Basic concepts in immunity and inflammation

Periodontal diseases are a result of complex interactions between host’s immune system and microorganisms. To understand the pathogenesis of periodontal diseases, one must have the basic understanding of the immune response generated against the microorganisms. In the previous chapter, we studied about various microorganisms that are involved in the etiopathogenesis of periodontal diseases. Let us now try to understand the body’s mechanisms to counter these microorganisms.

Immunity

Immunity is defined as the resistance to disease, specifically infectious disease. The immune system is a collection of cells, tissues, and molecules that function to defend us against infectious microbes. The main function of the immune system is to prevent or limit infections. This defense function of the immune system is essential for our ability to survive in an environment that harbors potentially deadly microbes. But, sometimes immune responses can also be damaging. Many autoimmune diseases are caused by the uncontrolled or excessive immune response against self-antigens (e.g. rheumatic fever, asthma, glomerulonephritis, many dermatological disorders which may manifest as desquamative gingivitis, multiple sclerosis and etc.)

Immunity can be classified as:

  • Innate (with which an individual is born)
  • Acquired/Adaptive (which a person acquires)

Immune system has mainly two Components:

  • Cell-mediated immunity
  • Antibody-mediated(humoral) immunity

Two other major components of immunity are:

  • Phagocytes
  • Complement system

Before we study the cell-mediated immunity (CMI) and antibody-mediated immunity (AMI), here is a brief description of the lymphoid system,

Lymphoid system

The body uses the lymphoid system to enable lymphocytes to encounter antigens and it is here that adaptive immune responses are initiated. The organs involved in specific as well as non-specific immunity are classified as primary (central) lymphoid organs and secondary (peripheral) lymphoid organs 1 (Figure 6.1). The blood and lymphatic vessels that carry lymphocytes to and from the other structures can also be considered as lymphoid organs. The primary lymphoid organs are the sites of antigen-independent lymphocyte proliferation. Here, B- and T-lymphocytes differentiate from lymphoid stem cells into mature albeit naïve effector cells. These include bone marrow and thymus. The secondary or peripheral lymphoid organs provide an environment that enables lymphocytes to interact with each other, with accessory cells, and with antigens, resulting in the initiation of antigen-specific primary immune responses. These include lymph nodes, spleen, Peyer’s patches, tonsils, adenoids, appendix and mucosa-associated lymphoid tissue (MALT).

Here is a brief description of these lymphoid organs:

Bone marrow:

Bone marrow is the primary source of all the cells of the immune system. During fetal development hematopoiesis occurs initially in the yolk sac and para-aortic mesenchyme and later in the liver and spleen. Then it gradually shifts to bone marrow.  Bone marrow is the site of origin of all T- and B- cells, mononuclear phagocytes, platelets, erythrocytes, and other leukocytes in an adult humn being. It is divided into wedge-shaped hematopoietic compartments filled with proliferating and differentiating blood cells in the connective tissue matrices bordered by venous sinuses.

Figure 6.1 General distribution of lymphatics in human body 

Lymphatics of human body

Thymus:

This lymphoepithelial organ develops from ectoderm derived from the third branchial cleft and endoderm of the third branchial pouch including mesenchymal components derived from cells of the cephalic neural crest which all migrate from the neck to the anterior mediastinum. It reaches its greatest size just prior to birth, then atrophies with age. The major function of the thymus is the production of T-cells or thymocytes. Cells from the bone marrow migrate to the thymus as precursors and develop into mature peripheral T-cells. The majority of T-cell production occurs before puberty. After puberty, the thymus shrinks and the production of new T-cells in the adult thymus drops away. Children with no development of thymus suffer from Di George syndrome that is characterized by a deficiency in T-cell development but normal numbers of B-cells 2.

 

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Lymph nodes:

There are numerous lymph nodes disseminated all over the body. They measure 1-25 mm in diameter and play a very important and dynamic role in the initial or inductive states of the immune response.

Anatomical structure of a lymph node:

The lymph nodes are circumscribed by a connective tissue capsule (Figure 6.2). They receive afferent lymphatics draining peripheral interstitial spaces and also receive blood from the systemic circulation through the hilar arteriole. A lymph node has two main regions: the cortex and the medulla. The cortex and the deep cortex (also known as paracortical area) are densely populated by lymphocytes, in constant traffic between the lymphatic and systemic circulation. In the cortex, at low magnification, one can distinguish roughly spherical areas containing densely packed lymphocytes, termed as follicles or nodules 3. It is important to note that T- and B-lymphocytes occupy different areas in the cortex. B-lymphocytes predominate in the follicles (hence, the follicles are designated as T-independent area), which also contain macrophages, follicular dendritic cells, and some T-lymphocytes.

 

Figure 6.2 Structure of a lymph node

Structure of a lymph node

Lymphatic circulation:

The blood circulating through the body leaks into tissues through the thin walls of the capillaries. This portion of blood plasma that escapes is called as interstitial fluid 4. Lymphatic capillaries reabsorb the excessive tissue fluid and transport the fluid through the lymphatic pathway, and ultimately dispose it into the blood (Figure 6.3). When this extracellular fluid enters the lymphatic system, it is called as lymph. It drains into larger vessels called the lymphatics. These vessels converge to form one of the two large vessels called lymphatic trunks, which are connected to veins at the base of the neck. One of these trunks, the right lymphatic duct, drains the upper right portion of the body, returning lymph to the bloodstream via the right subclavian vein. The other trunk, the thoracic duct, drains the rest of the body into the left subclavian vein. The lymphatic pathway is an open circuit where lymphatic capillaries in body tissues reabsorb excessive tissue fluid which is derived from blood plasma.

Figure 6.3 Diagrammatic representation of Lymphatic circulation

Lymphatic circulation 1

This lymph ultimately returns to the blood plasma (i.e. blood plasma in capillaries → interstitial fluid → lymph in lymphatic pathway →lymph returns to blood plasma).

With this basic description of the lymphoid system, now let us discuss the humoral- and cell-mediated immunity.

Humoral immunity

Humoral immunity is the type of host defense that is mediated by antibodies, the products of B-cells. Antibodies are secreted into mucosal lumens, blood, and interstitial fluids, and combat microbes at all these sites. As already stated, B-cells are the source of antibodies, let us first understand the development and maturation of B-cells. During embryogenesis, B-cells are first recognized in the liver. From there they migrate to the bone marrow, they don’t require thymus 5.

Phases of B-Cell development and maturation:

The development and maturation of B-cells occur in multiple steps. The stages of B-Cell development start with stem cells which differentiate into the pre B-cell and then into mature B-cell (explained later in the chapter). During B-cell maturation, the first phase of maturation is an antigen independent phase consisting of stem cells, pre B-cells and B-cells. The second phase of maturation is an antigen dependent phase, which comes into play due to the interaction of antigen with B-cell. IgM (sometimes IgD) is first displayed on the B-cell surface, which act as a receptor for antigens. B-cells constitute about 30% of the circulating lymphocytes 6. Their life span ranges from days to weeks. Approximately 10B-cells are produced each day. There is a large pool of B-lymphocytes approximately 10with different specificities.

Activation of B-Cells:

Multivalent antigens directly attach to IgM and IgD on B-cell surface. It is due to cross-linkage of antigen with antibodies on B-cell surface. Another way is via T-cells, through the secretion of IL-4 and IL-5. For activation of T-cells, the co-stimulatory interaction between CD40-CD40L and B7-CD28 is necessary (discussed later).

Antibody Structure and Humoral Response

Soluble proteins that circulate in the body and perform the major functions of the immune system are antibodies. Initially, when found they were called as Ï’ globulins (migration on the field) but today they are called immunoglobulins. Two most important features of immunoglobulins are specificity and biological activity. These can identify large numbers of antigens. One part of the antibody is adaptable to a large number of epitopes and the other part participates in biological activities 7.

Isolation of Immunoglobulins:

Electrophoresis of serum gives five major components: Albumin, α1, Î±2, β and Ï’ globulins. Out of these, Ï’ globulin is least migrating. It forms a very small peak so was difficult to identify. However, in the case of multiple myeloma, immunoglobulins are elevated, so this peak is particularly high. In 1845, Henry Bence Jones discovered immunoglobulin proteins in urine of multiple myeloma patients. These proteins are known as Bence Jones proteins.

