Two types of free radicals are produced by neutrophils,
macrophages, endothelial and other cells. The first type is
represented by reactive oxygen intermediates which are formed in
neutrophils by the activity of NADPH oxidase, the enzyme of the
respiratory burst. The second type includes reactive nitrogen
intermediates, the first member of them, nitric oxide being
produced by nitric oxide synthase.
Reactive oxygen intermediates (ROI)
Upon activation neutrophils and mononuclear phagocytes have
increased oxygen consumption, a process known as the
respiratory burst. During this, oxygen is univalently reduced by
NADPH oxidase to superoxide anion or its protonated form,
perhydroxyl radical, which then is catalytically converted by
action of superoxide dismutase to hydrogen peroxide:
NADPH oxidase is an electron transport chain found in
the wall of the endocytic vacuole of professional phagocytes and
in B and T lymphocytes. It is so called because NADPH is used as
an electron donor to reduce oxygen to superoxide and hydrogen
peroxide. NADPH oxidase is a complex enzyme composed at least of
five members. Two of them
are p21
and gp91
subunits of
a very unusual flavocytochrome b
in
the cytoplasmic membrane.
Two cytosolic proteins
(p47
, p67
), a qiunone, and
a Rac-related GTP-binding protein are thought to be the other
functional components of this electron transport system. (
phox meas ''
phagocyte
oxidase'', p -- protein, and gp
-- glycoprotein). The NADPH oxidase system is dissociated and thus
inactive in dormant neutrophils. While some components are
membrane bound, others are stored in the cytosol. Upon activation,
the cytosolic components translocate to the plasma membrane to
assemble the active oxidase. The absence of, or an abnormality in,
any one of these components result in chronic granulomatous
disease (CGD) characterized by the absence of respiratory burst
from neutrophils and monocytes of these patients. The children
suffer from repeated infections that respond poorly to
conventional therapy and almost invariably lead to early death.
Superoxide anion (
is both a one-electron reductant
and a one-electron oxidant that can pass through cell membrane via
anion channels. It apears that superoxide does not have direct
toxic effects on targets but, rather exerts its toxicity by
penetration to important sites where it subsequently is converted
to other ROI. Hydrogen peroxide
(
), hydroxyl radical (
) and
singlet oxygen are of the first importance of them.
Hydrogen peroxide interacts with myeloperoxidase (MPO),
contained in neutrophil azurophil granules to produce hypochlorous
acid, which is metabolized to hypochlorite (bleach) and chlorine:
Hydroxyl radical (
) is formed by several ways from
which decomposition of
catalyzed by 
is the most
important:
This reaction is supposed to be involved, for instace, in
asbestosis because asbestos contains high concentrations of iron.
The toxicity of 
is believed to
result from the ability of 
to serve as a powerful one-electron oxidant capable of abstracting
electrons from a large variety of compounds with the formation of
a new radical, which can oxidize other substances:
Hydroxyl radical and hypochlorite are the most powerfull
substances involved in microbicidal and cytotoxic reactions. HOCl
is 100 to 1000 times more effective
than 
. Furthermore,
HOCl-induced cell death occurs very rapidly in comparison to that
mediated by 
.
Singlet oxygen (
)
is an oxygen form whose electrons
are excited at a higher energy level compared to the normal
(ground) triplet oxygen. When returning to the ground state they
emit light (chemiluminiscence) which may have antimicrobial and
cytotoxic effects.
These oxidants also promote the margination of neutrophils by triggering the expression of adhesion molecules on endothelial cells.
ROI are involved in a variety of pathological conditions. For example pulmonary diseases in which oxygen radicals are thought to be involved include ARDS, hyperoxia, asbestosis, silicosis, paraquat toxicity, bleomycin toxicity, cigarette smoking, ionizing radiation and others.
ROI are highly toxic also for producing cells. Therefore
neutrophils have to contain large reserves of endogenous
antioxidants such as glutathione and ascorbate. Their ability to
maintain these antioxidants in the reduced state during
phagocytosis may prevent death from oxidative suicide.
Reactive nitrogen intermediates (RNI)
They are sometimes also called reactive oxynitrogen
intermediates (RONI). The pathway by which they are originated is
an oxidative process in which short-lived
nitric oxide (
)
is derived from the guanidino nitrogen in the conversion of
L-arginine to L-citrulline. This reaction
is catalysed by 
synthase and, like the respiratory burst, it involves oxygen
uptake.
Three distinct isoform of nitric oxide synthase (NOS) representing three distinct gene products have been isolated and purified. The three isoforms vary considerably in subcellular location, structure, kinetics, regulation, and hence functional roles (Table 1.4).
