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.