Structures of Light and Heavy Chains:

Porter in 1959 found that proteolytic treatment of enzyme papain splits immunoglobulins into three parts with almost equal molecular weight 8. Two of these parts can bind to antigen but no longer precipitate. These two parts are called as fragment antigen binding (Fab). The third fragment could be crystallized due to the property, indicative of its apparent homogenicity, so it was called as fragment crystallizable (Fc). It could not bind to antigen but it could participate in biological functions. His data led to the proposal of a Y-shaped structure of an antibody (Figure 6.4). At the same time, Edelman in the US discovered that Ï’ globulins were extensively reduced by treatment with mercaptoethanol (an agent that breaks disulfide bonds). It broke Immunoglobulin molecule into four chains, two heavy chains (H chains) of identical molecular weight up to 53,000 Daltons (D) and two light chains (L chains) having a molecular weight 22,000 D 9-11. On the basis of these findings, they proposed a structure of Immunoglobulins and got a noble prize in 1972.

Figure 6.4 Antibody structure

Antibody structure

Basic structure of Immunoglobulins:

  1. As already stated, the structure of immunoglobulins consist of light and heavy chains. A light chain contains about 211 to 217 amino acids and a heavy chain contains approximately 450 amino acids.
  2. All the species have only two types of light chains λ and κ. Every individual in a species synthesizes both of these chains, but ratio, i.e. Î»/κ varies from species to species (in mouse 95% and in human 60% are κ chains). But in any immunoglobulin molecule, both light chains are the same (either λ or κ). In humans, the ratio of immunoglobulin containing Îº chains to those containing λ chains is approximately 2:1.
  3. Heavy chains of virtually all species consist of five different classes (isotypes) that differ in their structure. H chains of different Immunoglobulins are IgM-µ, IgG-γ, IgA-α, IgE-ϵ, IgD-δ. Two heavy chains in an immunoglobulin molecule are the same. For example, IgG molecule can have κ2γ2 or Î»2γ2.
  4. Heavy chains confer on an immunoglobulin molecule its unique property such as half-lifein circulation, its ability to bind to certain receptors and activate enzymes.
  5. Further subdivision of these molecules gives subclasses. For example, IgG has been divided into IgG1, IgG2, IgG3, IgG4. In the same way, IgA has been divided into IgA1 and IgA2 . These sub-isotypes vary in number and arrangement of inter-chain disulfide bonds.
  6. L and H chains are subdivided into variableand constant regions. There are disulfide bonds present within a chain and between two chains. In a chain, these disulfide bonds make loops, called as domains. Most of the H-chains contain one variable (VH) and three constant (CH) domains. IgG and IgA have three CH domains, whereas IgM and IgE have four CH domains. Each domain contains about 110 amino acids.
  7. VHand VL are responsible for antigen binding, whereas constant region is responsible various biological functions, such as complement fixation and binding to the cell surface receptors. Certain amino acids in hypervariable regions are very variable. Only 5 to 10 amino acids in each hypervariable region form the binding There are three hypervariable regions present in both VH and VL. If we count from amino acid terminal these are found in and around 30, 50 and 95 amino acid regions which are called as complementary determining regions (CDR’S). Antigen-antibody binding involves electrostatic and van der Waals forces.
  8. Immunoglobulins of one species are antigenic to other species.

Individual Immunoglobulins:

Immunoglobulin G:

It is the predominant Immunoglobulin in blood that makes about 75% of the total immunoglobulins. The molecular formula is H2L2. It is a monomer and is divalent (has two identical antigen binding sites). It has got four subclasses with a ratio IgG1 : IgG2 : IgG3 : IgG4 = 66 : 23 : 7 : 4. It is synthesized in the fetus as early as 20 weeks of gestation and has a molecular weight of about 150000 D. Its sedimentation coefficient is 7S. Electrophoretically, IgG molecule is least anodic and migrate to gamma range. Except for the variable region, all immunoglobulins in one class have 90% homology. Thus, the antiserum is active against all classes of a particular immunoglobulin. The half-life of IgG1, IgG2 and IgG4 is 23 days, whereas for IgG3 it is 7days. IgG1 is responsible for erythroblastosis fetalis. The main functions of IgG include,

  1. Antibody-dependent cell-mediated cytotoxicity (ADCC): Virus infected cells can be destroyed by a combination of IgG and phagocytic cells. The antibody binds to the surface of the infected cell. This antibody is recognized by its receptor present on the phagocytic cell (macrophages or NK cells). The infected cell is killed.
  2. Activation of the complement
  3. Neutralization of toxins.
  4. Immobilization of bacteria by clumping their flagella and cilia.
  5. Neutralization of viruses (by attaching to surface of the virus and preventing its attachment to target cell)

Immunoglobulin M:

IgM is the first immunoglobulin to be produced after immunization. It is found in monomer or pentamer structure. For pentameric structure, the molecular weight is 900000 D and has a sedimentation coefficient of 19s. Five units are joined to each other by disulfide bonds. A polypeptide chain joins the Fc portion of monomer units called as J chain. This J chain is synthesized in B-cell or plasma cells.  It has a molecular weight of 15000 D. Valancy of IgM is 5 instead of 10, which is because of the congested structure of this immunoglobulin. Its Fab portion cannot open fully allowing attachment only to 5 antigen epitopes. If IgM is found to be raised in newborn, it is indicative of intrauterine infections of rubella, syphilis, toxoplasmosis, cytomegalovirus infections. It is an active activator of the complement system by classical pathway (Table 6.1).

Immunoglobulin A:  

It is the major immunoglobulin present in the external secretions such as saliva, mucous, sweat, gastric fluid and milk. It provides the neonate with protection against intestinal infections. Molecular weight is around 165000 D. Its sedimentation coefficient is 7s and it migrates to Î² or fast Î³ region. It may exist as a monomer or dimer. In secretions, it is secreted as a dimer. The dimers are united together by a J-chain. These units are covalently joined by J chain. It also has another polypeptide chain called the secretory component. Joining chain is a product of plasma cells. Secretory component is a product of epithelial cells and plays an active role in the transportation of IgA into various secretions.

Table 6.1 Description of various properties of antibodies

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Immunoglobulin D:

IgD is present in serum in very low concentration and its amount is variable. It is probably not secreted by plasma cells. It has not been designated for any definite function. But, it is said to be associated with IgM as a surface component of many B-cells. It is a monomer with a molecular weight around 150000 D and a sedimentation coefficient of 7S.

Immunoglobulin E:

It has an extra CH domain. Structurally, it is a monomer with a molecular weight around 190000 D. Its sedimentation coefficient is 8S and migrates to fast Î³ globulin region. IgE is called as â€œreaginic- antibody”. It is present in serum in lowest concentration. It has a specific domain on its H chain which attaches to the mast cells with high affinity. It attaches to mast cells and basophils through its receptors in Fc region. There are many cell surface receptors for FcR1 (10on basophils and 106 on mast cells). When two IgE molecules are cross-linked on these cells, they become activated and secrete its contents.

 

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Some facts about antibodies:

  • In blood highest concentration is that of IgG.
  • Fetal synthesis of IgM and IgA begin during the 5th
  • Plasma cells can secrete IgM, IgG, IgA, IgE (all except IgD).
  • IgA has 2 portions: J chain and S component.
  • IgM also has J chain.
  • Concentration in mg/ml = IgG: IgA: IgM: IgD: IgE = 12 : 1.8 : 1: 0-0.04: 0.00002

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  • Immunoglobulinpresent on the lymphocyte surface is IgD, and on basophils and mast cells is IgE.
  • Half-lives:
    • IgG1, IgG2, IgG4- 23 days
    • IgG3- 7 days
    • IgA – 5.5 days
    • IgM- 5 days
    • IgD – 2.8 days
    • IgE – 2 days
  • The placentalpassage is only for IgG.
  • IgA is primarily present in body secretions.
  • IgG and IgA are found in milk.
  • Complement activation is mainly by IgM but also by IgG.
  • Antiviral activity is highest with IgGs and IgA (IgM also)
  • Antibacterial activity is by IgG, IgA, IgM.
  • Antitoxin activity is only with IgG.
  • Allergic activity is with IgE.

