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).

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

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

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:

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:

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.

Fig. 8.2 Toll-like receptors (TLRs). LPS, lipopolysaccharide.

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 below) 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. 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.

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

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. interleukins-1 and -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:

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), β₁H globulin (factor H), CR1 and factor I.

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.

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.

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.

Fig. 8.5 Classical pathway of complement activation.

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 × 10⁶, forms transmembrane channels, which permit osmotic influx so that the target cell swells up and bursts.

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

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.

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

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:

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.

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 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 non-encapsulated 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 non-linear antigenic determinants (epitopes) and does not require MHC presentation.