Immune defences in action

14 Immune defences in action


The barrier effects of the skin and mucous membranes and their adjuncts such as cilia have already been referred to (see Ch. 9). We now turn to the back-up mechanisms called rapidly into play when an organism has penetrated these barriers – namely, complement, the phagocytic and cytotoxic cells, and a variety of cytotoxic molecules. While they lack the dramatic specificity and memory of adaptive (i.e. lymphocyte-based) immune mechanisms, these natural defences are vital to survival – particularly in invertebrates, where they are the only defence against infection (adaptive responses only evolved with the earliest vertebrates).

In addition to these non-specific mechanisms, the immune system enables the specific recognition of antigens by T and B cells as part of adaptive immunity. Broadly speaking, antibodies are particularly important in combating infection by extracellular microbes, particularly pyogenic bacteria, while T-cell immunity is required to control intracellular infections with bacteria, viruses, fungi or protozoa. Their value is illustrated by the generally disastrous results of defects in T and/or B cells, or their products, discussed in more detail in Chapter 30. This chapter gives examples of how these different types of immunity contribute to the body’s defences against microbes.

Antimicrobial peptides protect the skin against invading bacteria

A number of proteins that are expressed at epithelial surfaces, and by polymorphonuclear leukocytes (PMNs), can have a direct antibacterial effect. These include β-defensins, dermicidins and cathelicidins. Defensins form 30–50% of neutrophil granules, and disrupt the lipid membranes of bacteria. Dermicidin is made by sweat glands and secreted into sweat; it is active against E. coli, Staphylococcus aureus and Candida albicans. The precursor cathelicidin protein is cleaved into two peptides, one of which, LL37, is not only toxic to microorganisms but also binds LPS. Mice whose PMNs and keratinocytes are unable to make cathelicidin become susceptible to infection with group A streptococcus. Cathelicidin also plays a role in immunity to M. tuberculosis, through its action on vitamin D.

An interesting innate defence mechanism is the formation of neutrophil extracellular traps or NETS. Neutrophil serine proteinases such as cathepsin G, neutrophil elastase and proteinase 3 can be exocytosed by neutrophils to form neutrophil extracellular traps with chromatin that bind both Gram-positive and Gram-negative bacteria (Fig. 14.1) Of course, the bacteria can fight back, in this case through secreting DNAases or by having capsules to prevent entrapment.

Lysozyme is one of the most abundant antimicrobial proteins in the lung. Genetically engineered transgenic mice that had a lot more lysozyme activity than control mice in their bronchoalveolar lavage were much better at killing group B streptococci, and Pseudomonas aeruginosa (Fig. 14.2).


The alternative pathway and lectin binding pathways of complement activation are part of the early defence system

The basic biology of the complement system and its role in inducing the inflammatory response and promoting chemotaxis, phagocytosis and vascular permeability have been described in Chapter 9. Here we are concerned with its ability to directly damage microorganisms as part of the early response to infection. Contrary to what might be expected from the dramatic lysis of many kinds of bacteria in the test tube, the action of complement in vivo is restricted mainly to the Neisseria. Patients deficient in C5, C6, C7, C8 or C9 are unable to eliminate gonococci and meningococci, with the increased risk of developing septicaemia or becoming a carrier.

It should be emphasized that only the alternative pathway of complement activation or the mannan-binding lectin pathway form part of this natural ‘early defence’ system. Activation through the classical pathway occurs only after an antibody response has been made. It is not surprising to learn, therefore, that the alternative pathway appears to have evolved first.

Acute phase proteins and pattern recognition receptors

C-reactive protein is an antibacterial agent produced by liver cells in response to cytokines

Among the acute phase proteins produced in the course of most inflammatory reactions, C-reactive protein (CRP) is particularly interesting in being an antibacterial agent, albeit of very restricted range. CRP is a pentameric β-globulin, somewhat resembling a miniature version of IgM (molecular weight 130 000 compared with 900 000 for IgM). It reacts with phosphorylcholine in the wall of some streptococci and subsequently activates both complement and phagocytosis. CRP is produced by liver cells in response to cytokines, particularly interleukin-6 (IL-6, see Ch. 11), and levels can rise as much as 1000-fold in 24    h – a much more rapid response than that of antibody (see Ch. 9). Therefore, CRP levels are often used to monitor inflammation, for example, in rheumatic diseases. Most of the other acute phase proteins are produced in increased amounts early in infection and have not just antimicrobial activity but can act as opsonins, antiproteases, play an immunomodulatory role or be involved in the fibrinolytic or anticoagulant pathways. For example, many of the complement components are acute phase proteins. Those with a role in protection against infection are also termed pattern recognition receptors, such as mannose-binding lectin. Some acute phase proteins such as lipopolysaccharide binding protein may reduce pathology by binding toxic bacterial products such as lipopolysaccharide.

