The Innate Immune System

THE INNATE IMMUNE SYSTEM

1) Innate Barriers At Epidermal And Mucosal Surfaces

Innate defence against the passage of potentially pathogenic microorganisms across epidermal and mucosal barriers involves a range of non-specific mechanisms. Commensal microorganisms living on mucosal surfaces and on membranes such as skin and conjunctiva constitute one such mechanism). These commensals are, under normal circumstances, non-pathogenic, and help prevent colonization by pathogenic strains. There are also a number of physical and chemical barriers against microbial entry, including the flow of fluid secretions from tear ducts, the urogenital tract and the skin. Many of these secretions possess bacteriostatic or bactericidal activity due to their low pH or the presence of hydrolytic enzymes such as lysozyme (a peptidoglycan hydrolase). Similarly, the mucus barrier covering mucosal surfaces such as the epithelium of the lung serves as a false binding platform for microorganisms, preventing them from interacting with the underlying host cells. In the normal state the hydrated mucus barrier is efficiently cleared under the driving force of beating cilia. The serious lung infections seen in cystic fibrosis arise because patients are unable to clear the bacteria-laden dehydrated mucus effectively.

2) Innate Defence Once Epidermal Or Mucosal Barriers Have Been Compromised

The function of the innate defence system against microorganisms that have penetrated into interstitial tissues and the vascular compartment relies largely on the processes of phagocytosis and of activation of the alternative complement pathway. However, the functions of the innate system when exposed to microbial infection are also critical in the recruitment and activation of cells of the adaptive immune response.

The main cells mediating phagocytosis are the mononuclear phagocytic cells and granulocyte cell populations; of the latter, neutrophils are particularly important. For such cells to function, they must possess receptors to sense signals from their environment. In executing their effector functions they need to secrete a range of molecules that will recruit or activate other immune cells to a site of infection.

Before consideration of the process of phagocytosis, an overview of the mononuclear phagocytic cell and granulocyte cell populations is useful.

a) Mononuclear phagocytic cells

The mononuclear phagocytic cells include monocytes and macrophages. Monocytes make up approximately 5% of the circulating blood leucocyte population and are short-lived cells (circulating in blood for ≤ 8 hours), but migrate into tissue to give rise to tissue macrophages. The macrophages constitute a long-lived, widely distributed heterogeneous population of cell types which bear different names within different tissues, such as the migrating Kupffer cell within the liver or the fixed mesangial cell within the kidney glomerulus.

The mononuclear phagocytic cells secrete a wide range of molecules too numerous to list in full here. However, these secretions include:

•         Molecules which can break down or permeabilize microbial membranes and thereby mediate extracellular killing of microorganisms, e.g. enzymes (lysozyme or cathepsin G), bactericidal reactive oxygen species and cationic proteins.

•         Cytokines which can provide innate protective antiviral (e.g. interferon (IFN)-α or-β) and antitumour (e.g. TNF-α) activity against other host cells. A group of cytokines termed chemokines can also serve to chemoattract other leucocytes into an area of ongoing infection or inflammation, for example IL-8 which attracts neutrophils. Yet another group of cytokines has proinflammatory actions (e.g. IL-1 and TNF-α) which, among other outcomes, leads to activation of endothelial and leucocyte cells promoting increased leucocyte extravasation into tissues and, in the case of IL-1, activation of T-lymphocyte populations.

•         Bioactive lipids (e.g. thromboxanes, prostaglandins and leukotrienes), which further promote the inflammatory response through actions to increase capillary vasodilation and permeability.

•         The mononuclear phagocytic cells also possess numerous receptors that interact with their environment. These cells possess, among others:

•         Receptors for chemotaxis toward microorganisms, e.g. receptors for secreted bacterial peptides such as formylmethionyl peptide.

•         Receptors for complement proteins that serve as leucocyte activators (e.g. C3a and C5a; see section 2.2.4) or complement proteins that serve to coat (opsonize) microorganisms (e.g. C3b). An opsonized microbial surface more readily adheres to a phagocyte membrane, with the opsonin triggering enhanced activity of the phagocyte itself.

•         Receptors for promoting adherence, such as lectin receptors interacting with carbohydrate moieties on the surface of the microorganism, or receptors for Fc domains (non-antigen-recognition domains) of antibodies which opsonize microorganisms (e.g. the receptor for the Fc domain of IgG is Fcg), or integrin receptors for cell-cell adhesion (e.g. promoting interaction between a macrophage and T-lymphocyte).