 

Light and heavy chains of human antibodies:

Antibodies can also be explained in terms of isotypes, allotypes, and idiotypes (Figure 6.5).

Figure 6.5 The light and heavy chains making antibodies, their allotypes and isotypes

Immunoglobulins light and heavy chains

Isotypes:

Isotypes of antibodies are formed due to the differences in the amino acid sequence in their constant region. For example, IgG and IgA are isotypes as their heavy chains are different antigenically. Individual immunoglobulin can have its subtypes, such as IgG has its subtypes IgG1IgG2,  IgG3,  and  IgG4 (based on the antigenic difference on their heavy chains).

Allotypes:

Some features of antibodies vary from person to person, for example, IgG has 2H and 2L chains. Genes that code for γ chain are polymorphic. Every individual is inherited with different alleles which change the amino acid sequence from person to person.

Idiotypes:

These are antigenic determinants formed by specific amino acids present in the hypervariable region. Each idiotype is unique for an antibody producing cell.

Hence, it can be summarized that,

  • The constant region of heavy chain determines the immunoglobulin class.
  • The constant region of heavy and light chains determine the allotypes.
  • The variable region of light and heavy chains determine idiotypes.
  • The constant region of the heavy chains binds IgG to macrophages.
  • Fixation of complement is also in the constant region of the heavy chains.
  • Variable regions of light and heavy chains make the antigen binding sites.
 

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Immunodeficiency:

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Congenital B-Cell Immunodeficiency:

  • Burton’s agammaglobulinemia:This is an X- linked disease, so only males are affected.  In this condition, all Immunoglobulins are decreased to very low levels and deficiency of B-cell is also there. Pre B-cells are present, but they don’t form mature B-cells. This is due to lack of the enzyme tyrosine kinase. Cell-mediated immunity is normal. The patient starts having pyogenic infections after 6 months of birth as maternal IgG protection is reduced at this time.
  • Selective Immunoglobulin deficiency: The most common deficiency seen is that of IgA. IgG & IgM deficiencies are very rare. Patient with IgA deficiency has recurrent sinus & lung infection.

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Cellular Basis of Immune Response

During embryonic development, the blood cell precursors originate mainly in the yolk sac and fetal liver 12. These cells differentiate into myeloid and lymphoid series. Lymphoid series leads to the development of B-lymphocytes, T-lymphocytes, and NK cells, and myeloid series leads to the formation of monocytes and macrophages, erythrocytes, neutrophils, basophils, eosinophils, megakaryocytes /platelets and dendritic cells. The normal ratio of T-lymphocytes: B -lymphocytes is 3:1. Lymphocytes, the cells competent to initiate immune responses, can be divided into two major groups: thymus-derived (T-cells), responsible for “cellular immunity” (e.g. delayed hypersensitivity reactions) and bursa (or bursa-equivalent) derived (B-cells), which produce immunoglobulin (antibody) molecules and are involved in “humoral immunity”. “Accessory” cells, such as monocytes (or macrophages), polymorphonuclear leukocytes and mast cells act in an auxiliary manner by facilitating antigen processing or presentation, or by liberating factors which modify various manifestations of the immune response.

To understand the development and differentiation of lymphocytes, it is important to have the basic knowledge regarding major histocompatibility complex (MHC). Let us first discuss MHC before we read about T-cell and B-cell development.

Major histocompatibility complex

Cellular interactions are very important for the recognition and presentation of antigens to the immune system by antigen presenting cells 13, 14. The primary components of these interactions are T-cell antigen receptor (TCR) and the Major histocompatibility complex (MHC) / human leukocyte antigen (HLA) molecules. The major function of the TCR is to recognize the antigen in the correct context of MHC and to transmit an excitatory signal to the interior of the cell.

MHC classes:  

The MHC is highly polymorphic from individual to individual and segregates in families in a Mendelian co-dominant fashion.

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For MHC proteins, each individual has haplotypes i.e., one set of genes is inherited from each parent. These chains are very diverse (polymorphic) and there are many alleles of class I and class II MHC molecules. We have more than 47 alleles for HLA -A, 88 for HLA -B, 29 for HLA -C and 300 for HLA-D genes. An individual inherits only single allele from each locus and can make only one MHC-I and MHC-II protein. The expressions of these genes are co-dominant and are expressed. Each person can have 12 HLA proteins: 3 for class I and 3 for Class II from each parent. In addition to HLA genes which are encoding major antigen, there are several minor antigens encoded by several genes other than HLA. These genes lead to a slow antigen reaction, i.e., slows graft rejection. But a cumulative effect of these genes can produce more rapid graft rejection. Between Class I and Class II MHC, there is a third locus called as Class III. It encodes for two cytokines (TNF and lymphotoxins) and two complement components (C2 and C4), but it does not have any gene encoding for histocompatibility antigen.

Figure 6.6 Schematic map of human MHC on short arm of chromosome 6

smplified map of HLA region

Class I MHC:

The Class I MHC molecules are glycoproteins, which are found on the surface of virtually all nucleated cells. These are composed of two separate polypeptide chains, the heavier (44-47 KDa) α chain and the lighter (12 KDa) β chain. The α chain is identical to immunoglobulin and has three globular domains α1, α2, and α3. It has a hypervariable region at its N-terminal region. These regions are responsible for the recognition of self and non-self antigens. The carboxyl end of α chain resides inside the cell, while the amino end projects on the surface of the cell with a short intervening hydrophobic segment, which traverses the membrane.

Structure of Class I MHC molecules:

As already stated, structurally MCH I molecule consists of two polypeptide chains, a long α chain and a short β chain (β2-microglobulin) (Figure 6.7). The α chain has following four regions:

  • A cytoplasmic region, containing sites for phosphorylation and binding to cytoskeletal elements.
  • A transmembrane region, containing hydrophobic amino acids by which the molecule is anchored in the cell membrane.
  • A highly conserved α3 immunoglobulin-like domain to which CD8 binds.
  • A highly polymorphic peptide binding region formed from the α1 and α2 domains.

The β2- microglobulin in an association with α chain helps in maintaining the proper conformation of the molecule.

Figure 6.7 Structure of MHC molecule

Major histocompatibility complex

Class II MHC:

These are glycoproteins found primarily on the surface of the antigen-presenting cells, such as macrophages, B-cells, dendritic cells, cells of the spleen and Langerhans cells of the skin. MHC Class II molecules comprise of two non-identical and non-covalently associated polypeptide chains (α and β). These two chains have amino ends on the surface, a short transmembrane stretch, and intracytoplasmic carboxyl ends. Like MHC I, they have hypervariable regions. As already discussed,  the MHC I molecule is made up of two chains where chain I (α) is encoded by MHC and chain II (β2-microglobulin) is encoded by chromosome 15. In MHC II molecules both chains are encoded by MHC II. Two polypeptide chains have a constant region where the CD4 cell is attached.

Structure of Class II molecule:

Class II MHC molecules are composed of two polypeptide chains a α and a β chain of approximately equal length. Both chains have four regions:

  • A cytoplasmic region, containing sites for phosphorylation and binding to cytoskeletal elements
  • A transmembrane region, containing hydrophobic amino acids by which the molecule is anchored in the cell membrane.
  • A highly conserved α2 domain and a highly conserved β2 domain to which CD4 binds
  • A highly polymorphic peptide binding region formed from the α1 and β1 domains

Biological importance of MHC:

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These cells bear the responsibility to find out self and non-self cells. This is called as MHC-restriction. B-cells do not have this requirement as they can recognize the soluble antigens present in plasma with their surface associated IgG and/or IgM, acting as receptors.

With this basic description of MHC molecules, let us now discuss the T-cells and B-cells in detail.