Macrophages can recognize bacteria as foreign using Toll-like receptors

Another family of surface receptors, called Toll-like receptors, on macrophages and other cells, bind conserved microbial molecules such as lipopolysaccharide (LPS) (endotoxin), bacterial DNA, double-stranded RNA or bacterial flagellin. Pattern recognition receptors recognize these repeated structures (pathogen-associated molecular patterns, see Ch. 9) and this leads to release of proinflammatory cytokines such as tumour necrosis factor alpha (TNFα), IL-1 and IL-6. Signalling through the Toll-like receptors also leads to the increased expression of major histocompatibility complex (MHC) molecules and of co-stimulatory molecules, thus enhancing antigen presentation and usually leading to the activation of T-helper 1 (Th1) cells. It was recently suggested that a number of rare single nucleotide polymorphisms within the TLR4 gene (TLR4 binds endotoxin) were more common in people with meningococcal disease compared with controls.

Microbes in the cytosol of a cell can also be recognized as foreign, using another family of pattern recognition receptors, called nucleotide-binding and oligomerization leucine-rich repeat receptors (NLR). Some NLRs can sense bacterial or viral DNA, leading to activation of inflammasomes, which are complexes of proteins, ultimately leading to the secretion of IL-1β and other proinflammatory cytokines. NLRs can also induce a process called autophagy, in which normal cytoplasmic contents are degraded after fusion with autolysosmes.

Collectins and ficolins

Collectins are proteins that bind to carbohydrate molecules expressed on bacterial and viral surfaces. This results in cell recruitment, activation of the alternative complement cascade, and macrophage activation. Two collectins, the surfactant proteins A and D, are able to directly inhibit bacterial growth and opsonize bacteria, leading to phagocytosis and activation of complement. Surfactant protein A, has been shown to play a role in the innate defence of the lung against infection with group B streptococci. Mice deficient in surfactant protein A were much more susceptible to infection, developing greater pulmonary infiltration and dissemination of bacteria to the spleen, compared with those able to produce the collectin. Polymorphisms in the surfactant A and D genes have also been linked to susceptibility to respiratory syncytial virus (RSV), as these surfactants act as opsonins for the virus.

Mannose-binding lectin (MBL) is another collectin found in serum. Binding of MBL to carbohydrates containing mannose on microorganisms leads to complement activation, through the mannan-binding lectin pathway. Bacteria opsonized by MBL bind to the C1q receptor on macrophages, leading to phagocytosis. Many individuals have low serum concentrations of MBL due to mutations in the MBL gene or its promoter. A recent study of children with malignancies showed that MBL deficiency increased the duration of infections. Lung surfactant proteins A and D, and MBL, bind to the surface spikes or S protein of the SARS virus (see Ch. 19), and so people with low MBL genotypes may be at increased risk of SARS infection.

Ficolins are plasma proteins with a similar structure to collectins, and bind N-acetyl glucosamine and lipotechoic acid from the cell walls of Gram-positive bacteria.

Natural killer cells

Natural killer cells are a rapid but non-specific means of controlling viral and other intracellular infections

Natural killer (NK) cells provide an early source of cytokines and chemokines during infection, until there is time for the activation and expansion of antigen-specific T cells. NK cells can provide an important source of interferon-gamma (IFNγ) during the first few days of infection. NK cell cytokine production can be induced by monokines such as IL-12 and IL-18 that are in turn induced by macrophages in response to LPS or other microbial components. As well as IFNγ, NK cells can make TNFα and, under some conditions, the down-regulatory cytokine IL-10. Some tissues like the gut need their own special populations of NK-like cells. NK-like cells express some but not all the usual NK cell markers – but they do make large amounts of the cytokine IL-22, which helps defend the gut against certain intestinal pathogens.

NK cells can also act as cytotoxic effector cells, lysing host cells infected with viruses and some bacteria, as they make both cytotoxic granules and perforin. They recognize their targets by means of a series of activating and inhibitory receptors that are not antigen-specific. The inhibitory receptors recognize the complex of MHC class I and self peptide; if both this inhibitory receptor and another NK-cell-activating receptor are engaged, the NK cell will not be activated. However, if there is insufficient MHC class I on the cell surface, the inhibitory receptor is not engaged and the NK cell is activated to kill the target cell. This is an effective strategy, as some viruses inhibit MHC class I expression on the cells they infect. NK cells are therefore a more rapid but less specific means of controlling viral and other intracellular infections. The importance of NK cells is highlighted by the ability of mice lacking both T and B cells (severe combined immunodeficiency, SCID) to control some virus infections, and humans with NK cell defects are also susceptible to certain viruses (Table 14.1).

Table 14.1 Natural killer cells play an important role in controlling infections

Infections where NK cells have been shown to help control infection
Human Mouse
Human cytomegalovirus (HCMV; human herpesvirus 5) Mouse cytomegalovirus (MCMV)
Vesicular stomatitits virus (VSV) Herpes simplex virus
Herpes simplex virus (HSV) Vaccinia virus
Human papilloma virus (HPV) Influenza virus

NK cells form a bridge between the innate and adaptive immune responses, and their function may be enhanced by components of adaptive immunity. Some recent work even suggests that some NK cells can show some immunological memory, so perhaps their full functions are not yet fully appreciated!


