Constitutive barriers to infection

The tightly packed, highly keratinised cells of the skin constantly undergo renewal and replacement, which physically limits colonisation by microorganisms. Microbial growth is inhibited by physiological factors, such as low pH and low oxygen tension, and sebaceous glands secrete hydrophobic oils that further repel water and microorganisms. Sweat also contains lysozyme, an enzyme that destroys the structural integrity of bacterial cell walls; ammonia, which has antibacterial properties; and several antimicrobial peptides such as defensins. Similarly, the mucous membranes of the respiratory, gastrointestinal and genitourinary tract provide a constitutive barrier to infection. Secreted mucus acts as a physical barrier to trap invading pathogens, and immunoglobulin A (IgA) prevents bacteria and viruses attaching to and penetrating epithelial cells. As in the skin, lysozyme and antimicrobial peptides within mucosal membranes can directly kill invading pathogens, and additionally lactoferrin acts to starve invading bacteria of iron. Within the respiratory tract, cilia directly trap pathogens and contribute to removal of mucus, assisted by physical manœuvres, such as sneezing and coughing. In the gastrointestinal tract, hydrochloric acid and salivary amylase chemically destroy bacteria, while normal peristalsis and induced vomiting or diarrhoea assist clearance of invading organisms.

Endogenous commensal bacteria provide an additional constitutive defence against infection (p. 136). They compete with pathogenic microorganisms for space and nutrients, and produce fatty acids and bactericidins that inhibit the growth of many pathogens. In addition, commensal bacteria help to shape the immune response by inducing specific regulatory T cells within the intestine (p. 78).

These constitutive barriers are highly effective, but if external defences are breached by a wound or pathogenic organism, the specific soluble proteins and cells of the innate immune system are activated.

Phagocytes

Phagocytes (‘eating cells’) are specialised cells which ingest and kill microorganisms, scavenge cellular and infectious debris, and produce inflammatory molecules which regulate other components of the immune system. They include neutrophils, monocytes and macrophages, and are particularly important for defence against bacterial and fungal infections.

Phagocytes express a wide range of surface receptors that allow them to identify microorganisms. These pattern recognition receptors include the Toll-like receptors, NOD (nucleotide-oligomerisation domain protein)-like receptors and mannose receptors. They recognise generic molecular motifs not present on mammalian cells, including bacterial cell wall components, bacterial DNA and viral double-stranded RNA. While phagocytes can recognise microorganisms through pattern recognition receptors alone, engulfment of microorganisms is greatly enhanced by opsonisation. Opsonins include acute phase proteins such as C-reactive protein (CRP), antibodies and complement. They bind both to the pathogen and to phagocyte receptors, acting as a bridge between the two to facilitate phagocytosis (Fig. 4.1).

Neutrophils

Neutrophils, also known as polymorphonuclear leucocytes, are derived from the bone marrow (Fig. 4.2). They are short-lived cells with a half-life of 6 hours in the blood stream, and are produced at the rate of approximately 10¹¹ cells daily. Their functions are to kill microorganisms directly, facilitate the rapid transit of cells through tissues, and non-specifically amplify the immune response. This is mediated by enzymes contained in granules which also provide an intracellular milieu for the killing and degradation of microorganisms.

The two main types of granule are primary or azurophil granules, and the more numerous secondary or specific granules. Primary granules contain myeloperoxidase and other enzymes important for intracellular killing and digestion of ingested microbes. Secondary granules are smaller and contain lysozyme, collagenase and lactoferrin, which can be released into the extracellular space. Granule staining becomes more intense in response to infection (‘toxic granulation’), reflecting increased enzyme production.

