Phagocytes Employ An Array Of Killing Mechanisms
Killing by reactive oxygen intermediates
Trouble starts for the invader from the moment phagocytosis is initiated. There is a dramatic increase in activity of the hexose monophosphate shunt, generating reduced nicotinamide adenine dinucleotide phosphate (NADPH). Electrons pass from the NADPH to a flavine adenine dinucleotide (FAD) containing membrane flavoprotein and thence to a unique plasma membrane cytochrome (cyt b558). This has the very low midpoint redox potential of −245 mV that allows it to reduce molecular oxygen directly to superoxide anion (Figure 1.29a). Thus the key reaction catalyzed by this NADPH oxidase, which initiates the formation of reactive oxygen intermediates (ROI), is:
NADPH + O2 ® NADP+O2
The superoxide anion undergoes conversion to hydrogen peroxide under the influence of superoxide dismutase, and subsequently to hydroxyl radicals (·OH). Each of these products has remarkable chemical reactivity with a wide range of molecular targets, making them formidable microbicidal agents; ·OH in particular is one of the most reactive free radicals known. Furthermore, the combination of peroxide, myeloperoxidase, and halide ions constitutes a potent halogenating system capable of killing both bacteria and viruses (Figure 1.29a). Although H2O2 and the halogenated compounds are not as active as the free radicals, they are more stable and therefore diffuse further, making them toxic to microorganisms in the extracellular vicinity.
Killing by reactive nitrogen intermediates
Nitric oxide surfaced prominently as a physiologic mediator when it was shown to be identical with endothelium‐derived relaxing factor. This has proved to be just one of its many roles (including the mediation of penile erection, would you believe it!), but of major interest in the present context is its formation by an inducible NO· synthase (iNOS) within most cells, but particularly macrophages and human neutrophils, thereby generating a powerful antimicrobial system (Figure 1.29b). Whereas the NADPH oxidase is dedicated to the killing of extracellular organisms taken up by phagocytosis and cornered within the phagocytic vacuole, the NO· mechanism can operate against microbes that invade the cytosol; so, it is not surprising that the majority of nonphagocytic cells that may be infected by viruses and other parasites are endowed with an iNOS capability. The mechanism of action may be through degradation of the Fe–S prosthetic groups of certain electron transport enzymes, depletion of iron, and production of toxic · ONOO radicals. The N‐ramp gene, linked with resistance to microbes such as bacille Calmette–Guérin (BCG), Salmonella, and Leishmania that can live within an intracellular habitat, is now known to express a protein forming a transmembrane channel that may be involved in transporting NO· across lysosome membranes.
Killing by preformed antimicrobials
These molecules, contained within the neutrophil granules, contact the ingested microorganism when fusion with the phagosome occurs (Figure 1.29c). The dismutation of superoxide consumes hydrogen ions and raises the pH of the vacuole gently, so allowing the family of cationic proteins and peptides to function optimally. The latter, known as defensins, are approximately 3.5–4 kDa and invariably rich in arginine, and reach incredibly high concentrations within the phagosome, of the order of 20–100 mg/mL. Like the bacterial colicins described above, they have an amphipathic structure that allows them to insert into microbial membranes to form destabilizing voltage‐regulated ion channels (who copied whom?). These antibiotic peptides, at concentrations of 10–100 µg/mL, act as disinfectants against a wide spectrum of Gram‐positive and Gram‐negative bacteria, many fungi, and a number of enveloped viruses. Many exhibit remarkable selectivity for prokaryotic and eukaryotic microbes relative to host cells, partly dependent upon differential membrane lipid com- position. One must be impressed by the ability of this surprisingly simple tool to discriminate large classes of nonself cells (i.e., microbes) from self.
As if this was not enough, further damage is inflicted on the bacterial membranes both by neutral protease (cathepsin G) action and by direct transfer to the microbial surface of BPI, which increases bacterial permeability. Low pH, lysozyme, and lactoferrin constitute bactericidal or bacteriostatic factors that are oxygen independent and can function under anerobic circumstances. Interestingly, lysozyme and lactoferrin are synergistic in their action.
Finally, the killed organisms are digested by hydrolytic enzymes and the degradation products released to the exterior (Figure 1.26 h).
Neutrophils and macrophages can also deploy extracellular traps for microbes through releasing DNA
Recent discoveries have also revealed quite a surprising strategy that neutrophils (as well as their close granulocyte relatives) engage in for the purpose of immobilizing and killing extracellular bacteria and yeast: the formation of NETs (neutrophil extracellular traps). It appears that activated neutrophils can activate a self‐destruction pathway, the details of which are only emerging, that results in the release of the intracellular contents of the activated neutrophil into the extracellular space to act as a spider’s web‐like structure that can enmesh microbes and kill them in situ (Figure 1.30). The NETs themselves appear to be largely composed of neutrophil DNA with associated histones, along with high concentrations of neutrophil granule proteases such as cathepsin G, elastase, and proteinase‐3. The NET is thought to act as a depot for the latter proteases, helping to restrain their off‐target activities and also increase their local concentration. Interestingly, histone proteins have also been reported to have potent antimicrobial properties, although how this is achieved is unclear. Macrophages have also been reported to be able to deploy NET‐like structures under certain circumstances. Does the immune system have no end to the strategies it will engage in to protect us from harm?
By now, the reader may be excused a little smugness as she or he shelters behind the impressive antimicrobial potential of the phagocytic cells. But there are snags to consider; our formidable array of weaponry is useless unless the phagocyte can: (i) “home onto” the microorganism; (ii) adhere to it; and (iii) respond by the membrane activation that initiates engulfment. Some bacteria do produce chemical substances, such as the peptide formyl. Met. Leu. Phe, which directionally attract leukocytes, a process known as chemotaxis; many organisms do adhere to the phagocyte surface and many do spontaneously provide the appropriate membrane initiation signal. However, our teeming microbial adversaries are continually mutating to produce new species that may outwit the defenses by doing none of these. What then? The body has solved these problems with the effortless ease that comes with a few million years of evolution by developing the complement system.