T-Lymphocytes

T-lymphocytes arise from pluripotent stem cells in the bone marrow. Unlike B-lymphocytes, which undergo continued maturation in bone marrow, T-lymphocytes differentiate in the thymus. B-lymphocytes have immunoglobulins on their cell surface through which they recognize antigens but T-lymphocytes are surface-negative for immunoglobulins and do not exhibit DNA rearrangements in immunoglobulin genes. Rather, T-lymphocytes express a distinct membrane receptor for an antigen that recognizes antigen in conjunction with membrane glycoproteins encoded in the major histocompatibility complex. T-lymphocytes play a major role in the initiation and regulation of immune responses and are the key elements of cell-mediated immune responses against viruses, intracellular bacteria, and tumors. T-lymphocytes express different surface membrane antigens at various stages of their development and/or cell activation. These surface markers have been useful in the identification of phenotypic and functional diversity among T-lymphocytes. These markers previously were identified by means of monoclonal antibodies, which recognize specific antigenic determinants (termed epitopes) within a given surface membrane protein. So, a leukocyte surface marker, which is reactive toward a group (or cluster) of monoclonal antibodies, is identified according to a cluster of differentiation (CD) number (e.g. CD2, CD8). Certain CD markers are expressed by virtually all peripheral blood T-lymphocytes. This is the case with respect to CD2, a 50-KD glycoprotein through which T-lymphocytes form rosettes with sheep red blood cells. Other CD markers are useful in segregating T-lymphocytes into a number of distinct subpopulations. Thus, approximately 60 percent of peripheral blood T-lymphocytes express CD4, a glycoprotein expressed on T-cells whose activation is dependent upon the recognition of antigen in conjunction with Class II MHC molecules. Approximately 30 percent of peripheral blood T-lymphocytes express CD8; a membrane protein expressed by T-cells whose activation is dependent upon the recognition of antigen in conjunction with Class I MHC molecules. The majority of circulating T-lymphocytes express either CD4 or CD8, but not both. These two surface markers define subsets of T-lymphocytes with significantly different effector functions. Notably, CD4 T-lymphocytes typically function as helper (designated TH) cells, providing “help” to other T-lymphocytes as well as to immunoglobulin-producing B-lymphocytes. On the other hand, CD8 T-cells exhibit cytotoxic /suppressor (Tc or Ts) activity. CD8 T-lymphocytes also display cytotoxic activity toward numerous virally infected or tumor cells. It is not clear whether the suppressor and cytotoxic activities are mediated by the same or distinct subpopulations of CD8 T-cells.

T-lymphocyte ontogeny:

T-cell development occurs in the thymus. T-cell precursors, however, arrive at the thymus from the bone marrow. The stages of T-cell development are identified by the expression of specific cell surface markers, such as TCR (T-Cell Receptor), CD3 (which serves as the signal transduction component of TCR), and CD4/CD8. In the cortex of thymus within the cortical epithelium, T-cells progenitors differentiate under the influence of thymic hormones (Thymosin and Thymopoietin) into T-cell subpopulation. All T-cells have a CD3 receptor on their surface in association with antigen receptors. CD3 is a complex of 5 trans-membranous proteins which take information from the outside to the inside of the cell. Signal transduction is by Zeta-chain (transmembranous protein) which has tyrosine kinase activity. CD4 has single trans-membranous polypeptide, whereas, CD8 has 2 transmembrane polypeptides. These receptors may signal intracellularly by tyrosine kinase.

T-lymphocytes undergo differentiation in the thymus, irrespective of whether they express the CD4 or CD8 phenotype. The process of T-lym­phocyte maturation begins with the migration of T-cell precursors from the bone marrow to the cor­tical regions of the thymus. It is thought that these cells are attracted to the thymus by chemical signals provided by thymic epithelial cells. The earliest recognizable thymocyte committed to the T-cell lineage is the pro T-cell. These cells bear CD2 (the sheep erythrocyte receptor) but do not express the T-cell antigen receptor. Pro T-cells are also surface-negative for CD4 and CD8 (Double Negative or DN cells) (Figure 6.8).

During the next phase of development, maturing cortical thymocytes proceed with one of the two alternative pathways. In one instance, the thymocytes begin to re-arrange DNA segments encoding for the variable and constant regions of an alternative form of the T-cell antigen receptor (designated λ / δ TCR) and express this receptor in conjunction with a tightly associated complex of five membrane glycoproteins that form the CD3 complex. Most cells expressing the λ / δ TCR fail to express either CD4 or CD8 during further development and are released into the peripheral circulation as “double-negative” T-lymphocytes. The precise function of these λ / δ TCR-expressing cells have not been defined. The majority of pro T-cells follow a separate pathway of differentiation. These pre T-cells co-express both CD4 and CD8 (double positive cells) but do not yet express an antigen receptor. Subsequently, these cells undergo DNA rearrangements of genes encoding the constant and variable regions of the α / β TCR, which is expressed by the majority of mature T-lymphocytes in the periphery. At this stage, the thymocytes are both CD4 and CD8 positive and express the α / β TCR in conjunction with the CD3 complex. During the first stages of development, which occur in the thymic medulla, two important events take place, called as thymic education.

Figure 6.8 Development and maturation of T-cells from their precursors in bone marrow

Development and maturation of T-cells

Thymic education:

It is an important step in the maturation of T-cells. First, the cells progressively lose either CD4 or CD8. Secondly, these cells are “educated” by thymic epithelial cells to learn to differentiate between “self’ and “non-self’ MHC gene products. Those cells, which are auto-reactive toward self-MHC molecules, are eliminated by a process of clonal deletion, an important mechanism of self-tolerance. As a consequence of this selection process, only about 10 percent of the immature T-cells, which enter the thymus eventually reach the peripheral circulation.

Positive selection:

Since TCR’s recognize antigen only in the context of MHC’s, T-cells must be tuned to recognize host MHC first. During positive selection double-positive T-cells that can recognize self MHC’s are selected for proliferation and those T-cells that do not recognize self MHC die via apoptosis. The positive selection also assures that the right TCR selection will go with the appropriate CD4 or CD8. For example, TCR’s specific for MHC II need to retain CD4 and lose CD8. If the reverse occurs, they will die via apoptosis. The same is true for the T-cells that are specific for MHC-I, which need to retain CD8, and lose CD4

Negative selection:

At this point, those T-cells that are strongly activated by self MHC and/or self-peptides need to be eliminated in the thymus. If they escape this elimination, they may subsequently react against self-antigens, and cause autoimmune disease.
In summary, positive selection selects for those T-cells that react with MHC: self-antigen. Negative selection eliminates those that react strongly with MHC: self-antigen. Thus, successful T-cell differentiation selects for MHC-restricted TCR’s with low affinity for self-antigens. Cells that fall outside this range primarily die via apoptosis. The rationale here is that a T-cell that binds weakly to self-MHC/self-antigen will not be activated, but will be activated by a stronger binding to self-MHC/ foreign antigen complex.

Mature T-cells, which survive this selection process leave the thymic medulla through the walls of postcapillary venules. After circulating for a time, these T-lymphocytes distribute among various peripheral lymphoid tissues, including thymus-dependent regions of the inner cortex of lymph nodes, spleen, and mucosa-associated lymphoid tissue (e.g., Peyer’s patches in the colon). It is presently unknown what signal(s) drives proliferation and differentiation of immature thymocytes. These cells do express receptors for certain cytokines, which exhibit growth factor activity, including interleukin-2 (IL-2) and interleukin-7 (IL-7), produced by thymocytes and stromal cells, respectively. Alternatively, thymic epithelial cells may induce thymocyte activation via CD2 molecule, which recognizes a cell adhesion molecule (LFA-3) expressed on the epithelial cells.

Naïve Th cell:

Naïve Th cell is a T-cell that has differentiated in bone marrow and has successfully undergone the positive and negative selection processes in the thymus. A naïve T-cell is considered to be mature and unlike activated T-cells or memory T-cells, it has not encountered its cognate antigen within the periphery. Upon activation, naïve Th cells become Th0, since they have both Th1 and Th2 characteristics, with further stimulation Th0 cells deviate either towards Th1 or Th2. Th1 and Th2 cells are classified based on the pattern of cytokines that they secrete. If the Th0 cell primarily secretes IL2 and INF-γ, it is termed as Th1 cell. If the Th0-cell primarily secretes IL4, IL10, and IL13, it is termed as Th2 cell.