When tissues are changed or damaged, they trigger the local production of inflammatory molecules and cytokines. These stimulate the production and maturation of neutrophils in the bone marrow, and their release into the circulation. The neutrophils are recruited to the inflamed site by chemotactic agents and by activation of local endothelium. Transit of neutrophils through the blood stream is responsible for the rise in leucocyte count that occurs in early infection. Once within infected tissue, activated neutrophils seek out and engulf invading microorganisms. These are initially enclosed within membrane-bound vesicles which fuse with cytoplasmic granules to form the phagolysosome. Within this protected compartment, killing of the organism occurs through a combination of oxidative and non-oxidative killing. Oxidative killing, also known as the respiratory burst, is mediated by the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase enzyme complex. This converts oxygen into reactive oxygen species such as hydrogen peroxide and superoxide that are lethal to microorganisms. When combined with myeloperoxidase, hypochlorous ions (HOCl⁻, analogous to bleach) are produced, which are highly effective oxidants and antimicrobial agents. Non-oxidative (oxygen-independent) killing occurs through the release of bactericidal enzymes into the phagolysosome. Each enzyme has a distinct antimicrobial spectrum, providing broad coverage against bacteria and fungi.

The process of phagocytosis depletes neutrophil glycogen reserves and is followed by neutrophil cell death. As the cells die, their contents are released and lysosomal enzymes degrade collagen and other components of the interstitium, causing liquefaction of closely adjacent tissue. The accumulation of dead and dying neutrophils results in the formation of pus, which, if extensive, may result in abscess formation.

Dendritic cells

Dendritic cells are specialised antigen-presenting cells which are prevalent in tissues in contact with the external environment, such as the skin and mucosa. They can also be found in an immature state in the blood. They sample the environment for foreign particles, and once activated, carry microbial antigens to regional lymph nodes, where they interact with T cells and B cells to initiate and shape the adaptive immune response.

Cytokines

Cytokines are small soluble proteins that act as multipurpose chemical messengers. Examples are listed in Box 4.2. They are produced by cells involved in immune responses and by stromal tissue. More than 100 cytokines have been described, with overlapping, complex roles in intercellular communication. Their clinical importance is demonstrated by the efficacy of ‘biological’ therapies (often abbreviated to ‘biologics’) that target specific cytokines (pp. 1102 and 18).

Complement

The complement system is a group of more than 20 tightly regulated, functionally linked proteins that act to promote inflammation and eliminate invading pathogens. Complement proteins are produced in the liver and are present in the circulation as inactive molecules. When triggered, they enzymatically activate other proteins in a rapidly amplified biological cascade analogous to the coagulation cascade (p. 995).

There are three mechanisms by which the complement cascade may be triggered (Fig. 4.3):

Activation of complement by any of these pathways results in activation of C3. This, in turn, activates the final common pathway, in which the complement proteins C5–C9 assemble to form the membrane attack complex. This can puncture target cell walls, leading to osmotic cell lysis. This step is particularly important in the defence against encapsulated bacteria, such as Neisseria spp. and Haemophilus influenzae. Complement fragments generated by activation of the cascade can also act as opsonins, rendering microorganisms more susceptible to phagocytosis by macrophages and neutrophils (see Fig. 4.1). In addition, they are chemotactic agents, promoting leucocyte trafficking to sites of inflammation. Some fragments act as anaphylotoxins, binding to complement receptors on mast cells and triggering release of histamine, which increases vascular permeability. The products of complement activation also help to target immune complexes to antigen-presenting cells, providing a link between the innate and the acquired immune systems. Finally, activated complement products dissolve the immune complexes that triggered the cascade, minimising bystander damage to surrounding tissues.

Mast cells and basophils

Mast cells and basophils are bone marrow-derived cells which play a central role in allergic disorders. Mast cells reside predominantly in tissues exposed to the external environment, such as the skin and gut, while basophils are located in the circulation and are recruited into tissues in response to inflammation. Both contain large cytoplasmic granules which contain preformed vasoactive substances such as histamine (see Fig. 4.9, p. 89). Mast cells and basophils express IgE receptors on their cell surface (see Fig. 4.5). On encounter with specific antigen, the cell is triggered to release preformed mediators and synthesise additional mediators, including leukotrienes, prostaglandins and cytokines. These trigger an inflammatory cascade which increases local blood flow and vascular permeability, stimulates smooth muscle contraction, and increases secretion at mucosal surfaces.

