Essential Microbiology for Dentistry. 5th ed.

Chapter 8. The immune system and the oral cavity

Contributed by Professor Glen C Ulett

Centre for Medicine and Oral Health, Griffith University, Australia

The immune system: general considerations

Immunology is the branch of biology concerned with the body's defence reactions. The word 'immunity' is derived from the Latin word immunis, meaning 'free of burden'. In essence, the immune system exists to maintain the integrity of the body by excluding or removing the myriad of potentially burdensome or threatening microorganisms, which could invade from the environment. Internally derived threats, mutant cells with malignant potential, may also be attacked by the immune system.

There are two kinds of immunological defence:

1. natural or innate immunity, comprising mainly pre-existing antigen-non-specific defences

2. adaptive or acquired immunity, during which the immune system responds in an antigen-specific manner to neutralize the threat efficiently, and retains a memory of the threat so that any future encounter with the same threat will result in an accelerated and heightened protective response.

During its development, the immune system must be educated specifically to avoid reacting against all normal components of the body (tolerance). Immunology can be considered 'the science of self-non-self discrimination'.

The vital importance of the immune system is evident in the life-threatening infections suffered by patients with immune defects (immunodeficiency). In other situations, there may be too much immunity. A by-product of a successful immune response may be damage to normal 'bystander ' cells, but this is normally limited by stringent immune regulatory mechanisms. Deficiencies of immunoregulation may be the root causes of hypersensitivity diseases such as autoimmunity and allergy.

These concepts are summarized in Fig. 8.1.

The innate immune system

These intrinsic defence mechanisms are present at birth prior to exposure to pathogens or other foreign macromolecules. They are not enhanced by such exposures and are not specific to a particular pathogen.

Mechanical and chemical barriers

Intact skin is usually impenetrable to microorganisms. Membranous linings of the body tracts are protected by mucus, acid secretions and enzymes such as lysozyme, which breaks down bacterial cell wall proteoglycan. In the lower respiratory tract, the mucous membrane is covered by hair-like protrusions of the epithelial cell membrane called cilia. The movement of cilia can propel mucus-entrapped microorganisms from the tract (mucociliary escalator). Although most pathogens enter the body by binding to and penetrating mucous membranes, several defence mechanisms, including saliva, tears and mucous secretions, are involved in preventing this entry. Apart from acting to wash away potential invaders, these secretions also contain antibacterial or antiviral substances.

Defensins and cathelicidins

Defensins and cathelicidins are two major families of mammalian antimicrobial proteins. They contribute to host innate antimicrobial defences by disrupting the integrity of the bacterial cell membrane. Further, several members of defensins and cathelicidins have been shown recently to have chemotactic effects on host cells. Their capacity to mobilize various types of phagocytic leukocytes, immature dendritic cells and lymphocytes, together with their other effects, such as stimulating interleukin-8 production and mast cell degranulation, provides evidence for their participation in alerting, mobilizing and amplifying innate and adaptive antimicrobial immunity of the host (Table 8.1). In brief, upon microbial invasion, epithelial cells/keratinocytes and tissue macrophages are induced to produce β-defensins (especially HBD2 and 3) and cathelicidin/ LI-37. The defensins and cathelicidin form gradients that, in tandem with other chemotactic mediators (e.g., chemokines), lead to extravasation of various types of leukocytes to the site of infection in order to overcome the invading pathogens (Table 8.2).

Fig. 8.1 Normal and aberrant immunity.

Table 8.2 Cathelicidin and defensins, their sources and actions

Peptide

Name(s)

Major cell and tissue sources

Actions

Cathelicidin

LL-37/hCAP18

Neutrophils, mast cells, epithelia (skin, lung, gastrointestinal, urogenital, oral), sweat, seminal fluid

Antimicrobial,

chemotactic

a-Defensins

a-Defensins 1-4 (HNP-1 to HNP-4), HD- 5, HD-6

Neutrophils

Antimicrobial

β-Defensins

HBDs1-4

Neutrophils, epithelia (skin, oral, mammary, lung, urinary, eccrine ducts, ocular)

Antimicrobial,

chemotactic;

induces

histamine

release

HBDs, Human β-defensins; HD, human defensin; HNP human neutrophil peptide.

Table 8.1 Antigen-non-specific defence chemicals in oral secretions

Chemical

Antimicrobial

function(s)

Major cell source(s)

Calprotectin

Divalent cation chelator, restricts microbe nutrition

Oral epithelial cells and neutrophils

Defensins (a and β types)

Membrane pore-forming peptides, cause osmotic lysis

Leukocytes and epithelial cells

Cathelicidins

Lysosomal antimicrobial polypeptides

Macrophages and neutrophils

Saliva

Ig, lysozyme, lactoferrin, peroxidases and GCF

Salivary acinar cells

Lysozyme

Muramidase activity, aggregates microbes and amphipathic sequences

Macrophages, epithelial cells and neutrophils

Peroxidase

Oxidizes bacterial enzymes in glycolytic pathways

Salivary acinar cells, neutrophils, eosinophils

Histatins,

Cistatins

Various effects

Salivary acinar cells

SLPI, PRP

Antiviral activities

Various cell types

GCF

Provides blood components

Various cell types

Mucins

Aggregates bacteria, various effects, homotypic and heterotypic complexes

Salivary acinar cells

GCF, Gingival crevicular fluid; Ig, immunoglobulin; PRP proline-rich proteins; SLPI, secretory leukocyte protease inhibitor.

Phagocytosis

Phagocytosis is a process by which phagocytic cells ingest extracellular particulate material, including whole pathogenic microorganisms. If the mechanical defences are breached, the phagocytic cells become the next barrier. These include polymorphonuclear leukocytes (polymorphs) and macrophages. The former are short-lived circulating cells, which can invade the tissues, while the latter are the mature, tissue-resident stage of circulating monocytes.

Macrophages are found in areas of blood filtration where they are most likely to encounter foreign particles, e.g., liver sinusoids, kidney mesangium, alveoli, lymph nodes and spleen. Phagocytes attach to microorganisms by non-specific cell membrane 'threat' receptors, after which pseudopodia extend around the particle and internalize it into a phagosome. Lysosomal vesicles containing proteolytic enzymes fuse with the phagosome, and oxygen and nitrogen radicals are generated, which kill the microbe. The phagocytes have several ways of dealing with the phagocytosed material. For example, macrophages reduce molecular oxygen to form microbicidal-reactive oxygen intermediates that are secreted into the phagosome.

Pathogen-associated molecular patterns, pattern-recognition receptors and toll-like receptors

Unlike adaptive immunity, innate immunity does not recognize every possible antigen. The cells involved in innate immune

responses such as phagocytes (neutrophils, monocytes, macrophages) and cells that release inflammatory mediators (basophils, mast cells and eosinophils) are designed to recognize only a few highly conserved structures present in many different microorganisms. These cells recognize microbial structures called pathogen-associated molecular patterns (PAMPs) in order to activate the innate immune response. PAMPs are molecular components common to a variety of microorganisms but not found as a part of eukaryotic cells and include:

 lipopolysaccharide (LPS) from the Gram-negative cell wall

 peptidoglycan, lipoteichoic acids from the Gram-positive cell wall

 mannose (common in microbial glycolipids and glycoproteins but rare in humans)

 bacterial DNA

 N-formylmethionine found in bacterial proteins

 double-stranded RNA from viruses

 glucans from fungal cell walls.

This promotes the attachment of microbes to phagocytes and their subsequent engulfment and destruction. Most defence cells (macrophages, dendritic cells, endothelial cells, mucosal epithelial cells, lymphocytes) have on their surface a variety of receptors called pattern-recognition receptors (PRRs) capable of binding specifically to conserved portions of PAMPs so there is an immediate response against invading microbes. These receptors enable phagocytes to attach to microbes so they can be engulfed and destroyed by lysosomes. There are two functionally different classes of PRRs:

 endocytic PRRs (mannose receptors, scavenger receptors, opsonin receptors, and N-formyl Met receptors)

 signalling PRRs.

Signalling PRRs bind a number of microbial molecules such as flagellin, pilin, glycolipids, zymosan from fungi and viral double-stranded RNA. A major class of signalling PRRs is Toll-like receptors (TLRs), so named because of their similarity to the protein coded by the Toll gene identified in Drosophila melanogaster.