Natural killer (NK) cells

Natural killer (NK) cells are granular lymphocytes, which do not pass through the thymus. They don’t have CD4 or CD8 proteins and an antigen receptor. They kill virus-infected and tumor cells without the requirement of antigen presentation or MHC proteins 15. NK cells are activated by IL -2.  These are lymphocytes of the innate immune system that are involved in early defenses against 

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Dendritic cells:

Dendritic cells (DCs) were originally identified by Steinman and his colleagues in 1972 16. They are potent antigen presenting cells (APCs) that possess the ability to stimulate naïve T-cells. They comprise of a system of leukocytes widely distributed in all the tissues, especially in those that provide an environmental interface. DCs are derived from bone marrow progenitors and circulate in the blood as immature precursors prior to the migration into peripheral tissues. Within different tissues, DCs differentiate and become active in the taking up and processing of antigens and their subsequent presentation on the cell surface linked to MHC molecules. Upon appropriate stimulation, DCs undergo further maturation and migrate to secondary lymphoid tissues where they present antigen to T-cells and induce an immune response.

B-lymphocytes

B-lymphocytes are generated from the common lymphoid progenitors, which are originated from the differentiation of the hematopoietic stem cells. The yolk sac, fetal liver, and the adult liver are the sites in the body where these cells are generated and developed 17-19. B-lymphocytes play crucial roles in host defense against infection via a series of highly coordinated processes that include cell homing, antigen recognition, antibody secretion, antigen presentation, and/or cytokine release. The steps of maturation of B-cells are as follows:

In the bone marrow in early stages, the B-cell precursors (pre-pro B-cells) must interact physically with the stromal cells for their proliferation and maturation to occur. Later stages (late pro B-cells) merely need the soluble growth factors produced by stromal cells. Stromal cells produce several necessary growth factors and cell-cell adhesion molecules. One key growth factor for B-lymphopoiesis is interleukin-7 (IL-7).

The earliest identifiable stage of B-cell differentiation is the pre-pro B-cell. At this stage, the process of immunoglobulin gene rearrangement begins. The first re-arrangement entails the joining of the D segment to the J segment of the immunoglobulin heavy chain gene (IgH). Subsequent rearrangements bring the V region juxtaposed to the DJ portion. After this, surrogate light chains can combine with the μ heavy chain protein in pro- and pre- B-cells, forming a structure referred to as the pre B-cell receptor (Figure 6.9).

Figure 6.9 Diagrammatic representation of B-cell lineage development from its precursors in bone marrow

B-cell maturation stages

A newly formed B-cell displays IgM on its cell surface. At this stage, the B-cell is still immature and responds to antigen differently than a mature B-cell. Immature B-cells can be functionally removed by interaction with self-antigen, either by undergoing programmed cell death (apoptosis) or by anergy, in which the cell is rendered non-responsive in the presence of the antigen. Thus, similar to T-cells, immature B-lymphocytes undergo a process of “negative selection” to delete cells that are reactive to “self” antigen.

Immature B-cells that are not removed by the processes of negative selection leave the bone marrow and migrate to peripheral or secondary lymphoid tissues such as spleen and lymph nodes. Here, further maturation takes place and the newly formed B-cells express IgD, in addition to IgM, on the cell surface. The mature B-cells are now fully responsive to antigens and interaction with T-cells.

Macrophages

Macrophages are a part of the innate immune response. Unlike T- and B-cells, they do not contain any specific receptors. Macrophages have the important function of homeostasis as they continuously phagocytose self-proteins and cells in their vicinity, during normal tissue repair and aging (e.g. old red blood cells). All of these proteins are degraded and presented on MHC-II. As they are self-proteins so they do not activate T-cells, because, in the absence of infection, macrophages express low levels of MHC-II, and almost no co-stimulator (B7). Further, T- cells with the high-affinity receptor for self-peptides have been deleted during T-cell development in the thymus.
When there is an infection, macrophages possess certain types of receptors that recognize differential carbohydrate patterns on foreign cells. They also have receptors for specific bacterial products such as lipopolysaccharide (LPS) (endotoxin). When these molecules bind their bacterial ligands, macrophages become strong antigen-presenting cells because of upregulation of MHC-II and B7. They also start to secrete cytokines that aid in their functions (IL-1, 6, 8, 12 and TNF-α). It is at this point that antigen presentation by MHC II will activate Th cells 20.

The complement system

Term complement means to augment the effect of other components of the immune system. The complement system is a group of more than 23 proteins that interact with each other to opsonize the pathogen and induce a series of inflammatory responses that help to generate an immune response against that pathogen. Many of these proteins are proteases that are themselves activated by proteolytic cleavage. The principal participants in this system are 11 proteins designated C1 through C9, B, and D.

Historical aspect:

The complement system is an important component of the innate immune response. Buchner (1891) 21 found a heat labile factor in blood that was capable of killing bacteria and named it ‘alexin’ (in Greek, means ‘to ward off’). In 1895 Bordet discovered that this heat-labile activity in serum was responsible for the lysis of bacteria. Vibrio cholerae were lysed within minutes when serum from an immunized animal was added to the bacteria. However, when this serum was incubated at 56°C for a few minutes, the lytic activity was lost even though the antibody activity remained. Untreated serum, when added to the reaction restored the lytic activity. Therefore, the lysis of bacteria required both the antibody and this heat labile substance 22. Ehrlich (1899) named this heat labile substance as ‘Complement’ 23.

Bordet described the antibody dependent arm of the complement system, referred to as ‘classical pathway’.  An antibody-independent mechanism of complement system was discovered by Pillemer in 1954 and he named it “properdin system” 24, 25. All components of the classical pathway and the membrane-attack complex (MAC) are designated by the letter C followed by a number. The components were numbered in the order of their discovery rather than the sequence of reactions, which is C1, C4, C2, C3, C5, C6, C7, C8, and C9. The complement system involves cleavage reactions, where complement proteins are cleaved into a larger fragment which is designated as ‘b’ and the smaller fragment which is designated as ‘a’ fragment.

Activation of complement system:

Components of the complement systems are pro-enzymes. These components are cleaved to form active enzymes. Complement system can be activated by immune complexes and immunologic molecules like endotoxins. It occurs by 3 mechanisms (Figure 6.10),

  1. Classical pathway.
  2. Alternative pathway.
  3. Lectin pathway.

Pathways of complement system:

Classical pathway:

The binding of the antibody to its antigen triggers the complement system through the so-called classical pathway. It can occur in solution or when the antibodies have bound to antigens on a cell surface. Only IgM and IgG can activate the complement cascade. One molecule of IgM can activate the complement, but activation by IgG is with the help of cross-linking of two molecules. C1 binds to the region located in the FC region of the heavy chain. Out of all IgG’s only IgG1, IgG2, and IgG3 can fix the complement and not IgG4.

Step by step activation of classical pathway:

C1 activation:
C1, binds to the Fc region of IgG or IgM antibody molecules that have interacted with the antigen.  C1 binding does not occur to antibodies that have not been complexed with the antigen and binding requires the presence of calcium and magnesium ions (in some cases C1 can bind to aggregated immunoglobulin [e.g. aggregated IgG] or to certain pathogen surfaces in the absence of an antibody).  The binding of C1 to the antibody is via C1q and C1q must cross-link at least two antibody molecules before it is firmly fixed.  The binding of C1q results in the activation of C1r which in turn activates C1s.  The result is the formation of an activated “C1qrs”, which is an enzyme that cleaves C4 into two fragments C4a and C4b.

C4 and C2 activation (generation of C3 convertase):
The C4b fragment binds to the membrane and the C4a fragment is released into the microenvironment.  Activated “C1qrs” also cleaves C2 into C2a and C2b.  C2a binds to the membrane in association with C4b, and C2b is released into the microenvironment. The resulting C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and C3b.

C3 activation (generation of C5 convertase):
C3b binds to the membrane in association with C4b and C2a, and C3a is released into the microenvironment.  The resulting C4bC2aC3b is a C5 convertase.  The generation of C5 convertase is the end of the classical pathway. It results in the formation of C5a and C5b where C5a diffuses away and C5b participates in the formation of ‘membrane attack complex’ (MAC). MAC that is responsible for the lysis of the bacterial cell is formed by C5b, C6, C7, C8 and C9.

Alternative pathway:

This pathway is activated in the presence of immunogenic bacterial, fungal and viral cell surface substances. There is no antibody present, as it is a new infection. The cell wall polysaccharides and endotoxins possess binding to C3 and factor B. This complex is cleaved by a protease, factor D to produce C3bBb. The C3bBb thus formed, acts as C3 convertase to form C3b. It must be noted here that alternate pathway is more important when we first time get infected by microorganisms and the antibodies against them, required to trigger the classical pathway are not present.