•         Receptors for cytokines including those involved in macrophage activation (e.g.IFN.-γ) or limiting macrophage mobility (e.g. macrophage inhibitory factor, MIF) and hence increasing cell retention at a site of infection.

 b)    Granulocyte cell populations

The granulocyte cell populations include the neutrophils, basophils and eosinophils. The short-lived (2–3 days) neutrophil is the most abundant granulocyte (comprising > 90% of all circulating blood granulocytes) and is the most important in terms of phagocytosis; indeed, this is the main function of the neutrophil. The receptors and secretions of the neutrophil are similar to those of the macrophage, although notably the neutrophil does not present antigen via MHC class II proteins (see later). The neutrophil is recruited to sites of tissue infection or inflammation by a neutrophil-specific chemotactic factor (IL-8) and is also chemoattracted and activated by some of the same factors described for mononuclear phagocytic cells, including complement protein C3a, bacterial formylmethionyl peptides and leukotrienes. Like macrophages, neutrophils undergo a respiratory burst and are very effective generators of reactive oxygen species.

Eosinophils are poor phagocytic cells and have a specialized role in the extracellular killing of parasites such as helminths, which cannot be physically phagocytosed. Basophils are non-phagocytic cells.

c) Phagocytosis

Macrophages and neutrophils in particular demonstrate a high capacity for the physical engulfment of particles such as microorganisms or microbial fragments from their immediate extracellular environment. This process (Figure 9.2) is made up of a number of steps:

•         Chemotaxis of the phagocyte toward the microorganism through signals arising from the microorganism itself (e.g. formylmethionyl peptide), signals arising from complement proteins (e.g. C3a and C5a) generated as part of the activation of the alternative complement pathway (see section 2.2.4), or signals due to release of inflammatory factors (e.g. leukotrienes) secreted by other leucocyte cells situated at the site of an infection.

•         Adherence of the microorganism to the surface of the phagocyte (step A in Figure 9.2), involving adhesion through lectin receptors present on the surface of the phagocyte which interact with carbohydrate moieties on the surface of the microorganism; adhesion through complement C3b receptors present on the surface of the phagocyte interacting with C3b molecules that have opsonized the surface of the microorganism; and adhesion through Fc receptors which interact with the Fc domain of antibodies that have opsonized the surface of a microorganism.

•         Membrane activation of the phagocyte actin-myosin contractile network to extend pseudopodia around the attached microorganism (step B in Figure 9.2). Membrane activation will also lead to the generation of a ‘respiratory burst’ by the phagocyte which involves an increase in the activity of the phagocyte membrane NADPH oxidase which converts molecular oxygen into bactericidal reactive oxygen species such as superoxide anion (• O−), hydrogen peroxide (H2O₂), and in particular hydroxyl radicals (•O H) and halogenated oxygen metabolites (HOCl⁻).

•         The enclosure of phagocytosed material, initially within a membranous vesicle termed a phagosome. Here, cationic proteins such as defensins and reactive oxygen species begin microbial membrane degradation. This is followed within minutes by fusion of the phagosome with a lysosome to form a phagolysosome, whose contents are at an acidic pH of about 5 which is optimal for the continued active breakdown of microbial structural components (step C in Figure 9.2).

d) Alternative complement pathway

The alternative complement pathway fulfils a critical role in innate immune defence. The complement system comprises at least 20 different serum proteins; many are known by the letter C and a number, e.g. C3. Many of the complement proteins are zymogens, i.e. proenzymes requiring proteolytic cleavage to be enzymically active themselves; some are regulatory in function. The cleavage products of complement proteins are distinguished from their precursor by the suffix ‘a ’ or ‘b ’, e.g. C3a and C3b, with the suffix ‘b’ generally denoting the larger fragment that stays associated with a microbial membrane, and the suffix ‘a’ generally denoting the smaller fragment that diffuses away. The activation of the complement pathway occurs in a cascade sequence, with amplification occurring at each stage, such that each individual enzyme molecule activated at one stage generates multiple activated molecules at the next. In the ‘resting’ state, in the absence of infection, the complement proteins are inactive or have only a low level of spontaneous activation. The cascade is tightly regulated by both soluble and membrane-bound associated proteins. The regulation of the complement pathway prevents inappropriate activation of the cascade (i.e. when no infection is present) and also minimizes damage to host cells during an appropriate complement response to a microbial infection. Complement activation is normally localized to the site(s) of infection.

There are three main biological functions of the alternative complement pathway.

• Opsonization of microbial membranes. This involves the covalent binding of complement proteins to the surface of microbial membranes. This opsonization or coating by complement proteins promotes adherence of the opsonized microbial component(s) to the cell membranes of phagocytic cells. The complement protein C3b is a potent opsonin.

• Activation of leucocytes. This involves complement proteins acting on leucocytes, either at the site of infection or at some distance away, with the result of raising the level of functioning of the leucocytes in immune defence. For example, C3a is a potent leucocyte chemoattractant and also an activator of the respiratory burst.