Binding of PAMPs to signalling PRRs promotes the synthesis and secretion of regulatory molecules such as cytokines that are crucial to initiating innate immunity. Various types of TLRs bind different PAMPs and initiate different types of innate immune responses (Fig. 8.2). PAMPs can also be recognized by a series of soluble PRRs in the blood that function as opsonins and initiate the complement pathway.

Natural killer cells

Natural killer (NK) cells are non-phagocytic lymphocytes that account for up to 15% of blood lymphocytes and have a special role in the killing of virus-infected and malignant cells (Fig. 8.3). These cells have two kinds of receptors with opposing action: antigen receptors able to recognize specific molecules on target cells, through which activation signals are transmitted, and receptors that recognize self major histocompatibility complex I (MHC I) antigens (see later) through which inactivation signals are transmitted. Activation of NK cells can only occur when there is no inactivation signal, so virus-infected and tumour cells with downregulated MHC I antigens are susceptible to NK cytotoxicity, but normal MHC I-positive cells are protected.

Fig. 8.2 toll-like receptors (TLRs). LPS, Lipopolysaccharide.

Fig. 8.3 Killing of major histocompatibility complex (MHC) I-deficient cells by natural killer (NK) cells.

The killing mechanism is activated by cytokines released by virus-infected cells, tissue cells, lymphocytes and NK cells themselves. The NK cells are also important in the adaptive immune response, being the effector cells for killing antibody- coated microorganisms.

Acute-phase proteins

Acute-phase proteins are serum proteins produced by the liver in response to tissue-damaging infections and other inflammatory stimuli such as cytokines (e.g., interleukin-1 and interleukin-6). Although the physiological role of the acute-phase proteins is not fully understood, it has been recognized to enhance the efficiency of innate immunity. Positive acute-phase proteins increase in plasma concentration in the acute-phase response to inhibit or kill microbes through opsonization, coagulation, antiprotease activity and/or complement activation. Negative acute-phase proteins including human serum albumin and transferrin are reduced in concentration in the acute-phase response and act to limit inflammation. Together acute-phase proteins provide immediate defence and enable the body to recognize and react to foreign substances prior to more extensive activation of the immune response. The concentration of the following positive acute-phase proteins in body fluids increases rapidly during tissue injury or infection:

 C-reactive protein functions as a soluble PRR and can bind to bacteria to promote their removal by phagocytosis. It is a major acute-phase protein, so named as it binds to the C-polysaccharide cell wall component on a variety of bacteria and fungi. This binding activates the classical complement system, resulting in increased clearance of the pathogen.

 a1-Antitrypsin neutralizes proteases released by bacteria, activated polymorphonuclear leukocytes or damaged tissue to limit damage caused by excessive enzyme activity.

 Mannose-binding protein functions as a soluble PRR and activates the lectin complement pathway to promote inflammation and attract phagocytes.

Interferon

Interferon, produced by virus-infected cells, comprises a group of cytokines that mediate innate immunity and includes those that protect against viral infection and those that initiate inflammatory reactions that protect against bacterial pathogens.

Complement

The complement system is very much involved in the inflammatory response and is one of the key effector mechanisms of the immune system. It consists of at least 30 components— enzymes, regulators and membrane receptors—which interact in an ordered and tightly regulated manner to bring about phagocytosis or lysis of target cells.

Complement components are normally present in body fluids as inactive precursors. The alternative pathway of complement activation can be stimulated directly by microorganisms and is important in the early stages of the infection before the production of antibody. It is part of the innate immune system. The classical pathway requires antibody, which may take weeks to develop. Both pathways can lead to the lytic or membrane attack pathway. During the course of complement activation, numerous split products of complement components, with important biological effects, are produced.

Alternative activation

Complement factor C3 is the central component of both the classical and alternative pathways (Fig. 8.4). Products of C3 activation, C3b and inactivated C3b (iC3b) bind to microorganisms and are recognized by complement receptors (CRs) on phagocytes. If any C3b molecules bind to a normal host cell surface, they can then bind the next component in the sequence, factor B. Factor D (the only complement factor present in body fluids as an active enzyme) splits off a small fragment, Ba, leaving an active C3 convertase, C3bBb, on the cell surface. However, the normal host cell is able actively to dissociate and inactivate C3bBb. This is achieved by the concerted action of regulatory proteins decay-accelerating factor (DAF), membrane cofactor protein (MCP), p1H globulin (factor H), CR1 and factor I.

Activator surfaces are those that inhibit the regulatory proteins, allowing C3bBb to remain intact. For example, bacterial endotoxins and LPSs inhibit factor H. The enzyme C3bBb converts C3 into C3a and C3b. The latter is incorporated, along with properdin (factor P), to form PC3bBbC3b. This is a stable enzyme whose substrates are C3 and C5. It amplifies C3b production and activates the membrane attack pathway.

Fig. 8.4 alternative pathway of complement activation. B, Factor B; CR, complement receptor; D, factor D; DAF, decay-accelerating factor; H, β1H-globulin; I, C3 inactivator; MCP membrane cofactor protein; P, properdin.

Fig. 8.5 Classical pathway of complement activation.

Classical activation

Classical pathway of complement activation (Fig. 8.5) is mainly initiated by complexes of antigen with antibody. Antibodies of the immunoglobulin (Ig) IgG1, IgG2, IgG3 and IgM classes, but not IgG4, IgA, IgD or IgE, can activate the classical pathway.

The first component of the classical pathway, C1, is actually a complex of C1q, C1r and C1s. This complex can bind very weakly to monomeric IgG, but when IgG complexes with antigen in such a way that adjacent IgG molecules are close together, C1q binds firmly between the two molecules. The C1 complex can bind strongly to a single molecule of pentameric IgM, but only after the conformation of the latter has been altered by binding to antigen.

Activated C1 reacts with fluid-phase C4 and C2, splitting off small peptides C4a and C2a. The resulting C4b2b is deposited on a surface and performs a similar job to C3bBb of the alternative pathway: it can convert C3 into C3a and C3b, and the latter can either opsonize particles for phagocytosis or bind to C4b2b. Cell-bound C4b2b3b is more stable than C4b2b, being somewhat protected from the regulatory proteins DAF and C4-binding protein. Like PC3bBbC3b, it activates the membrane attack pathway.

Membrane attack

The peptides Bb and C2b, bound into their respective alternative (PC3bBbC3b) and classical (C4b2b3b) pathway enzymatic complexes, initiate membrane attack (Fig. 8.6) by splitting a small peptide, C5a, from C5 to form C5b. This molecule binds C6 and C7. Cell-bound C5b67 acts as a template for the binding of one molecule of C8 and up to 18 molecules of C9. Normal cells in the body are largely protected from bystander lysis by homologous restriction factor (HRF), which intercepts C8 and C9 before they can be properly assembled into the membrane attack complex (MAC). The MAC, with a molecular weight of 1-2 x 106, forms transmembrane channels, which permit osmotic influx so that the target cell swells up and bursts.

Biological effects of complement activation

Probably the most important function of the complement system is to opsonize antigen-antibody (immune) complexes, microorganisms and cell debris for phagocytosis (Fig. 8.7). This is achieved by deposition of C3b and iC3b on the particle. Phagocytes bind to the particle via CR1, CR3 and CR4. Also, CR1 is found on erythrocytes, which can bind immune complexes coated with C3b and transport them to the spleen or liver for digestion by macrophages.

The peptides C3a, C4a and C5a are anaphylatoxins that cause mast cell degranulation and smooth-muscle contraction. They increase vascular permeability, which permits cells and fluids to enter the tissues from the circulation. They are regulated by anaphylatoxin inactivator, which splits off the C-terminal arginine so that binding to cellular receptors can no longer occur. Further important properties of C5a are:

 inducing adherence of blood phagocytes to vessel endothelium, following which they are able to migrate into the tissues

 upregulating CR1, CR3 and CR4

 attracting phagocytes (chemotaxis) towards the site of complement activation.

Certain microorganisms, notably Gram-negative bacteria, can be lysed directly by the MAC. Gram-positive bacteria, however, are protected by their thick peptidoglycan cell walls.