Phagocytes engulf, kill and digest would-be parasites

Perhaps the greatest danger to the would-be parasite is to be recognized by a phagocytic cell, engulfed, killed and digested (Fig. 14.3). A description of the various stages of phagocytosis is given in Chapter 9. Phagocytes (principally macrophages) are normally found in the tissues where invading microorganisms are more likely to be encountered. In addition, phagocytes present in the blood (principally the PMNs) can be rapidly recruited into the tissues when and where required. Only about 1% of the normal adult bone marrow reserve of 3    ×    1012 PMNs is present in the blood at any one time, representing a turnover of about 1011 PMNs/day. Most macrophages remain within the tissues, and well under 1% of our phagocytes are present in the blood as monocytes. PMNs are short lived, but macrophages can live for many years (see below).

Oxidative killing

Non-oxidative killing

Non-oxidative killing involves the use of the phagocyte’s cytotoxic granules

Oxygen is not always available for killing microorganisms; indeed, some bacteria grow best in anaerobic conditions (e.g. the Clostridia of gas gangrene), and oxygen would in any case be in short supply in a deep tissue abscess or a TB granuloma. Phagocytic cells therefore contain a number of other cytotoxic molecules. The best studied are the proteins in the various PMN granules (Table 14.3), which act on the contents of the phagosome as the granules fuse with it. Note that the transient fall in pH accompanying the respiratory burst enhances the activity of the cationic microbicidal proteins and defensins. Neutrophil serine proteinases have homology to the cytotoxic granzymes released by cytotoxic T cells.

Table 14.3 Contents of polymorphonuclear leukocyte (PMN) and eosinophil granules

PMN and eosinophil granule contents  
PMN Eosinophil
Primary (azurophil) Specific (heterophil) Cationic
Myeloperoxidase Lysozyme Peroxidase
Acid hydrolases Lactoferrin Cationic proteins
Cathepsins G, B, D Alkaline phosphatase ECP
Defensins NADPH oxidase MBP
BPI Collagenase Neurotoxin
Cationic proteins Histaminase Lysophospholipase

BPI, bactericidal permeability increasing protein; ECP, eosinophil cationic protein; MBP, major basic protein; NADPH, nicotinamide adenine dinucleotide phosphate.

Another phagocytic cell, the eosinophil, is particularly rich in cytotoxic granules (Table 14.3). The highly cationic (i.e. basic) contents of these granules give them their characteristic acidophilic staining pattern. Five distinct eosinophil cationic proteins are known and seem to be particularly toxic to parasitic worms, at least in vitro. Because of the enormous difference in size between parasitic worms and eosinophils, this type of damage is limited to the outer surfaces of the parasite. The eosinophilia typical of worm infections is presumably an attempt to cope with these large and almost indestructible parasites. Both the production and level of activity of eosinophils is regulated by T cells and macrophages and mediated by cytokines such as interleukin 5 (IL-5) and tumour necrosis factor alpha (TNFα).

Monocytes and macrophages also contain cytotoxic granules. Unlike PMNs (Table 14.4), macrophages contain little or no myeloperoxidase, but secrete large amounts of lysozyme. Lysozyme is an antibacterial molecule maintained at a concentration of about 30    mg/mL in serum, though this concentration can increase to as high as 800    mg/mL in rare cases of monocytic leukaemia. Macrophages are extremely sensitive to activation by bacterial products (e.g. LPS) and T-cell products, e.g. IFNγ. Activated macrophages have a greatly enhanced ability to kill both intracellular and extracellular targets.

Table 14.4 The major phagocytic cells – PMNs and macrophages – differ in a number of important respects

Polymorphonuclear leukocytes and macrophages compared
  PMN Macrophage
Site of production Bone marrow Bone marrow
Duration in marrow 14    days 54    h
Duration in blood 7–10    h 20–40    h (monocyte)
Average life span 4    days Months–years
Numbers in blood (2.5–7.5)    ×    109/L (0.2–0.8)    ×    109/L
Marrow reserve 10        ×    blood  
Numbers in tissues (Transient) 100        ×    blood
Principal killing mechanisms Oxidative, non-oxidative Oxidative, nitric oxide, cytokines
Activated by TNFα, IFNγ, GM-CSF, microbial products TNFα, IFNγ, GM-CSF, microbial products (e.g. LPS)
Important deficiencies CGD
Lipid storage diseases
Major secretory products Lysozyme Over 80, including: lysozyme, cytokines (TNFα, IL-1), complement factors

CGD, chronic granulomatous disease; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; TNFα, tumour necrosis factor alpha.

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Jul 9, 2017 | Posted by in MICROBIOLOGY | Comments Off on Immune defences in action

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