Natural killer cells

Natural killer (NK) cells are large granular lymphocytes which play a major role in defence against tumours and viruses. They exhibit features of both the adaptive and innate immune systems: they are morphologically similar to lymphocytes and recognise similar ligands, but they are not antigen-specific and cannot generate immunological memory.

NK cells express a variety of cell surface receptors. Some recognise stress signals, while others recognise the absence of human leucocyte antigen (HLA) molecules on cell surfaces (down-regulation of HLA molecules by viruses and tumour cells is an important mechanism by which they evade T lymphocytes). NK cells can also be activated by binding of antigen–antibody complexes to surface receptors. This physically links the NK cell to its target in a manner analogous to opsonisation, and is known as antibody-dependent cellular cytotoxicity (ADCC).

Activated NK cells can kill their targets in various ways. Pore-forming proteins, such as perforin, induce direct cell lysis, while granzymes are proteolytic enzymes which stimulate apoptosis. In addition, NK cells produce a variety of cytokines, such as tumour necrosis factor (TNF)-α and interferon-γ (IFN-γ), which have direct antiviral and antitumour effects.

Lymphoid organs

Humoral immunity

Immunoglobulins

Immunoglobulins (Ig) are soluble proteins made up of two heavy and two light chains (Fig. 4.5). The heavy chain determines the antibody class or isotype, i.e. IgG, IgA, IgM, IgE or IgD. Subclasses of IgG and IgA also occur. The antigen is recognised by the antigen-binding regions (F_ab) of both heavy and light chains, while the consequences of antibody-binding are determined by the constant region of the heavy chain (F_c) (Box 4.3).

4.3   Classes and properties of antibody

Antibody/properties	Concentration in adult serum	Complement activation*	Opsonisation
IgG
4 subclasses: IgG1, IgG2, IgG3, IgG4 Distributed equally between blood and extracellular fluid, and transported across placenta IgG2 is the predominant antibody produced against polysaccharides	8.0–16.0 g/L	lgG1 +++ lgG2 + lgG3 +++	IgG1 ++ IgG3 ++
IgA
2 subclasses: IgA1, IgA2 Highly effective at neutralising toxins Particularly important at mucosal surfaces	1.5–4.0 g/L	−	−
IgM
Highly effective at agglutinating pathogens	0.5–2.0 g/L	++++	−
IgE
Mostly bound to mast cells, basophils and eosinophils Important in allergic disease and defence against parasite infection	0.003–0.04 g/L	−	−
IgD
Function unknown	Not detected	−	−

^*Refers to activation of the classical complement pathway, also called ‘complement fixation’.

Antibodies can initiate a number of different actions. They facilitate phagocytosis by acting as opsonins (see Fig. 4.1), and can also facilitate cell killing by cytotoxic cells (ADCC, p. 75). Binding of antibodies to antigen can trigger activation of the classical complement pathway (see Fig. 4.3). In addition, antibodies may act directly to neutralise the biological activity of toxins. This is a particularly important feature of IgA antibodies, which act predominantly at mucosal surfaces.

The humoral immune response is characterised by immunological memory: that is, the antibody response to successive exposures to antigen is qualitatively and quantitatively different from that on first exposure. When a previously unstimulated (naïve) B lymphocyte is activated by antigen, the first antibody to be produced is IgM, which appears in the serum after 5–10 days. Depending on additional stimuli provided by T lymphocytes, other antibody classes (IgG, IgA and IgE) are produced 1–2 weeks later. If, some time later, a memory B cell is re-exposed to antigen, the lag time between antigen exposure and the production of antibody is decreased (to 2–3 days), the amount of antibody produced is increased, and the response is dominated by IgG antibodies of high affinity. Furthermore, in contrast to the initial antibody response, secondary antibody responses do not require additional input from T lymphocytes. This allows the rapid generation of highly specific responses on pathogen re-exposure.