Fig. 8.6 Membrane attack pathway. HRF, Homologous restriction factor; P properdin.

Fig. 8.7 Biological effects of complement. CR, Complement receptor; MAC, membrane attack complex; RBC, red blood cell.

Inflammation

The local inflammatory response is usually accompanied with a systemic response known as the acute-phase response. The manifestation of this response includes the induction of fever and increased production of leukocytes, and the production of soluble factors, including acute-phase proteins in the liver. Injured or infected tissues become inflamed in order to direct components of the immune system to where they are needed. The blood supply to the tissues is increased, capillaries become more permeable to soluble mediators and leukocytes, and leukocytes migrate towards the site of infection as a result of the production of chemotactic factors.

The adaptive immune system

The defence mechanisms in adaptive immunity can specifically recognize and selectively eliminate pathogens and foreign macromolecules. In contrast to innate immunity, adaptive immune responses are reactions to specific antigenic challenge and display four cardinal features: specificity, diversity, immunological memory and discrimination of self and non-self.

Adaptive immune responses are specific for distinct antigens. This unique specificity exists because B and T lymphocytes express membrane receptors that specifically recognize different antigens. Importantly, adaptive immunity is not dependent on innate immunity. Through delicately modulated interactions, the two types of defence mechanisms work synergistically to produce more effective immunity.

Fig. 8.8 Cells and organs of the immune system. NK, Natural killer.

Cells of the immune system

All the cells of the immune system (Fig. 8.8) are derived from self-regenerating haematopoietic stem cells present in bone marrow and foetal liver. These differentiate along either the myeloid or the lymphoid pathway. Myeloid precursor cells give rise to mast cells, erythrocytes, platelets, dendritic cells, polymorphs (eosinophils, basophils, neutrophils) and mononuclear phagocytes (monocytes in the blood, macrophages in the tissues). Lymphoid precursor differentiation gives rise to T (thymus-dependent) lymphocytes, B (bone marrow-derived) lymphocytes and NK lymphocytes.

During post-natal life, B cell genesis takes place in the bone marrow. Each newly formed B cell expresses a unique B cell receptor (BCR) on its membrane for antigen-binding. Although T lymphocytes also arise in the bone marrow, they migrate to the thymus to mature. During its maturation, the T lymphocyte expresses a specific antigen-binding molecule known as the T cell receptor (TCR) on its membrane.

The B lymphocytes are responsible for secreting Ig antibodies and can also function as highly efficient antigen-presenting cells (APCs) for T lymphocytes. The latter are divided into two major subsets: T-helper cells, which usually bear the 'cluster of differentiation' marker CD4, and T-cytotoxic cells, which usually carry CD8. The T-helper cells are required for activating the effector function of B cells, other T cells, NK cells and macrophages. They do this by transmitting signals via cell-to-cell contact interactions and/or via soluble hormone-like factors called lymphokines. The T-cytotoxic cells kill target cells such as virus-infected host cells. Another functional property of some T lymphocytes is to downregulate immune responses. These T-suppressor cells are usually CD8-positive. Dendritic cells and monocytes/macrophages play key roles in the immune system as APCs.

The lymphoid organs

The primary sites of lymphocyte production are the bone marrow and thymus. Immature lymphocytes produced from stem cells in the bone marrow may continue their development within the bone marrow (B lymphocytes, NK cells) or migrate to the thymus and develop into T lymphocytes. 'Education' within the primary lymphoid organs ensures that emerging lymphocytes can discriminate self from non-self. They migrate through the blood and lymphatic systems to the secondary lymphoid organs—spleen, lymph nodes and mucosa-associated lymphoid tissue (MALT) of the alimentary, respiratory and urogenital tracts. Here, lymphocytes encounter foreign antigens and become activated effector cells of the immune response.

The spleen acts as a filter for blood and is the major site for clearance of opsonized particles. It is an important site for production of antibodies against intravenous antigens. The lymph nodes form a network of strategically placed filters, which drain fluids from the tissues and concentrate foreign antigen on to APCs and subsequently to lymphocytes. Spleen and lymph nodes are encapsulated organs, whereas MALT is nonencapsulated dispersed aggregates of lymphoid cells positioned to protect the main passages by which microorganisms gain entry into the body. Gut-associated lymphoid tissue (GALT) includes Peyer's patches of the lower ileum, accumulations of lymphoid tissue in the lamina propria of the intestinal wall and the tonsils.

Mature lymphoid cells continuously circulate between the blood, lymph, lymphoid organs and tissues until they encounter an antigen, which will cause them to become activated (see Chapter 9).

Antigen recognition

The T and B lymphocytes are responsible for specificity in the immune response. They have cell surface receptors whose purpose is to recognize foreign antigens. Each receptor usually binds only to a single antigen, though there may be a degree of cross-reactivity with other antigens of very similar structure. Since all antigen receptors on a given lymphocyte are identical, each B or T cell can usually recognize only one antigen. A single cell, on encountering its specific antigen, must proliferate to form a clone of identical cells able to deal with the offending antigen (clonal selection).

The TCR recognizes linear peptides bound to MHC molecules on the surface of APCs. The BCR binds directly to often nonlinear antigenic determinants (epitopes) and does not require MHC presentation.

Major histocompatibility complex

In humans, products of the highly polymorphic MHC genetic loci on chromosome 6 are known as histocompatibility locus antigens (HLAs). Their function is to bind APC-processed short antigenic peptides and present them on the APC surface to T cells. HLA phenotype is responsible for tissue transplant rejection when the recipient and donor are not HLA-matched. There are two classes of HLA molecules:

1. HLA-A, -B and -C (class I) are found on all nucleated cells in the body.

2. HLA-DQ, -DR and -DP (class II) molecules are usually only found on monocytes/macrophages, B cells, dendritic cells (i.e., APCs), some epithelial cells and activated T cells.

One HLA-A, -B, -C, -DQ, -DR and -DP antigen is inherited from each parent, so each individual expresses up to six class I and six class II antigens. Each HLA molecule can bind a large number of different antigenic peptides. However, the complement of HLA antigens possessed by an individual will determine the range of antigenic peptides that can be presented by APCs. Class I molecules present peptides to CD8+ T lymphocytes, while CD4+ T cells are restricted to MHC class II.

The TCR and generation of T cell diversity

The TCR is a two-chain structure comprising polypeptides derived from TCR a and TCR β genes. Less frequently, a subset of T cells will use TCR у and TCR 8 instead. Each chain consists of a variable (V) region and a constant (C) region. The two adjacent V regions make contact with antigenic peptides and the presenting MHC. The genetic template for the a-chain is created by joining one of many Va genes with one of the more than 40 J (joining) a genes and a single C gene. The в-chain template is similarly created by joining one of the large number of Vβs, one of two D (diversity) βs, one of 2 Jβs and one of the two Cβ genes. The number of different аβ V regions that can be created is high, and the repertoire is further increased by the random addition of small numbers of template-independent nucleotides.

The BCR, generation of B cell diversity and isotype selection

The BCR is a cell membrane-bound form of Ig antibody and recognizes the same antigenic specificity as the antibody that will eventually be secreted by the B cell. It is a four-chain structure comprising two identical heavy (H) chains, which anchor the receptor in the plasma membrane, and two identical light (L) chains. The whole molecule projects out from the B cell surface in the shape of a Y. Like TCR chains, each H and L chain consists of V and C regions. The antigen-binding site is created by the juxtaposition of V regions from one H and one L chain, and there are two such sites per BCR. Their tertiary structure creates a pocket that accommodates an epitope with the mirror-image configuration.

The VL region genetic template is created by rearranging V and J genes, while the VH chain is derived from the recombination of V, D and J genes. Additional diversity is created by n-region additions. Furthermore, point mutations can be introduced into V genes after antigenic stimulation, which tend to increase the strength of binding of an antibody or BCR to its antigen.

There are nine CH genes on chromosome 14q32 arranged in the order 5'-μ-δ-у3112-у4-ε-а2-3'. The class, or isotype, of Ig depends on which CH gene is used: ц gives IgM, 8 IgD, y3 IgG3, a1 IgA1, ε IgE. Immature B cells use only ц and express IgM, while mature but unstimulated B cells express IgM and IgD. Following stimulation by antigen, B cells can delete 5' genes, for example, μ, δ, y3, and express the next most 5' CH gene, in this case γ1 (IgG1). Switching to particular CH genes is largely under the control of regulatory T cells.