Lectin binding pathway:

The lectin binding pathway is very similar to the classical pathway. It is initiated by the binding of mannose-binding lectin (MBL) to the bacterial surfaces with mannose-containing polysaccharides (mannans). Binding of MBL to a pathogen results in the association of two serine proteases, MASP-1 and MASP-2 (MBL-associated serine proteases). MASP-1 and MASP-2 are similar to C1r and C1s, respectively, and MBL is similar to C1q. Formation of the MBL/MASP-1/MASP-2 tri-molecular complex results in the activation of the MASPs and subsequent cleavage of C4 into C4a and C4b. The C4b fragment binds to the membrane and the C4a fragment is released into the microenvironment. Activated MASPs also cleave C2 into C2a and C2b. C2a binds to the membrane in association with C4b and C2b is released into the microenvironment. The resulting C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and C3b. C3b binds to the membrane in association with C4b and C2a and C3a is released into the micro-environment. The resulting C4bC2aC3b is a C5 convertase. The generation of C5 convertase is the end of the lectin pathway. The biological activities and the regulatory proteins of the lectin pathway are the same as those of the classical pathway.

Figure 6.10 The complement system

Complement activation pathways

Regulation of the complement system:

At antibody level: Normally complement binding site located in the heavy chain on Fc region of antibodies is not available for C1 component of the complement system. It is only after the binding of antigen, conformational changes take place which allows C1 complement to bind to the antibody. That is why the complement is not activated even when a high amount of IgM and IgG are present blood.

Regulation by serum proteins: C1 Inhibitor (C1-INH) is an important regulator of the complement system (Table 6.2). It inhibits the C1r and C1s serine proteases and prevents the auto-activation of the C1qrs complex. C1-INH is also a biologically significant inhibitor of kallikrein and coagulation factor XII. Its synthesis is stimulated by interferon-γ, interleukin 1 (IL-1) and IL-6.

C3a inactivator (C3a-INA; Carboxypeptidase B):  It inactivates C3a.

Factor H: Its principal function is to regulate the alternative pathway of the complement system. It accelerates the decay of the alternative pathway C3 convertase (C3b,Bb) and is also a cofactor for factor I-mediated cleavage and inactivation of C3b. In the absence of factor H, spontaneous activation of the alternative pathway of complement occurs in plasma, which leads to consumption of complement components C3 and factor B.

Decay accelerating factor [DAF]: DAF is a glycol protein located on the surface of human cells that protects them from lysis by the MAC. It acts by destabilizing C3 convertase and C5 convertase.

C4 binding protein (C4-BP) and Factor I: C4-BP facilitates the degradation of C4b by Factor I. C4-BP also prevents the association of C2a with C4b, thus blocking the formation of C3 convertase.

Protein S (vitronectin): After the formation of MAC, some of the C5bC6C7 complexes formed can dissociate from the membrane and enter the fluid phase. It can bind to other nearby cells and lead to their lysis. This damage is prevented by Protein S (vitronectin). Protein S binds to the soluble C5b67 and prevents its binding to other cells.

Table 6.2 Components of complement system, their actions, and their controls

[table “124” not found /]

Biological effects of the complement components:

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Basic concepts in inflammation

In simple words, inflammation is the response of the living tissue to injury. The word inflammation is derived from Latin word ‘inflammare’ (to set on fire). It is a well-organized sequence of events that take place in a living tissue following an injury due to mechanical trauma, chemical trauma, toxins and infection with pathogens which produce products that are harmful to the host.

The credit for the first documentation of the four cardinal signs of inflammation goes to a Roman, Aulus Cornelius Celsus, more commonly known as Celsus (1st century AD) (Table 6.3). He was the first one to highlight first four signs of inflammation rubor et tumor cum calore et dolore (redness and swelling with heat and pain) 26, 27. The fifth sign of inflammation, function laesa (loss of function) was added by Virchow in 1871 28. With advances in microscopy, inflammation was defined at the cellular level. The historical milestones in our understanding of inflammation are given in the table…

Table 6.3 Key historical events in the research on inflammation

[table “125” not found /]

Inflammatory response:

The inflammatory response consists of various changes in the tissue, including circulatory (hemodynamic) changes, changes in the vessel wall permeability, response of white blood cells and release of soluble chemical mediators of inflammation. Let us discuss these changes in detail,

Circulatory (hemodynamic) changes:

The vascular changes during the acute inflammatory reaction were described by Lewis in 1927 as ‘the triple response to injury’ following application of a blunt instrument drawn firmly across the skin. These include flush (a dull red line follows due to capillary dilatation), flare (a red, irregular, surrounding zone then develops, due to arteriolar dilatation) and wheal (a zone of edema develops due to exudation into the extra-vascular space). A lot of research has been done since then.

The microcirculation of the soft tissue is made up of a network of small capillaries lying between arterioles, which have a thick muscular wall, and thin-walled venules. There is no smooth muscular layer in capillaries to control their diameter and these are quite narrow in diameter. Thus, the smooth muscle wall present in the arterioles controls the blood flow through the capillary bed by acting as a sphincter. Under normal conditions, the blood flow through the capillaries is intermittent. There are some preferential channels through which blood flows, while others are usually not used for blood flow. The blood flow in vessels larger than capillaries is different in that the blood cells flow mainly in the center of the lumen while plasma flows close to the vessel wall. This feature keeps the blood cells away from the vessel walls and facilitates the smooth blood flow. The vascular changes that occur during inflammation are as follows,

Increased vascular permeability:

Blood vessels are mostly lined by a single layer of endothelial cells, which form a layer of uniform thickness around the vessel wall. These vessels, thus act as a micro filter, allowing the passage of water and solutes, but also act as a barrier for the passage of blood cells. Along with the transfer of fluid and solutes through ultra-filtration, there is diffusion of gasses like oxygen and carbon dioxide. The fluid that passes out of the vessels returns back to the vascular compartment because of high osmotic pressure inside the blood vessels due to the presence of plasma proteins. Thus, under normal conditions the fluid that is forced out of the blood vessels at the arteriolar end due to high hydrostatic pressure returns back into the blood vessels at the venous end due to low hydrostatic pressure. This balance between the hydrostatic and osmotic pressure in the blood vessels is essential for the maintenance of soft tissue health.

During acute inflammation, vasodilation at the arteriolar end is one of the earliest changes observed. It causes opening of new capillary beds in that area. This results in increased blood flow, causing heat and redness. Vasodilation is induced by the action of several mediators, notably histamine, and nitric oxide, on vascular smooth muscles (discussed later). Furthermore, the increased hydrostatic pressure at the arteriolar end results in the escape of more fluid along with plasma proteins into the extravascular compartment. The escape of plasma proteins into the extravascular compartment further facilitates fluid accumulation due to increase in osmotic pressure outside the vascular compartment. Thus, more fluid leaves the blood vessels than what is returned to them. This fluid, which contains plasma proteins is called as exudate.

Accumulation of cells and formation of cellular exudate:

The accumulation of polymorphonuclear cells (PMN’s) in the extracellular space is the hallmark of acute inflammation. There is a specific manner in which PMN’s come out of the blood vessels and migrate towards the site of injury. The process of neutrophil migration from the blood vessels is termed as ‘trans-endothelial migration of neutrophils’. Now, let us discuss this procedure in detail.

Transendothelial migration of neutrophils

As already stated, under normal condition, the blood cells are confined to the central stream in the blood vessel and not in the peripheral or plasmatic zone. However, following an injury, the acute inflammatory reaction is initiated which results in the loss of intravascular fluid and increase in plasma viscosity with slowing of flow at the site of acute inflammation. The reduced flow rate of the blood allows neutrophils to flow in the plasmatic zone. Once, the neutrophils come near the wall of the blood vessel, the process of trans-endothelial migration is initiated. There are a number of receptor molecules that are involved in the process of leukocyte migration from inside the blood vessel to outside in the connective tissue. The knowledge of these receptors is essential to understand the mechanism of trans-endothelial migration.

Molecules involved in trans-endothelial migration:

The role of the majority of following molecules in the trans-endothelial migration of leukocytes has been demonstrated by blocking the respective receptors by antibodies.