Table 1.4: Isoforms of human NO
synthase and their
characteristics
Two of the enzymes are constantly present and termed
constitutive NOS (cNOS). The endothelial cNOS is mostly
membrane bound and formed only in endothelial cells. The
neuronal cNOS was identified in the cytosol of central and
peripheral neurons. 
derived from the cNOS isoform act as
a physiologic regulator by relaxing vascular smooth muscle or by
functioning as a neurotransmitter. These isoforms produce small
amounts of 
for short periods in a calcium/calmodulin dependent
manner upon stimulation. Endothelial cNOS with the endothelial
cell acting as a signal transducer,
releases 
continuously in
varying amounts to regulate blood vessel tone and thus also the
blood flow and pressure. Large amounts of 
produced in
a prolonged time may cause vasodilalation and hypotension, whereas
insufficient 
formation may be involved in hypertension. It
seems that 
plays a fundamental role in the regulation of the
cardiovascular system. The organic nitrates used as vasodilatation
drugs for many years spontaneously release or are biotransformed
to the active form which is 
Within the CNS, 
is released in
response to increases in intracellular 
that follow
stimulation of glutamate receptors and may be classified as
a mediator of slow synaptic transmission. A second function for
within the CNS may relate to the toxic effects because its
increased release may lead to epileptic seizures and brain damage.
The third isoform of NOS is not present in resting cells but
instead the cells must be induced to express the enzyme, thus the
name
inducible NOS (iNOS). Stimuli typically include
cytokines and/or lipopolysaccharide (LPS), and once expressed the
enzyme generates large amounts of NO. A number of cytokines is
involved in the production of iNOS.
Among them IFN-
, IL-1, IL-6,
THF-
, GM-CSF (granulocyte-macrophage colony stimulatory factor)
and PAF (platelet activating factor) exert the stimulatory effect
whereas the suppression has been observed in the case of IL-4,
IL-8, IL-10, TGF-
(transforming growth factor), PDGF
(platelet-derived growth factor) and MDF (macrophage deactivating
factor).
may react with superoxide to form highly toxic
peroxynitrite anion:
which may be transformed in an acid milieu to peroxynitrite acid and then to hydroxyl radical:
Independent pathways are involved in the sythesis of ROI and
RNI. Dormant neutrophils produced 
continuously but activation
arrest this pathway in favor of the oxidative burst. Thus,
although the ROI and RNI pathways are independent, they may
compete for common substrates such as NADPH
and 
and exert other
modulating effects on each other. The steady-state production of
these species may dictate the anti/proinflammatory balance.
Microbial killing appears to ROI dependent in normal neutrophils
but RNI may play a role in cells with deficiences in the NADPH
oxidase/MPO pathways. Nitric oxide may also contribute to the
microbicidal activity of neutrophils by reacting with ROI to form
secondary cytotoxic species such as peroxynitrite.
The main role of neutrophil-derived 
may be to facilitate
the migration of neutrophils from blood vessels to surrounding
tissue by causing vasodilatation. 
facilitates relaxation of
vascular smooth muscle, and ROI initiate vasoconstriction through
the production of superoxide, which removes NO. In addition 
inhibits neutrophil adhesion to vascular endothelium and this may
prevent inflammatory and ischemia-reperfusion injuries.
The basis of the functional activity of 
is its dual
actions on some enzymes of target cells. The small amount of 
released by cNOS isoforms is adequate to activate the known
NO-sensitive enzymes (guanylate cyclase and
ADP-ribosyl-transferase) and participate in NO signaling pathways.
The larger amounts of 
generated by iNOS may also activate the
NO-sensitive enzymes, but in many cell types the high output of
also exceed the necessary concentration threshold to inhibit
the action of certain iron-containing enzymes, namely aconitase,
NADPH-ubiquinone oxidoreductase, succinate-ubiquinone
oxidoreductase, ribonucleotide reductase, NADPH oxidase and
glyceraldehyd-3-phosphate dehydrogenase.
Activation of soluble guanylate cyclase by 
leads to the
synthesis of cGMP, which leads to relaxation of vascular smooth
muscle cells, inhibition of platelet adherence, aggregation,
inhibition of neutrophil chemotaxis, and signal transduction in
the central and peripheral nervous system.
causes autoribosylation of glyceraldehyde-3-phosphate
dehydrogenase, which inactivates this glycolytic enzyme. 
also
inhibits three mitochodrial enzymes: aconitase of the Krebs cycle
and NADPH ubiquinone oxidoreductase and succinate-ubiquinone
oxidoreductase of the electron transport chain.
Induced 
synthesis was reported in inflammatory responses
initiated by microbial products or autoimmune reactions and also
in the systemic inflammatory response,
also referred to as sepsis.
likely participates in the inflammatory reaction and
subsequent joint destruction in some types of arthritis. For
instace synovial fluid from patients with osteoarthritis exhibits
elevated nitrate concentrations (nitrate are end products of the
L-arginine-NO synthase pathway). There is also evidence for
chronic expression of iNOS in the smooth muscle in atherosclerotic
aortic aneurysms, a disease in which there is progressive
dilatation and destruction of the aortic wall leading often to
fatal rupture.