Deletion of anti-self reactivities

Random usage of all the possible TCR and BCR V gene combinations would result in a large fraction of the repertoire being directed against self. This fraction of the repertoire must be purged in order to prevent immune damage to the body. This is achieved largely during late embryonic and early neonatal development. Following seeding of the primary lymphoid organs by lymphoid precursors, differentiation along defined developmental pathways occurs, accompanied by rapid cell proliferation and also massive cell loss due to depletion of anti-self reactivities.

T cell differentiation

The most immature thymocytes are TCR-CD3-CD4-CD8-. These first differentiate into TCR-CD3-CD4+CD8+ and then rearrange TCR аβ or TCR yδ genes and express CD3; TCR+CD3+CD4+CD8+ are then selected for MHC reactivity. Thymocytes with TCRs that bind weakly to MHC antigens on thymic cortical epithelial or stromal cells are allowed to survive (positive selection); those with no MHC reactivity die 'of neglect'. Thymocytes with strong reactivity against self MHC + self peptides (there will have been little exposure to foreign peptides in utero) expressed on medullary dendritic cells and macrophages are signalled to undergo programmed cell death (PCD) by apoptosis (negative selection).

If the weak reactivity with MHC that results in positive selection is against MHC class I, the T cell, when fully mature, will only respond to peptides presented on class I. It will stop expressing CD4 but continue to express CD8, which itself has the ability to bind to a monomorphic site on MHC I and functions as an important coreceptor to strengthen adhesion between the T cell and the APC. The mature T cell will be TCR +CD3+CD4+CD8+ and function as a T-cytotoxic or T-suppressor cell. Alternatively, selection on MHC II will produce class

II-restricted TCR+CD3+CD4+CD8+ T-helper cells. CD4 strengthens the adhesion between the T cell and the APC by binding to MHC II.

Fewer than 10% of thymocytes survive the selection process. Those that do have the ability to bind weakly to MHC on APCs and the potential to bind strongly to MHC + non-self peptides will leave the thymus and enter the circulation.

B cell differentiation

The process of B cell development in the bone marrow occurs by the stepwise rearrangements of the V, D and J segments of the Ig H and L chain gene loci. During early B cell genesis, productive IgH chain gene rearrangement leads to assembly of the pre-B cell receptor (pre-BCR). The pre-BCR, transiently expressed by developing precursor B cells, comprises the Ig yH chain, surrogate light (SL) chains VpreB and 85, as well as the signal-transducing heterodimer Igа/Igβ. Signalling through the pre-BCR regulates allelic exclusion at the Ig H locus, stimulates cell proliferation and induces pre-B cells that further undergo the rearrangement of the IgL chain genes. Once H and L chains are produced, a complete BCR, consisting of IgM plus Igа and Igδ, will be expressed on the surface of immature B cells.

At this stage, the V genes of the BCR are in germ-line configuration; i.e., they have not incorporated any point mutations. Products of germ-line V genes generally have low affinity for antigen and can bind weakly to several different antigens (polyreactivity). Weak binding of antigen plus receipt of signals from T-helper cells induces low-affinity B cells to proliferate. The V gene point mutations introduced at cell division alter the strength of binding to antigen, with retention of B cells with higher-affinity BCRs (affinity maturation).

The need to delete anti-self BCRs is probably less than the need to delete anti-self TCRs, since B cells require T cell help to produce high-affinity antibodies, and deletion of anti-self T-helper cells should be sufficient to prevent activation of antiself B cells. Furthermore, it is desirable to have low-affinity autoantibodies able to opsonize tissue breakdown products for clearance by phagocytes, which would ensure removal of previously sequestered tissue antigens before they could activate T cells.

Peripheral tolerance

Thymic deletion of T cells bearing self-reactive TCRs is undoubtedly the most important mechanism for ensuring non-reactivity to self. Nevertheless, not all self antigens are represented in the thymus, so extrathymic tolerance induction is also needed.

Autoreactive T cells are most likely to encounter extrathymic self peptides on epithelial cells rather than professional APCs. The activation signal through the TCR will therefore not be followed by co-stimulatory signals required for full activation. Such an interaction may result either in apoptosis of the T cell, or it may become anergic; i.e., it survives but in a non-reactive state, often with downregulated expression of TCR, CD3 and CD4/CD8.

Regulatory T cells can suppress the responses of activated T cells, which are required to regulate anti-self reactions when there is failure of thymic or peripheral tolerance induction. Although their mechanism of action is not fully understood, regulatory T cells appear to operate mainly by producing immunosuppressive cytokines and inhibiting T-helper cells.

Disorders of the immune system

Hypersensitivity, also called an allergic reaction, is an exaggerated reaction of the immune system to an antigen to which there has been prior exposure (sensitized). Types include:

 anaphylactic reactions (type I): e.g., IgE antibody on basophils and mast cells binds with antigens causing release of histamine, prostaglandins and other effectors. These types of reactions can be localized, respiratory or gastrointestinal related, systemic, or associated with shock

 cytotoxic reactions (type II): e.g., activation of complement and lysis of red blood cells (RBC) (main Ig: IgM), which can involve drugs (haptens) binding to RBC and inducing antibodies against them

 immune complex reactions (type III): e.g., complement fixing antigen-antibody complexes (main Ig: IgA). These are usually phagocytosed, but if the complexes are too small for phagocytosis, they can attach to the basement membrane of blood vessels and trigger inflammation

 cell-mediated reactions (type IV, delayed hypersensitivity): e.g., contact allergy in the skin. This involves delayed hypersensitivity T cells and activation of memory cells.

Autoimmune reactions are damaging immunological reactions between the host and its own tissues as a result of breakdown in the mechanisms regulating immune tolerance. Types include:

 type I: mediated by anti-self antibodies, often due to microbial molecular mimicry

 type II: cytotoxic autoimmune reactions, in which antibody reacts with cell surface antigens without cell destruction (e.g., Grave's disease, where the thyroid gland is stimulated to produce large amounts of hormones resulting in an enlarged thyroid, goitre and bulging eyes)

 type III: immune complex autoimmune reactions, where IgG and IgM (and sometimes complement) form immune complexes that cause inflammation (e.g., rheumatoid arthritis, where IgG, IgM and complement immune complexes cause chronic inflammation and severe damage to the cartilage and bone joints)

 type IV: cell-mediated autoimmune reactions, which involve destruction of a particular cell type by T cells (e.g., insulin-dependent diabetes mellitus, where insulin-secreting cells of the pancreas are destroyed by T cells).

Immune deficiency is caused when there is a defect in one or more of the various points along the differentiation pathways of immunocompetent cells. Considering the complex cellular interactions involved in immune responses and the central role of T cells, immune deficiencies primarily involving T cells are also associated with abnormal B cell function. Immunodeficiency syndromes are associated with unusual susceptibility to infections and often associated with autoimmune disease and cancer. The types of infection occurring in patients with an immune deficiency can often provide the first clue as to the nature of the immune defect. Types include:

 congenital immune deficiency: these can involve humoural or cell-mediated immune components and are inherited as recessive traits

■ acquired immune deficiency: these can involve humoural or cell-mediated immune components and often result from drugs, illness, cancer or viruses.

Oral defence mechanisms

Innate immune mechanisms

As mentioned earlier, innate immunity encompasses all of the antigen-non-specific defence mechanisms that every person is born with and is the initial response used to eliminate microbes or prevent them from entering the body (Fig. 8.9). This includes:

 anatomical barriers

 mechanical removal

 antigen-non-specific defence chemicals

 microbial antagonism

 defence cells and their activation

 phagocytosis

 inflammation

 fever

 the acute-phase response

 complement.

Two major mechanisms of innate immunity in the oral cavity are immune exclusion and inflammation. Immune exclusion refers to the inactivation and clearance of microbes from the oral mucosal epithelium and enamel surfaces. Inflammation occurs when there is a need to remove infectious agents at sites of mucosal penetration and encompasses phagocytes, detection of PAMPs by PRRs and various inflammatory mediators.