Selectins:

Selectins belong to a family of transmembrane molecules, expressed on the surface of leukocytes and activated endothelial cells. They are of 3 types; L-selectin, P-selectin, and E-selectin. The smallest of these is L-selectin, which is found on most leukocytes. P-selectin, the largest selectin, is expressed on activated platelets and endothelial cells primarily. E-selectin is expressed on activated endothelium during chemically or cytokine-induced inflammation. These contain an N-terminal extracellular domain with structural homology to calcium-dependent lectins, followed by a domain homologous to epidermal growth factor, and two to nine consensus repeats (CR) similar to sequences found in complement regulatory proteins. Each of these adhesion receptors is inserted via a hydrophobic transmembrane domain and possesses a short cytoplasmic tail. During inflammation, the initial attachment of leukocytes from the blood stream is because of these molecules which cause slow downstream movement of leukocytes along the endothelium via transient, reversible, adhesive interactions, called leukocyte rolling.

Integrins:

These are transmembrane adhesive heterodimeric glycoproteins, made up of α and β subunits that function as receptors for the extracellular matrix.  β subunit is known as CD18 and the α subunit is known as CD11. The α subunit is found in 4 forms, namely a, b, c and d. So, based on α subunit variability, these glycoproteins are classified as,

  • CD11a/CD18, also known as lymphocyte function associated antigen-1 (LFA-1),
  • CD11b/CD18, also known as macrophage receptor-1 (MAC-1),
  • CD11c/CD18 (p150, 95/CR4) and
  • CD11d/CD18 (αDβ2).

The principal receptors for ICAM-1 are the β integrins LFA-1 and MAC-1, and those for VCAM-1 are the integrins α4β1 and α4β7.

Intercellular adhesion molecules (ICAM-1 and ICAM-2):

These molecules perform many important tasks during trans-endothelial migration. They belong to the immunoglobulin superfamily and represent endothelial ligands for the leukocyte β-2 integrin LFA-1. ICAM-1 is constitutively expressed on endothelial cells, platelets, and most leukocytes whereas, ICAM-2 appears to be concentrated at endothelial cell junctions.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) or CD-31:

These molecules help in the endothelial cell to cell adhesion via homophilic interactions and they help in transendothelial migration through endothelium-leukocyte interactions. PECAM-1 homophilic interactions allow leukocytes to emigrate through the endothelial barrier 29. These belong to the immunoglobulin gene superfamily and are expressed on leukocytes, platelets, neutrophils, monocytes, and selected T-cell subsets 30.

Junctional adhesion molecules (JAMs):

JAMs are the members of the immunoglobulin gene superfamily. JAM proteins are localized in the intercellular junctions of polarized endothelial and epithelial cells. They are found in three forms JAM-A, JAM-B, and JAM-C.  JAM-A is expressed at epithelial tight junctions and intercellular borders of endothelial cells, as well as on the surfaces of megakaryocytes. JAM-B and JAM-C are believed to be involved in leukocyte adhesion, transmigration, and interactions between different cell subsets during inflammation 31. Although JAM-A normally engages in homophilic adhesion, during inflammation it can bind to CD11a/CD18 on the leukocyte 32.

Vascular cell adhesion molecule-1 (VCAM-1):

It is an adhesion molecule that is not constitutively expressed on endothelial cells, but is upregulated by chemokines 33. This molecule has been shown to interact with monocytes and lymphocytes and participates in leukocyte transmigration during the inflammatory response 34.

VE-cadherin:

VE-cadherin is a transmembrane protein that establishes homotypic calcium-dependent interactions with its extracellular domain. It is one of the major components of adherence junctions. Although, many proteins have been implicated in endothelial cell-cell adhesion 34, 35; VE-cadherin has a central role in the regulation of the integrity of the endothelial barrier and leukocyte transmigration as evidenced in vitro and in vivo studies. The juxtamembrane domain binds p120 catenin, while the membrane distal domain binds β-catenin and plakoglobin in a mutually exclusive fashion that depends on cell-cell contact maturation. Finally, α-catenin alternately associates to β-catenin/plakoglobin or to the actin cytoskeleton.

CD99:

CD99 is a 32 kD, a highly O-glycosylated molecule that is expressed on the surfaces of most leukocytes and is concentrated at the borders between confluent endothelial cells 35. Similar to PECAM-1, CD99 functions in a homophilic manner in the transmigration of leukocytes but CD99 regulates a later step in this process as compared to PECAM.

CD99L2 (CD99-like molecule 2):

It represents a protein of unknown function with moderate sequence homology to CD99, which is expressed on leukocytes and endothelial cells 36. Similar to PECAM, the homophilic interaction between CD99 at the endothelial cell border and CD99 on monocytes 37 and neutrophils 37 is required for transmigration.

Steps in transendothelial migration of leukocytes:

  • Slow rolling,
  • Adhesion strengthening,
  • Intra-luminal crawling,
  • Transendothelial migration,
  • Migration through the basement membrane, and
  • Interstitial migration

Step by step mechanism of transendothelial migration:

  1. As already stated, inflammation is a protective response of the body to any insult, which is manifested by the release of a variety of pro-inflammatory mediators from resident leukocytes and mast cells, including cytokines like IL-1β, TNF-α etc. Complement components like C3a and C5a are also important initiators of trans-endothelial migration of leukocytes.
  2. These mediators stimulate endothelial cells to express P-selectin and E-selectin on their luminal surfaces 37. Initial tethering and rolling are mediated by P-, E- and L-selectins 38 (Figure 6.11). The initial rolling brings the leukocyte into proximity with endothelial cells, where it can be activated by luminal surface-bound chemokines or lipid chemoattractants (for example, platelet activating factor- PAF) 39. Recent works suggest that not only selectins, but also integrins such as LFA-1 and MAC-1 also support leukocyte rolling 40.
  3. After initial tethering and rolling the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) are expressed. Their binding to activated integrins probably contributes to adhesion stabilization and cell motility. In addition to mediating adhesion, integrins also generate intracellular signals that regulate various cellular functions 41, 42.
  4. Subsequently, leukocytes crawl inside the blood vessels in a MAC-1 and ICAM-1 dependent manner, seeking the preferred sites for diapedesis 43. After crawling, leukocytes migrate to a nearby endothelial border and squeeze between the tightly opposed endothelial cells to the underlying basement membrane in a next process of transendothelial migration (also called as diapedesis) 44. PECAM-1 (CD31) and CD99 act at sequential steps as the leukocyte crosses the endothelial barrier 45. Two main routes of leukocyte transendothelial migration are described in the literature:
    1. The trans-cellular route, when leukocytes emigrate through the body of endothelial cells, and
    2. The para-cellular route, when leukocytes emigrate through junctions between adjacent endothelial cells 46, 47.

In trans-cellular migration of leukocyte ICAM-1 on endothelial cell engages with LFA-1 on leukocyte and induces the formation of micro-villi like endothelial cell projections embracing the migrating leukocyte in a cup-like structure 37, 48.

Figure 6.11 Trans-endothelial migration of neutrophils

Transendothelial migration

Most of the recently published studies identify, however, the para-cellular migration route as the main mechanism by which leukocytes emigrate from the intravascular compartment into the interstitium 47. Many cell contact proteins such as PECAM-1, members of the JAM family (JAM-A, JAM-B, and JAM-C), CD99, and ICAM-2 etc. are involved in the para-cellular migration of leukocyte into the extracellular matrix.

  1. Ultimately, the leukocyte enters the extracellular matrix and under the chemoattractant gradient reaches the site of inflammation, where it performs its various functions.

Neutrophil-derived products and their functions:

The activated neutrophils synthesize and secrete cytokines, chemokines, leukotrienes and prostaglandins, and by virtue of their accumulation in large numbers within the inflammatory tissue, they may contribute significantly to the local production of inflammatory mediators. The activated neutrophils have been shown to produce IL-1, IL-1RA, IL-6, IL-12, TGF-β and TNF-α 49. Furthermore, neutrophils also synthesize and secrete leukotrienes and prostaglandins, especially leukotriene B4 (LTB4) and prostaglandin E2 (PGE2), which are synthesized from arachidonic acid by lipoxygenases and cyclo-oxygenases pathways, respectively 50. Neutrophils also secrete matrix metalloproteinases (MMP-8 and MMP-9) which are involved in the degradation of connective tissue 51. These mediators along with causing inflammatory changes in the tissue, also activate other immune cells. A detailed description of neutrophils and their role in periodontal diseases has been given in “Role of neutrophils in periodontal health and disease”.

