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.

Figure 14.1 Neutrophil extracellular traps can trap bacteria. These chromatin-containing complexes can trap bacteria such as Shigella (illustrated).

(Photograph courtesy of Dr. Volker Brinkmann, Max Planck Institute for Infection Biology, Berlin.)

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).

Figure 14.2 Transgenic mice making greater amounts of lysozyme are more resistant to infection with Pseudomonas aeruginosa. (A) The transgenic mice made 18-fold more lysozyme than the wild-type control mice. (B) The transgenic mice showed much greater killing of Pseudomonas aeruginosa in the lungs following intratracheal infection than the wild-type mice.

(Redrawn with data from Akinbi, H.T. et al. (2000) Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice. J Immunol 165:5760–5766.)

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.

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.

It is probably unwise to generalize about the benefit or otherwise of fever

Several microorganisms have been shown to be susceptible to high temperature. This was the basis for the ‘fever therapy’ of syphilis by deliberate infection with blood-stage malaria, and the malaria parasite itself may also be damaged by high temperatures, though it is obviously not totally eliminated. In general, however, one would predict that successful parasites were those that were adapted to survive episodes of fever; indeed the ‘stress’ or ‘heat-shock’ proteins produced by both mammalian and microbial cells in response to stress of many kinds, including heat, are thought to be part of their protective strategy. On the other hand, several host immune mechanisms might also be expected to be more active at slightly higher temperatures: examples are complement activation, membrane function, lymphocyte proliferation and the synthesis of proteins such as antibody and cytokines.

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

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).

Figure 14.3 (A) Electron micrograph and (B) diagrammatic representation of neutrophil containing phagocytosed Candida albicans (×    7000).

(Courtesy of H. Valdimarsson.)

Phagocytes kill organisms using either an oxidative or a non-oxidative mechanism

The mechanisms by which phagocytes kill the organisms they ingest are traditionally divided into oxidative and non-oxidative, depending upon whether the cell consumes oxygen in the process. Respiration in PMNs is non-mitochondrial and anaerobic, and the burst of oxygen consumption, the so-called ‘respiratory burst’ (Fig. 14.4) that accompanies phagocytosis represents the generation of microbicidal reactive oxygen intermediates (ROIs).

Figure 14.4 Oxygen-dependent microbicidal activity during the respiratory burst. The enzyme NADPH oxidase in the phagosome membrane reduces oxygen by the addition of electrons to form superoxide anion (). This can then give rise to hydroxyl radicals (^•OH), singlet oxygen (Δg′O₂) and H₂O₂, all of which are potentially toxic. If lysosome fusion occurs, myeloperoxidase or in some cases, catalase from peroxisomes, acts on peroxides in the presence of halides to generate toxic oxidants such as hypohalite. NADPH, nicotinamide adenine dinucleotide phosphate.

(Reproduced from: Male, D., Brostoff, J., Roth, D.B., Roitt, I. (2006) Immunology, 7th edn. Mosby Elsevier, with permission.)

Oxidative killing involves the use of ROIs

The importance of ROIs in bacterial killing was revealed by the discovery that PMNs from patients with chronic granulomatous disease (CGD) did not consume oxygen after phagocytosing staphylococci. Patients with CGD have one of three kinds of genetic defect in a PMN membrane enzyme system involving nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, PHOX (see Ch. 30). The normal activity of this system is the progressive reduction of atmospheric oxygen to water with the production of ROIs such as the superoxide ion, hydrogen peroxide and free hydroxyl radicals, all of which can be extremely toxic to microorganisms (Table 14.2).

Table 14.2 Some organisms killed by reactive oxygen and nitrogen species

CGD patients are unable to kill staphylococci and certain other bacteria and fungi, which consequently cause deep chronic abscesses. They can, however, deal with catalase-negative bacteria such as pneumococci because these produce, and do not destroy, their own hydrogen peroxide in sufficient amounts to interact with the cell myeloperoxidase, producing the highly toxic hypochlorous acid. The defective PMNs from CGD patients can be readily identified in vitro by their failure to reduce the yellow dye nitroblue tetrazolium to a blue compound (the ‘NBT test’, see Ch. 31).

The way in which ROIs actually kill microorganisms is controversial

ROIs can damage cell membranes (lipid peroxidation), DNA and proteins (including vital enzymes), but in some cases it may be the altered pH that accompanies the generation of ROIs that does the damage. Killing of some bacteria and fungi (e.g. E. coli, Candida) occurs only at an acid pH, while killing of others (e.g. staphylococci) occurs at an alkaline pH. There may also be a need for protease activity (e.g. cathepsins, elastase), with enzyme solubilization occurring as a result of the influx of H⁺ and K⁺ into the phagocytic vesicle.

Cytotoxic lipids prolong the activity of ROIs

As already mentioned, one of the targets of the toxic ROIs is lipid in cell membranes. ROIs are normally extremely short lived (fractions of a second), but their toxicity can be greatly prolonged by interaction with serum lipoproteins to form lipid peroxides. Lipid peroxides are stable for hours and can pass on the oxidative damage to cell membranes, both of the parasite (e.g. malaria-infected red cell) and of the host (e.g. vascular endothelium). The cytotoxic activity of normal human serum to some blood trypanosomes has been traced to the high-density lipoproteins, and in cotton rats to a macroglobulin.

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

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