Acquired immune mechanisms are also important in the oral cavity; summaries of both are given in Table 8.3.

The oral mucosal epithelium

The oral mucosa is an anatomical barrier that prevents entry of potentially harmful microbes. Oral health depends on the integrity of the mucosal barrier, which also provides a habitat for normal oral flora. Continuous sloughing (desquamation) of the oral mucosal epithelium continuously removes microbes that colonize the mucosa, and this minimizes the microbial biomass in the oral cavity. Stable colonization therefore requires a continual process of microbial attachment, growth and reattachment to exposed epithelial cells, or growth of microbes in saliva at a rate exceeding the salivary flow or dilution rate. When the oral mucosa is compromised (e.g., during chemotherapy), infections frequently develop. Constituents of the oral mucosa that prevent penetration of microbes into deeper tissues include saliva, keratin in some areas of the mouth (on the free and attached gingiva, hard palate, areas of the dorsum of tongue), a granular layer, which discharges membrane-coating granules, and a basement membrane that provides barrier function for immune exclusion.

Cells in the oral mucosa also express TLRs for immune surveillance. Resident professional phagocytes as well as circulating cells of the vasculature in the oral mucosal epithelium enable innate defence. Evidence of an intracellular lifestyle of some periodontal pathogens including Aggregatibacter actinomycetem-comitans and Porphyromonas gingivalis within buccal epithelial cells suggests that host cells may be used as a protective niche by some microbes to avoid extracellular defences such as antibodies, phagocytes and salivary antimicrobial components, as well as antibiotics.

Fig. 8.9 a diagrammatic representation of the natural defence mechanisms of the oral cavity. Ig, Immunoglobulin; PMNL, polymorphonuclear leukocyte; TLRs, Toll-like receptors.

Table 8.3 Non-specific host defence factors of the mouth 

Defence factors

Main function

Epithelial desquamation

Physical removal of microbes

Saliva flow

Physical removal of microbes

Mucin/agglutinins

Physical removal of microbes

Lysozyme

Cell lysis (bactericidal, fungicidal)

Lactoferrin

Iron sequestration (bactericidal, fungicidal)

Apolactoferrin

Iron sequestration (bactericidal, fungicidal)

Sialoperoxidase system

Hypothiocyanite production (neutral pH); hypocyanous acid production (low pH)

Histidine-rich peptides

Antibacterial and antifungal activity

Salivary leukocyte protease inhibitor (SLPI)

Blocks cell surface receptors needed for entry of HIV

Intraepithelial lymphocytes and Langerhans cells

Cellular barrier to penetrating bacteria and/or antigens

Secretory IgA

Prevents microbial adhesion and metabolism

IgG, IgA, IgM

Prevent microbial adhesion; opsonins; complement activators

Complement

Activates neutrophils

Neutrophils/macrophages

Phagocytosis

HIV, Human immunodeficiency virus; Ig, immunoglobulin.

Antigen-non-specific defence chemicals in oral secretions (Table 8.3)

Various antigen-non-specific defence chemicals promote innate immune defence in the oral cavity. These include calprotectin, defensins, saliva (and the enamel pellicle), gingival crevicular fluid (GCF) and mucins. Non-cellular mediators of antimicrobial defence help to protect the oral mucosa through potent antibacterial, antiviral, and antifungal activities, which can affect oral microbes in several ways:

 they can aggregate or agglutinate microbes,

 they can promote or inhibit microbial adhesion,

 they can directly kill or inhibit the growth of microbes, and/or

 they can contribute to microbial nutrition.

 Calprotectin is a calcium- and zinc-chelating antimicrobial peptide produced by non-keratinized oral epithelial cells. The chelating activities of calprotectin has an antimicrobial effect as this deprives microbes of essential divalent ions. Calprotectin is present in neutrophils, monocytes, macrophages and probably GCF.

 Defensins, in contrast, are a class of pore-forming cationic peptides that insert into the phospholipid bilayer of bacterial membranes causing osmotic instability and cell lysis. Defensins are divided into a- and β-defensins according to their pattern of disulphide bonds and cysteine spacing. Defensins in saliva are also active against fungi and enveloped viruses; cause degranulation of mast cells; and are chemotactic for neutrophils, dendritic cells and memory T cells. Eukaryotic cells resist the lytic action of defensins due to lower phospholipid content in the membranes of these cells. Formation of cell membrane-traversing ring structures by cationic peptides is comparable with the nature of the MAC of the complement cascade.

 Cathelicidins are a family of antimicrobial polypeptides found in lysosomes in macrophages and neutrophils that provide innate immune defence against bacteria. These are summarized in Table 8.1.

 Saliva contains secretions from the major and minor salivary glands, exfoliated epithelial cells, oral microbes and GCF. The antimicrobial actions of saliva are several-fold; salivary flow combined with the continuous swallowing that cleanses the mouth removes debris and unattached microbes; saliva also replenishes fluids in the oral cavity, which dilutes and clears microbes and acid from plaque; and saliva contains neutrophils as wells as several antigen-non-specific defence chemicals that kill microbes. These include secretory IgA, IgA, IgG (and sometimes IgM), lysozyme, peroxidases, lactoferrin and chromogranin A (an antifungal protein). These chemicals are synthesized by the salivary glands, the oral epithelium and leukocytes in the gingival crevice/pocket, or are derived from plasma through the GCF. Saturating levels of calcium and phosphorus in saliva, together with fluoride, help to remineralize white spot lesions and negatively charged salivary molecules, which have a high affinity for the tooth surface, and inhibit the precipitation of calcium phosphate salts.

 The persistent film of saliva that coats the teeth and the oral epithelium as the salivary (enamel) pellicle also helps to maintain a balance between tooth demineralization and remineralization. The pellicle includes many of the defence chemicals found in saliva as well as proline-rich proteins, albumin, histatins, cystatins, statherin, mucins, amylase and complement component C3.

These may serve as receptors for bacteria that adhere to the tooth surface; however, selective attachment of harmless normal resident oral flora probably restricts the attachment of potential pathogens. In conditions of low salivary flow, e.g., Sjogren's syndrome, individuals are more susceptible to colonization with potential pathogens, and severe caries is a frequent outcome of poor salivary protective function.

 Lysozyme present in saliva and derived from salivary glands and GCF is similar to lysozyme found in other bodily fluids in that it is bactericidal due to muramidase activity; i.e., it splits the β-1,4 glucosidic linkage between NAG (N-acetyl glucosamine) and NAM (N-acetyl muramic acid) in the peptidoglycan of bacterial cell walls causing osmotic lysis. Many oral microbes are resistant to muramidase action, but lysozyme also has other effects: it activates endogenous bacterial enzymes (autolysins) in the cell wall that can kill bacteria, it aggregates oral bacteria to facilitate their removal and it contains amphipathic sequences within the C-terminus that have antimicrobial properties. Lysozyme also synergizes with other defence chemicals including lactoferrin and peroxidase for antimicrobial effect.

 Peroxidase activity in saliva comprises peroxidases from salivary glands as well as myeloperoxidase from neutrophils and eosinophil peroxidase. These catalyse the peroxidation of thiocyanate and halides by hydrogen peroxide (from aerobic metabolism of glucose by normal oral flora), which causes the formation of hypothyocyanite. Hypothyocyanite oxidizes bacterial enzymes in glycolytic pathways, and this inhibits the growth of oral microbes. Hydrogen peroxide is also toxic to eukaryotic cells, but its reduction by salivary peroxidases probably helps to protect the oral mucosa. Salivary lactoperoxidase generates toxic superoxide radicals that also kill microbes.

 Histidine-rich proteins (histatins) are cationic proteins found in abundance in submandibular/sublingual and parotid saliva. They display various functions including the initiation of histamine release from mast cells, inhibition of hydroxyapatite crystal growth, neutralization of toxins, protease activity, fungicidal activity and bactericidal activity. Histatins also prevent bacterial coaggregation and serve as competitive inhibitors of certain proteases, which may affect the pathogenesis of periodontitis since it involves extensive proteolytic destruction of host tissues. Cystatins, in contrast, are a family of proteins secreted mainly by the submandibular and sublingual salivary glands, which inhibit cysteine proteases. This is considered important for antimicrobial defence because of the beneficial functions of cysteine proteases in many oral microbes. Cystatins also influence inflammation because of their effects on host proteolytic and cytokine activity.

