among basophil-tropic cytokines, IL-3 enhances basophil recruitment into lymphoid tissues, augments basophil secretion of IL-4 and promotes basophil expansion after parasite infection. However, some studies show that IL-3 is not required for the maintenance of basophils in vivo, probably because this function is also covered by the IL-7-like cytokine thymic stromal lymphopoietin (TSLP). Basophils release IL-4 and facilitate the differentiation of Th2 cells producing IL-4 in response to signals from IgE-binding antigens, cytokines (IL-3, GM-CSF, IL-33 or IL-18), microbial receptors (TLR2 and TLR4), and allergenic proteases (Sokol et al., 2009).
Eosinophils
Eosinophils, the second most frequent granulocyte subset in the circulation protects host from parasitic (particularly nematode) infections. Such infections induce antigen-specific IgE production, the antibodies coating the organism then eosinophils binds its low affinity receptors (FcRII). Eosinophils are not phagocytic, but have large granules containing major basic protein, eosinophilic cationic protein, eosinophil peroxidase, and eosinophil-derived neurotoxin, which are highly cytotoxic when released onto the surface of organisms (Puga et al., 2012). In recent years eosinophils have also been shown to modulate adaptive immunity as a result of their ability to up-regulate the expression of MHC-II molecules and secrete cytokines, chemokines, lipid mediators and growth factors (Puga et al., 2012).
Eosinophils modulate innate immune responses by regulating the activation of mast cells, basophils and neutrophils through MBP. In addition, eosinophils induce the expression of antigen-loading MHC-II and T cell costimulatory molecules after undergoing transendothelial migration and in the presence of appropriate cytokines (Akuthota et al., 2010). Eosinophil production of chemokines and cytokines such as TNF, IL-4 and IL-12 not only influences the recruitment and maturation of DCs, but also induces the differentiation of Th1 and Th2 cells.
1.3 Innate (Nonspecific) Immunity
Innate or nonspecific immunity which refers to the basic resistance to disease that an individual is born with, provide the first line of host defence against invading microbial pathogens and also protects against some tumour cells until an acquired immune response develops (Dhasarathan et al., 2010). Innate immunity can be envisioned as comprising four types of defensive barriers: anatomic; physiologic; endocytic and phagocytic; and inflammatory (Parkin and Cohen, 2001). The physiologic barriers that contribute to innate immunity include elevated temperature (e.g., fever), pH (e.g., acidity produced in stomach and within macrophages), oxygen tension, and various soluble factors (Kuby, 1997). Thera are also soluble proteins such as lysozyme, interferons (INF) and other cytokines and complement. A central feature of the innate reaction is recruitment and activation of neutrophils at the site of infection to eradicate pathogens. During the very early stages of infection or tissue damage, there is release of cytokines from activated macrophages. Two of these, granulocyte and granulocyte-macrophage colony stimulating factors, stimulate division of myeloid precursors in the bone marrow, releasing millions of cells into the circulation and causing a characteristic neutrophil leucocytosis (Wotherspoon, 2012). To home to a site of infection, neutrophils use a multistep process involving proinflammatory mediators, adhesion molecules, chemoattractants, and chemokines (Nathan, 2006). The recruited neutrophils phagocytose organsisms by making pseudopodia (projections of cytoplasmic membrane) which form a membrane-bound vesicle (phagosome) around the particle (Parkin and Cohen, 2001). In this protected compartment killing of the organism occurs by a combination of two mechanisms. The oxyg