• Lysis of the target cell membrane. This involves a collection of complement proteins associating on the surface of a microbial membrane to form a membrane attack complex (MAC), which leads to the formation of membrane pores and, ultimately, microbial cell lysis.

Figure 9.3 shows a highly schematized view of the activation cascade for the alternative complement pathway on a microbial membrane surface. The activation steps in the alternative pathway are also shown in Figure 9.7, which contrasts with the activation steps in the classical complement pathway involving antibody.

Figure 9.3 A highly schematized overview of the activation cascade for the alternative complement pathway on a microbial membrane surface. In the presence of a microbial membrane the C3b formed by C3 tickover deposits on the microbial membrane (step A). C3a diffuses away, leading to leucocyte activation. The deposited C3b leads to the generation of a stabilized C3 convertase (step B) which, through a positive feedback loop, leads to the amplified cleavage of more C3. Some C3b associates with the C3 convertase to generate a C5 convertase (step C) which will eventually lead to the generation of an MAC.

The pivotal protein in the alternative pathway is C3 (195 kDa). Under normal circumstances (in the absence of infection) C3 is cleaved very slowly through reaction with water or trace amounts of proteolytic enzyme to give C3b and C3a. The C3b formed is susceptible to nucleophilic attack by water and is rapidly inactivated to give iC3b. The C3a is not generated in sufficient amounts to lead to leucocyte activation and is rapidly inactivated. This normal low-level cleavage of the C3 molecule is termed ‘C3 tickover’ and it provides low levels of starting material, i.e. C3b, which will be required for full activation of the alternative complement pathway in the case of a microbial infection.

In the presence of a microbial membrane the C3b formed by C3 tickover will be susceptible to nucleophilic attack by hydroxyl or amine groups on the membrane surface, leading to the covalent attachment of C3b to the membrane (step A in Figure 9.3). Once C3b has attached to the membrane, factor B can bind to form a molecule termed C3bB. This complex is stabilized by a soluble protein called properdin. Factor D then enzymically cleaves the bound factor B to generate a molecule termed C3bBb which is the C3 convertase of the alternative pathway (Figure 9.3 inset).

This newly generated stable C3 convertase enzymically cleaves C3 to generate further C3b and C3a molecules, leading to leucocyte activation (by C3a) and greater deposition of C3b on the microbial membrane and hence further generation of C3 convertase molecules. In effect the microbial membrane has activated a positive feedback loop with cleavage of C3 to generate high amounts of C3b and C3a molecules.

The deposited C3b not only leads to the formation of the C3 convertase but also coats the microbial membrane as an opsonin and so promotes binding to phagocyte cell membranes. Some of the deposited C3b associates with the newly formed C3 convertase to generate a complex termed C3bBb3b, which is the C5 convertase of the alternative pathway (step B in Figure 9.3). This C5 convertase binds the complement protein C5 and cleaves it into C5a (a leucocyte activator) and C5b (an opsonin). The C5b remains associated with the membrane and acts as a plat-form for the sequential binding of complement proteins C6, C7, C8 (step C in Figure 9.3). The α-chain of the C8 molecule penetrates into the microbial membrane and mediates conformational changes in the incoming C9 molecules such that C9 becomes amphipathic (simultaneously containing hydrophilic and hydrophobic groups). In this form it is capable of insertion through the microbial membrane where it mediates a polymerization process that gives the MAC. The MAC generates transmembrane channels within the microbial membrane with the osmotic pressure of the cell leading to an influx of water and eventual microbial cell lysis.

Differences between host cell membranes and microbial cell membranes mean that the cascade is only activated in the prresence of microorganisms, so C3 tickover cannot give rise to full activation of the alternative pathway in the absence of microbial membrane. Stable deposition of a functional C3 convertase only occurs on the microbial cell surface. The differences that exist include, for example:

• lipopolysaccharide or peptidoglycan on microbial membranes that promote the binding of C3b

• the high sialic acid content of host cell membranes that promotes the dissociation of any C3 convertase formed on host surfaces

• the presence of specific host cell membrane proteins that also serve a key regulatory function.

Decay activating factor (DAF) or complement receptor type 1 (CR1) are host cell membrane proteins that serve to competitively block the binding of factor B with C3b and hence inhibit formation of a C3 convertase; they also promote disassembly of any C3 convertase formed. Membrane cofactor protein (MCP) and CR1 are further host cell membrane proteins that promote the displacement of factor B from its binding with C3b. Host cell membranes also possess a protein, CD59, which prevents the unfolding of C9—a required step for membrane insertion to form an effective MAC.