 Antiviral components in saliva include the secretory leukocyte protease inhibitor (SLPI) and several other proteins that have been demonstrated to possess activity against human immunodeficiency virus (HIV). SLPI is a small, cationic, acid-stable protein produced by serous acinar and epithelial cells. SLPI inhibits viral entry and/or uncoating in host cells and also displays serine protease inhibitor activity, which would protect the mucosal barrier from neutrophil-derived enzymes secreted during inflammation. SLPI also displays some bactericidal and fungicidal activity. Another class of salivary antiviral proteins is human parotid proline-rich proteins, which inhibit HIV activity most likely by interfering with the interactions between virus and host cell surfaces. Finally, thrombospondin 1 is an extracellular matrix glycoprotein secreted by submandibular and sublingual salivary glands that inhibits viral infection of monocytes and T cells. For HIV, this appears to occur via binding of thrombospondin 1 to viral gp120, which would inhibit the virus interacting with CD4 receptors on T cells.

 GCF is a vehicle by which blood components including leukocytes (estimated to consist of 95% neutrophils, 3% monocytes and 2% lymphocytes) can reach the oral cavity via flow of fluid through the junctional epithelium of the gingivae (gingival margin) into the gingival crevice. Normally, the flow of GCF is low but flow increases with inflammation to flush oral surfaces that are vulnerable to penetration by microbes. The composition of GCF also changes during inflammation from a transudate to a plasma-like inflammatory exudate, which can be collected from patients with oral disease. Various constituents of innate and acquired immunity reach sites of plaque accumulation from the blood via the GCF including neutrophils, plasma proteins (e.g., albumin and fibrin), monocytes, T and B lymphocytes, and Igs (IgG, IgM and IgA). Signalling molecules and inflammatory mediators including neutrophil elastase, collagenase-2, prostaglandin E2 and classical and alternative complement pathway components are also common in GCF. Other enzymes including lysozyme and proteases (a mixture of host and bacterial) have also been detected in GCF, and these have been shown to inactivate IgA. The functional significance of GCF is related to the antimicrobial properties of its constituents that impact oral microbial colonization and survival.

■ The mucus layer on intraoral surfaces exists as a sticky, slippery gel-like barrier composed of mucin glycoproteins, which prevent entry of microbes into underlying tissue. Mucus traps microbes and removes them from the oral cavity by sloughing. Mucus is also selectively permeable to allow transition of nutrients and waste products but not microbes. Mucins are derived from salivary glands and include the membrane-bound mucins MUC1 and MUC4, the gel-forming mucin MUC5B (MG1), and MUC7 (MG2). The gelatinous consistency of some mucins (e.g., MUC5B) is due to a thread-like structure rich in carbohydrates (up to 80%) and high molecular mass. In contrast, other mucins display low viscosity due to a smaller mass and relatively simplistic structure (e.g., MUC7); these different physicochemical properties enable distinct functions of different mucins. Mucins are distributed unevenly in the oral cavity; for example, they are rare in parotid secretions. Thus, saliva in the areas vestibular to the maxillary molars (derived from the parotid glands) is low in mucins. In contrast, saliva in the areas located vestibular to the upper incisors is derived from submandibular and sublingual glands and is rich in mucins. Similarly, more parotid agglutinin and other serous proteins such as amylase and proline-rich proteins are found in maxillary premolar pellicles compared with mandibular anterior pellicles. Unique patterns of mucin distribution probably influence oral microbial communities. Mucins can also aggregate bacteria via interactions between mucin saccharides and bacterial proteins. However, different sugars aggregate different oral bacteria, which may remove some microbes but allow other species to remain. Mucus also contains lysozyme, IgA, lactoperoxidase and lactoferrin to sequester iron from microbes. Mucins can form homotypic complexes (end-to-end oligomers) to enable lubrication properties and heterotypic complexes with S-IgA, lysozyme, cystatins and β-defensin to increase local concentrations of antimicrobial molecules. Low mucin production has been correlated with a higher microbial biomass suggesting a link between mucins and oral health.

Functionality of salivary defence constituents

The functions of individual components in saliva and GCF secretions are dynamic and related to molecular shape and enzymatic activity. The functions of these components may vary under different physicochemical conditions and are sometimes altered following absorption onto surfaces as opposed to in solution. For example, surface-absorbed proline-rich proteins promote bacterial adhesion; however, these molecules do not interact with bacteria when in solution.

Salivary amylase interacts with streptococci, but disruption of its disulphide bonds alters its molecular shape and abates this biological activity. Changes in conformation or epitope structure induced by binding to surfaces are the most likely explanation for divergent function in these components. Multiple overlapping functions are also common among salivary components. This enables redundancy in the activities of many salivary components. Functional redundancy may provide more dependable antimicrobial action for circumstances in which host components have been neutralized as a result of microbial activity. For example, agglutination of microbes is a shared function among many salivary components (i.e., mucins, S-IgA, parotid agglutinin, lysozyme, etc.), which would enable agglutination and clearance of microbes from the oral cavity even if one of the components were to be rendered non-functional (e.g., inactivation of S-IgA by microbial enzymes).

Amphifunctionality, i.e., both protective and detrimental effects, is also inherent in some salivary components. For example, statherin promotes remineralization of the tooth by inhibiting the formation of calcium and phosphate salts; however, when adsorbed to the enamel pellicle, statherin can also promote the adhesion of potentially cariogenic microbes to the tooth. Seemingly contradictory functions should be considered against the background that many salivary and pellicle components must act to promote the harmless normal resident oral flora but must also actively inhibit the adherence and growth of potential pathogens. Functional relationships between different salivary, pellicle and GCF components can be homotypic (same molecule) or heterotypic (different molecules) as in the case of mucins.

Microbial interactions and the normal oral flora

Colonization of the oral mucosal epithelium by normal resident oral flora is an important innate defence mechanism for immune exclusion because it prevents potential pathogens from colonizing the mouth. The normal flora secretes metabolic by-products such as antibiotics, competes for nutrients and receptors, and may alter the conditions in the microenvironment (e.g., pH, oxygen) to limit the growth of potential pathogens. Components of the normal flora such as LPSs may also stimulate non-specific innate immune defence mechanisms (e.g., activation of phagocytes, synthesis of crossprotective antibodies). When the normal oral flora is depleted (e.g., during broad-spectrum antibiotic therapy), the equilibrium between the oral mucosa and resident flora is disturbed, providing an opportunity for potential pathogens that may result in oral disease. One example is the infection by the oral fungal pathogen Candida albicans, where most of the commensal bacteria are killed by broad-spectrum antibiotics such as tetracycline.

The gingival sulcus, teeth and tongue harbour a normal flora, which includes several species of streptococci and other bacteria, now known to comprise more than 700-1000 species. Microbial relationships resulting, for example, from coaggregation between different species in mixed biofilms on teeth may encompass:

 microbial antagonism (one species harms, and can exclude, the other)

 synergism (two species co-operate to benefit both), e.g., co-operation between streptococci and gingivitis pathogens during disease

 symbiosis (a close ecological relationship of at least two species where at least one species benefits, the other may be unaffected or harmed)

 commensalism (one species benefits, the other is unaffected)

 mutualism (both species benefit), and

 parasitism (one species benefits, the other is harmed)

Adaptive immunity in oral health and disease

Acquired or adaptive immunity refers to all of the antigen- specific defence mechanisms that take several days to weeks to become protective and are designed to react with and remove specific antigens. Acquired immunity develops throughout one's life and is completely dependent on T and B lymphocytes. Acquired immunity in the oral cavity comprises both humoural and cellular mechanisms that involve GCF Igs (IgM, IgG and IgA) derived from plasma cells in the gingivae, effector T lymphocytes and, principally, S-IgA. The normal resident oral flora appears to be important in inducing a self-limiting humoural mucosal immune response that provides defence against potential pathogens. MALT that lies beneath the oral mucosal epithelium contains phagocytes for killing microbes and APCs, which sample antigens in the oral mucosa and provide the link between innate and acquired immune responses. Lymphoid cells around the basement membrane also help to eliminate any potential pathogens that overcome innate immune exclusion and pass through the intact oral mucosal epithelium.