Cellular immunity

T lymphocytes (also known as T cells) mediate cellular immunity and are important for defence against viruses, fungi and intracellular bacteria. They also play an important immunoregulatory role, orchestrating and regulating the responses of other components of the immune system. T-lymphocyte precursors arise in bone marrow and are exported to the thymus while still immature (see Fig. 4.6 below). Within the thymus, each cell expresses a T-cell receptor with a unique specificity. These cells undergo a process of stringent selection to ensure that autoreactive T cells are deleted. Mature T lymphocytes leave the thymus and expand to populate other organs of the immune system. It has been estimated that an individual possesses 10⁷–10⁹ T-cell clones, each with a unique T-cell receptor, ensuring at least partial coverage for any antigen encountered.

T cells respond to protein antigens, but they cannot recognise these in their native form. Instead, intact protein must be processed into component peptides which bind to a structural framework on the cell surface known as HLA (human leucocyte antigen). This process is known as antigen processing and presentation, and it is the peptide/HLA complex which is recognised by individual T cells. While all nucleated cells have the capacity to process and present antigens, specialised antigen-presenting cells include dendritic cells, macrophages and B lymphocytes. HLA molecules exhibit extreme polymorphism; as each HLA molecule has the capacity to present a subtly different peptide repertoire to T lymphocytes, this ensures enormous diversity in recognition of antigens within the population.

T lymphocytes can be segregated into two subgroups on the basis of function and recognition of HLA molecules. These are designated CD4⁺ and CD8⁺ T cells, according to the ‘cluster of differentiation’ (CD) antigen expressed on their cell surface. CD8⁺ T cells recognise antigenic peptides in association with HLA class I molecules (HLA-A, HLA-B, HLA-C). They kill infected cells directly through the production of pore-forming molecules such as perforin, or by triggering apoptosis of the target cell, and are particularly important in defence against viral infection. CD4⁺ T cells recognise peptides presented on HLA class II molecules (HLA-DR, HLA-DP and HLA-DQ) and have mainly immunoregulatory functions. They produce cytokines and provide co-stimulatory signals that support the activation of CD8⁺ T lymphocytes and assist the production of mature antibody by B cells. In addition, their close interaction with phagocytes determines cytokine production by both cell types.

CD4⁺ lymphocytes can be further subdivided into subsets on the basis of the cytokines they produce:

Investigations and management

Complement C3 and C4 are the only complement components that are routinely measured. Screening for complement deficiencies is performed using more specialised functional tests of complement-mediated haemolysis, known as CH50 and AP50 (classical haemolytic pathway 50 and alternative pathway 50). If abnormal, these haemolytic tests should be followed by measurement of individual complement components.

There is no definitive treatment for complement deficiencies. Patients should be vaccinated with meningococcal, pneumococcal and H. influenzae B vaccines in order to boost their adaptive immune responses. Life-long prophylactic penicillin to prevent meningococcal infection is recommended. At-risk family members should also be screened.

DiGeorge syndrome

This results from failure of development of the 3rd/4th pharyngeal pouch, usually caused by a deletion of 22q11. It is associated with multiple abnormalities, including congenital heart disease, hypocalcaemia, tracheo-oesophageal fistulae, cleft lip and palate, and absent thymic development. The immune deficiency is characterised by very low numbers of circulating T cells, despite normal development in the bone marrow.

Bare lymphocyte syndromes

These are caused by absent expression of HLA molecules within the thymus. If HLA class I molecules are affected, CD8⁺ lymphocytes fail to develop, while absent expression of HLA class II molecules affects CD4⁺ lymphocyte maturation. In addition to recurrent infections, failure to express HLA class I is associated with systemic vasculitis caused by uncontrolled activation of NK cells.

Autoimmune lymphoproliferative syndrome

This is caused by failure of normal lymphocyte apoptosis (p. 50), leading to non-malignant accumulation of autoreactive cells. This results in lymphadenopathy, splenomegaly and a variety of autoimmune diseases.