Know more……………..

Are neutrophils a component of innate or acquired immunity?

Neutrophils have long been considered as an important component of innate immunity by virtue of their ability to phagocytose the invaders and destroy them. These cells are equipped with various enzymes such as defensin, perforin, and granzymes etc., which destroy the invading microorganisms. Furthermore, neutrophils also synthesize certain cytokines and chemokines, which activate other immune cells. They interact with immune cells, particularly with dendritic cells and lead to the formation of IL-12 and TNF-α, deviating the immune response towards a Th1 phenotype.

Neutrophils have the capacity to degrade and process antigens as well as efficiently present antigenic peptides to lymphocytes. Therefore, it can be concluded that neutrophils along with their active role in innate immune response, also participate in adaptive immunity 52. However, most of the aspects of neutrophil participation in adaptive immunity still need to be investigated.

 

Chemical mediators of acute inflammation

The chemical mediators of inflammation can broadly be divided into systems based on their source and/or chemical composition. These are as follows,

Vasoactive amines:

  • Histamine (derived from mast cells and platelets)
  • Serotonin (derived from platelets)

Plasma protein systems:

  • Kinin system
  • Complement system
  • Clotting/Fibrinolytic System

Eicosanoids derived from arachidonic acid metabolism:

  • Prostaglandins
  • Leukotrienes

Platelet activating factor

Cytokines:

  • Interleukin 1 (IL-1)
  • Interleukin 6 (IL-6)
  • Interleukin 1 (IL-1)
  • Tumor necrosis factor (TNF)

Products of phagocytosis

  • Oxygen-derived free radicals
  • Cationic proteins
  • Neutral proteases

Nitric oxide (NO)

The chemical mediators of inflammation can also be classified as preformed and newly formed mediators. The preformed mediators are already present in the cells, while the newly formed are synthesized during the acute inflammatory response. The primary preformed mediators include histamine (derived from mast cells, basophils, platelets), Serotonin (platelets) and lysosomes (neutrophils, macrophages) while newly formed mediators include prostaglandins (mast cells, leukocytes), leukotrienes (mast cells, leukocytes), platelet-activating factor (leukocytes, endothelial cells), reactive oxygen species (all leukocytes), nitric oxide (macrophages) and cytokines (lymphocytes and macrophages) (Table 6.4, 6.5). Following is the brief description of these mediators,

Histamine:

It is a preformed mediator of inflammation and is the first one to be released during inflammation. The major sources of histamine include mast cells, basophils and eosinophil leukocytes, and platelets. The release of histamine is triggered by physical injury (trauma, heat, and cold), complement factors (C3a, C5a), neuropeptides (substance P), cytokines (IL-1, IL-8), an antigen binding to mast cells and by lysosomal proteins released from neutrophils, including cationic proteins. Histamine is also chemotactic for eosinophils. The primary action of histamine is vasodilation which is caused by its binding to specific H1 receptors in the vascular wall, inducing endothelial cell contraction, gap formation, and thus resulting in edema. This effect can be inhibited pharmacologically by H1-receptor antagonists.

Serotonin:

Serotonin (5-hydroxytryptamine) is a vasoactive amine, derived from the platelets. Its primary actions are vasodilation, increased vascular permeability, and platelet aggregation.

Prostaglandins:

These are the products of arachidonic acid metabolism. The four principal bioactive prostaglandins generated include prostaglandin E2 (PGE2), prostacyclin (PGI2), prostaglandin D2 (PGD2) and prostaglandin F2α (PGF2α). Out of these four prostaglandins, PGE2 is most abundant during acute inflammation, however, PGI2 also plays an important role in this process. PGD2 is primarily produced by mast cells. PGE2, PGI2, and PGD2 are powerful vasodilators individually and they also synergize with other inflammatory vasodilators like histamine and bradykinin. The vasodilatory effect of histamine and bradykinin is potentiated by PGE2, PGI2, and PGD2. Furthermore, these mediators do not produce pain themselves, but potentiate the afferent C fiber sensitization by histamine and bradykinin in causing pain.

Leukotrienes:

These mediators are synthesized from arachidonic acid by white blood cells through the action of soluble cytosolic enzymes. These are also synthesized by mast cells and platelets. 5-lipoxygenase oxidizes arachidonic acid to give an intermediary leukotriene (LTA4). Intracellular enzymes then convert LTA4 to either LTB4 or a series of cysteinyl-containing leukotrienes (LTC4, LTD4, and LTE4). Out of all the leukotrienes, LTB4 acts as a potent chemotaxin and is present in inflammatory exudate. The cysteinyl-containing leukotrienes (LTC4, LTD4, and LTE4) cause an increase in vascular permeability.

Cytokines:

123

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Chemokines:

Chemokines are a large family of cytokines that play a highly important role in orchestrating the exquisitely organized and regulated movement of cells to specific locations within the body 56.Two initially discovered chemokines were IL-8 and monocyte chemoattractant protein (MCP)-1. The primary function of chemokines is to create a chemical gradient along which various immune cells (such as neutrophils, monocytes) move towards the site of injury. Furthermore, by attachment of chemokines to their receptors on neutrophils, eosinophils, basophils, mast cells and other cells, they trigger granule exocytosis, oxidative burst with the release of superoxide, and nitric oxide, and can affect gene expression, proliferation, homeostasis and apoptosis 57.

Table 6.4 Important cells involved in inflammation, cytokines they produce and respond to

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Reactive oxygen species:

The reactive oxygen species are a short-lived group of incredibly cytotoxic molecules released during inflammation. These significantly contribute to the tissue damaging effects during inflammation 58-60. Although, certain levels of reactive oxygen species are required for normal metabolism, but during inflammation, they are produced in an excessive amount which causes tissue damage. The reactive oxygen species are primarily present in the form of superoxide, hydrogen peroxide, and lipid hydroperoxides. Neutrophils are the major source of free radicals at the site of inflammation.

Nitric oxide (NO):

This molecule has multiple modulating effects on inflammation and plays a key role in the regulation of immune responses. It is a short-lived molecule (half life around 6 seconds), produced by enzymes known as nitric oxide synthases (NOSs). In tissues, nitric oxide (NO) is generated enzymatically by NOSs, which oxidize L-arginine to L-citrulline 61, 62. Because of its small size, this molecule can penetrate the cell membrane and thus can significantly affect cellular functions. Nitric oxide may play regulatory roles in virtually every stage of the development of inflammation, primarily by increasing vascular permeability and vasodilatation.

 Components of complement system:

As already discussed, complement system along with killing the invading microorganisms also orchestrates and connects various responses during immune and inflammatory reactions 63. The components of the complement system are involved virtually in all phases of inflammation, including changes in vascular flow and caliber, the increase in vascular permeability, extravasation of leukocytes, and chemotaxis (Table 6.5).

Kinins:

Kinins are important chemical mediators that produce many of the cardinal manifestations of inflammation. There are two major kinin families: the slow acting bradykinins and the fast acting tachykinins. The bradykinin family consists of bradykinin and Lys-bradykinin (also known as kallidin). These are formed by proteolytic cleavage of their protein precursor, kininogen, by plasma and tissue proteases known as kallikreins 64, 65. The cellular effects of these vasoactive kinin peptides are executed via at least two different classes of receptors: B1 and B2.  Pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are potent up-regulators of B1 receptor expression 66. The tachykinin family includes the neuropeptides SP, neurokinin A (NKA) and neurokinin B (NKB) which perform important functions in the central nervous system (Table 6.5).

 

Table 6.5 Various chemical mediators of inflammation, their sources, and action

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Conclusion

The basic understanding of immunity and inflammation is essential to understand various aspects of periodontal disease progression. In the above discussion, we briefly discussed various aspects of innate as well as adaptive immunity. The interaction between microorganisms and host are complex and involve various chemical mediators. In the upcoming chapters, we shall read about the host-microbial interactions and various chemical mediators that are involved in the progression of periodontal diseases.

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References:

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