Oral lymphoid tissues

Extraoral lymph nodes and intraoral lymphoid tissues are present in the mouth. Four types of intraoral lymphoid tissues are palatine and lingual tonsils, salivary gland lymphoid tissue (which contributes to S-IgA production), gingival lymphoid tissue and scattered submucosal lymphoid cells. Networks of lymph capillaries and lymph vessels link the oral mucosa, gingivae, and pulp to other structures such as the tongue and drain into submandibular, retropharyngeal and other lymph nodes. Microbes that have overcome innate immune exclusion and penetrated through the oral mucosa may enter the lymphatics directly or be transported into the lymphatics by phagocytes. When microbial antigens reach lymphocytes in the MALT, an immune response is elicited. Activated lymphocytes that have encountered antigen leave the MALT via the efferent lymphatics and enter the circulation, after which they relocate to the lamina propria to drive acquired immune responses. T cells in the lamina propria are predominantly of CD4 and CD8 types but another type of T cell termed 'intraepithelial lymphocytes' (IELs) are located between the epithelial cells and basement membrane. These cells appear to be involved in immune surveillance, maintenance of mucosal integrity via synthesis of growth factors and the removal of epithelial cells that become infected. B cells in the lamina propria and associated with acini of the major and minor salivary glands synthesize IgA. Tonsils may also guard the entry into the digestive and respiratory tracts, while the gingival lymphoid tissue may help in the immune response to dental plaque.

S-lgA in oral defence

S-IgA is the predominant Ig in saliva. It prevents microbes from adhering to mucosal epithelial cells by binding to and agglutinating them, which promotes their removal from the oral cavity. In contrast to the IgA present in plasma that is almost always monomeric (and derived from plasma cells in the bone marrow), S-IgA is composed of an IgA dimer derived from the polymerization of two IgA molecules (derived from plasma cells in the salivary glands) by joining (J) chain glycoprotein. Tetramers of S-IgA are also common. Incorporation of a glycoprotein fragment of the polymeric Ig receptor termed the secretory component (SC; synthesized by epithelial cells of the salivary acini) into IgA dimers forms complete S-IgA. Receptors for the SC on oral epithelial cells bind to S-IgA, which enables capturing and shedding of opsonized oral microbes, and this contributes to immune exclusion. Antigen-specific inhibition of microbial adherence by S-IgA depends on B cell clones produced against unique oral microbial antigens. In contrast, S-IgA present in the enamel pellicle may promote attachment of microbes to the tooth surface. S-IgA can also neutralize microbial toxins, enzymes and viruses. However, unlike other Igs, S-IgA does not activate complement and is therefore regarded as a non-inflammatory Ig. This unique attribute enables S-IgA to maintain the integrity of the mucosal barrier since complement activation generates potent mediators of inflammation such as C3a and C5a. The SC also makes the normally susceptible hinge region of S-IgA resistant to proteolytic and acidic conditions that exist in the mouth. S-IgA also helps to prevent infection within the salivary glands. It is noteworthy that more IgA (plasma and secretory) is produced each day than the other four types of Igs combined. Finally, S-IgA also influences innate defence by synergizing with the antimicrobial activities of lysozyme and potentiating the activities of mucins by reducing the negative surface charge and hydrophobicity of oral bacteria (allows the bacteria to be coated with mucins). Some S-IgA displays pluri-specific action (polyreactive, i.e., binds a wide range of bacterial and host antigens), which is believed to protect the oral mucosa prior to the induction of highly antigen-specific S-IgA.

Pluri-specific S-IgA appears to be derived in a T-independent manner against commensal oral microbes, food and host tissue antigens. In contrast, T-dependent mechanisms probably impart extremely specific S-IgA through B cell somatic hypermutation to produce Igs directed against only a single unique antigen. It is notable that some oral pathogens produce proteases that cleave and subvert the function of S-IgA. Heterotypic associations between S-IgA and lactoferrin, and S-IgA and agglutinins have been demonstrated, but their role in oral defence is unclear. Enigmatically, humans with a selective IgA deficiency are not highly susceptible to mucosal infection, and this condition is largely asymptomatic. Functional redundancy of antimicrobial molecules at the oral mucosal surface probably explains this apparent contradiction in the acquired immune response to oral microbes. In many people, selective IgA deficiency correlates with increased transportation of IgM into external secretions, which would compensate for this immune deficiency at mucosal surfaces.

PCD in response to oral microbes

Apoptosis, also termed 'programmed cell death' (PCD), is an important physiological mechanism through which the immune system responds to diverse forms of cell damage. PCD occurs normally under many conditions to remove unwanted, damaged or dying host cells; e.g., it removes autoreactive lymphocytes by negative selection, and it regulates the size of T cell memory pools after resolution of infection. PCD can promote the removal of pathogens by killing the host cells that are infected with them. PCD is controlled by cytoplasmic cysteine-dependent aspartate-directed proteases termed caspases that exist in all human cells and direct two pathways of PCD: death receptor- independent deregulation of mitochondrial function (the intrinsic pathway) and activation of death receptors (the extrinsic pathway).

End-stage PCD involves cleavage of proteins required for cell integrity, DNA degradation, chromatin condensation, externalization of lipid phosphatidylserine, cell shrinkage and cell disassembly into 'apoptotic bodies'. Importantly, apoptotic bodies are actively phagocytosed by macrophages to prevent spillage of intracellular contents from dying cells, and this limits inflammation. PCD in gingival epithelial cells has important implications for mucosal barrier function because of effects on immune exclusion, inflammation, antigen processing and presentation, and the acquired regulatory responses of T and B lymphocytes. For example, PCD facilitates antigen presentation to T lymphocytes through MHC I during tuberculosis. A significant group of oral pathogens including P. gingivalis, A. actinomycetemcomitans, Candida albicans, and Treponema denticola have been shown to modulate PCD pathways in human cells; whether these PCD responses are part of the normal immune response to these microbes and beneficial to oral health is unclear. However, detection of PCD in chronically inflamed gingiva suggests that it may help to maintain homoeostasis in the gingival tissue. On the other hand, induction of PCD by subgingival pathogens may also contribute to local tissue destruction during periodontitis; for example, up to 10% of the total cell population in gingival biopsies from patients with chronic periodontitis has been shown to be apoptotic. PCD in bone-lining cells triggered in the acquired immune response to P. gingivalis also appears to contribute to deficient bone formation in periodontitis by reducing the coupling of bone formation and resorption. Finally, delayed PCD in neutrophils during periodontitis has also been observed, suggesting a mechanism of neutrophil accumulation at sites of oral disease.

Some examples of recently discovered cell death responses triggered by oral microbes and substances are given in Table 8.4.

Table 8.4 Recently discovered cell death responses triggered by oral microbes and substances

Oral microbe or substance

Target cell type(s) and PCD response

Apoptosis regulatory molecule

Porphyromonas gingivalis

Epithelial cells, inhibits PCD

Gingipain adhesin peptide A44

Candida albicans

Vascular endothelial cells

Unknown

Streptococcus salivarius

Epithelial cells, no PCD activity, homoeostatic

None detected

Aggregatibacter actinomycetemcomitans

T cells, induces PCD

Leukotoxin

Fusobacterium nucleatum

Human monocyte-derived macrophages, lacks PCD activity

LPS

Fluoride

Various cell types, induces PCD

Unknown

Mastic

Oral polymorphonuclear leukocytes, inhibits PCD; oral squamous cell carcinoma, induces PCD

Unknown

LPS, Lipopolysaccharide; PCD, programmed cell death.

Key facts

 The immune system exists to protect the body against threats from outside (pathogens) and inside (e.g., cancer).

 Various natural or innate defence mechanisms initiate protection, but specific or adaptive responses, with memory, are required to neutralize fully most threats.

 Deficient immunological function results in increased susceptibility to infection.

 The immune system must learn not to react against ‘self components’; otherwise, autoimmune disease results.

 Components of the innate immune system include phagocytes, natural killer (NK) cells, the alternative complement pathway and inflammation.

 The adaptive immune response requires antigen-presenting cells (macrophages, dendritic cells, B cells) to process antigen into peptides displayed on major histocompatibility complex (MHC) molecules on the cell surface.

 The T lymphocytes are of two types: T-helper cells, which are CD4+ and recognize peptides presented by MHC II molecules, and T-cytotoxic/suppressor cells, which are CD8+ and recognize MHC I-peptide complexes.

 Both B cells and T cells recognize antigen through specific receptors. These receptors have variable regions that are derived by selection and recombination of germ-line gene segments.

 Those T cells whose antigen receptors react strongly to self molecules in the thymus are deleted, while those that recognize self molecules outside the thymus are usually made non-reactive.

 In the oral cavity, innate immunity is mediated principally by immune exclusion and inflammation.

The oral mucosal epithelium provides a physical barrier that, when breached, renders individuals highly susceptible to infection.

Antigen-non-specific defence chemicals important in the oral cavity are calprotectin, defensins, saliva, lysozyme, peroxidases, histidine-rich proteins and cystatins.

Antiviral components in saliva include the secretory leukocyte protease inhibitor, parotid proline-rich proteins and thrombospondin 1.

GCF is a plasma-like inflammatory exudate containing neutrophils, plasma proteins, monocytes, lymphocytes and immunoglobulins (IgG, IgM and IgA), which collectively impede microbial colonization, persistence and survival. Functional redundancy provides dependable antimicrobial action in the oral cavity and is related to the dynamic nature of enzymatic activity and shape of individual molecules. Amphifunctionality, i.e., both protective and detrimental effects, is inherent in some salivary components.

Intraoral lymphoid tissues are palatine and lingual tonsils, salivary gland lymphoid tissue, gingival lymphoid tissue and scattered submucosal lymphoid cells.

S-IgA, the predominant immunoglobulin in saliva, prevents microbes from adhering to mucosal epithelial cells and can display pluri- or highly antigen-specific actions.

Apoptosis in gingival epithelial cells and leukocytes has important implications for mucosal barrier function, acquired immunity and disease pathogenesis in the oral cavity.

Review questions (answers on p. 363 & p. 364)

Please indicate which answers are true, and which are false.

8.1 Lymphocyte populations do not include:

A. B lymphocytes

B. phagocytes

C. CD4+ helper T cells

D. natural killer cells

E. CD8+ cytotoxic T cells

8.2 Innate immune mechanisms do not include:

A. mechanical barriers

B. phagocytosis

C. acute-phase proteins

D. antibody-mediated neutralization

E. complement activation

8.3 Which of the following is not a molecular event occurring during cell development in bone marrow?

A. immunoglobulin heavy-chain gene rearrangement

B. immunoglobulin light-chain gene rearrangement

C. μ heavy-chain expression in precursor B cells

D. expression of IgE on B cell surface

E. pairing of μ heavy chain with light chain to form IgM molecule

8.4 During T cell development in the thymus:

A. CD4+CD8+ cells differentiate into CD4-CD8- cells

B. positive selection takes place after negative selection

C. CD4-CD8- cells are located in the thymic medulla

D. mature, functional T cells are either CD4+CD8- or CD4-CD8+ cells

E. thymocytes undergo extensive immunoglobulin gene rearrangements

8.5 Two major mechanisms of innate immunity in the oral cavity are:

A. gingival crevicular fluid (GCF) and salivary agglutinins

B. mucins and peroxidases

C. calprotectin and lysozyme

D. complement and S-IgA

E. immune exclusion and inflammation

8.6 Which of the following oral mucosa constituents prevent microbial penetration:

A. saliva

B. keratin

C. granular layer

D. basement membrane

E. resident professional phagocytes

8.7 Antimicrobial actions of antigen-non-specific defence chemicals in oral secretions include but are not limited to:

A. aggregation of microbes

B. agglutination of microbes

C. promotion of microbial adhesion

D. inhibiting the growth of microbes

E. contributing to microbial nutrition

8.8 Defensins in oral secretions are:

A. pore-forming peptides that cause osmotic instability in microbes

B. divided into a and у types according to disulphide bond patterns

C. active against bacteria, fungi and some enveloped viruses

D. chemotactic for eosinophils and basophils

E. comparable in mode of action to the membrane attack complex

8.9 In addition to the defence chemicals normally found in saliva, the salivary pellicle contains which of the following defence chemicals:

A. proline-rich proteins

B. histatins and cystatins

C. calprotectin

D. complement component C3

E. cathelicidins

8.10 Antiviral components in saliva include:

A. complement component C5

B. peroxidases

C. lactoferrin

D. secretory leukocyte protease inhibitor

E. parotid proline-rich proteins

8.11 Constituents of acquired immunity that reach sites of plaque accumulation from the blood via the GCF include:

A. IgA

B. neutrophils

C. T and B lymphocytes

D. alternative complement pathway components

E. IgG and IgM

8.12 Mucins:

A. are distributed evenly in the oral cavity

B. aggregate bacteria via interactions between mucin proteins and bacterial saccharides

C. form homotypic complexes to enable lubrication

D. form heterotypic complexes to concentrate antimicrobial molecules locally

E. (production) has been correlated with lower microbial biomass

8.13 Which of the following statements regarding the functions of salivary and GCF antimicrobial components are true:

A. changes in conformation or epitope structure induced by binding to surfaces do not explain divergent function in relation to antimicrobial action

B. amphifunctionality refers to antimicrobial components with either protective or detrimental effects towards microbes

C. functional redundancy provides more wide-ranging antimicrobial activity for the control of many different classes of microbes

D. functional relationships between GCF antimicrobial components are always heterotypic

E. functions of salivary antimicrobial components are related to molecular shape and enzymatic activity

8.14 A microbial relationship between streptococci and fusobacteria leading to plaque biofilm formation can be regarded as:

A. microbial antagonism

B. microbial synergism

C. microbial symbiosis

D. microbial commensalism

E. microbial parasitism

8.15 Adaptive immunity in the oral environment:

A. encompasses non-antigen-specific defence mechanisms that take several days to weeks to become protective

B. is mediated largely by S-IgA

C. influences innate defence in the oral cavity by synergizing with lysozyme and mucins

D. utilizes immunoglobulin to neutralize microbial toxins, enzymes and viruses

E. inhibits microbial adherence using S-IgA produced against oral microbial antigens

8.16 Which of the following statements on programmed cell death in response to oral microbes are true:

A. it is controlled by cytoplasmic cysteine-dependent arginine-directed proteases termed caspases

B. it occurs normally in the oral cavity to remove unwanted, damaged or dying host cells

C. two pathways of programmed cell death are the intrinsic and the extrinsic pathways

D. apoptotic bodies promote inflammation in response to oral microbes

E. programmed cell death may be beneficial to oral health but may also contribute to local tissue destruction during periodontitis

Further reading

Diamond, G., Beckloff, N., Weinberg, A., et al. (2009). The roles of antimicrobial peptides in innate host defense. Current Pharmaceutical Design, 15(21), 2377-2392.

Gorr, S. U. (2009). Antimicrobial peptides of the oral cavity. Periodontology, 51, 152-180.

Janeway, C. A., Jr., Travers, P., Walport, M., et al. (2001). Immunobiology (5th ed.). New York: Garland Publishing.

Lamster, I. B., & Ahlo, J. K. (2007). Analysis of gingival crevicular fluid as applied to the diagnosis of oral and systemic diseases. Annals of the New York Academy of Sciences, 1098, 216-229.

Macpherson, A. J., McCoy, K. D., Johansen, F. E., et al. (2008). The immune geography of IgA induction and function. Mucosal Immunology, 1(1), 11-22.

Mestecky, J., Lamm, M. F., McGhee, J. R., et al. (2005). Mucosal immunology (3rd ed.). San Diego: Elsevier.

Roitt, I. M. (1997). Roitt's essential immunology (9th ed.). Oxford: Blackwell.

Roitt, I., Brostoff, J., & Male, D. (1998). Immunology (5th ed.). London: Mosby.

Staines, N., Brostoff, J., & James, K. (1994). Introducing immunology (2nd ed.). London: Mosby.

Ulett, G. C., & Adderson, E. E. (2006). Regulation of apoptosis by Gram-positive bacteria: Mechanistic diversity and consequences for immunity. Current Immunology Reviews, 2, 119-141.



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