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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 1
26 MOLECULAR AND CELLULAR
MEDIATORS OF THE
INFLAMMATORY RESPONSE
Vivienne M. Gough, M.B., CH.B., Constantinos Kyriakides, M.D., and Herbert B. Hechtman, M.D.
The Inflammatory Response MECHANISM OF NEUTROPHIL-MEDIATED INJURY
The inflammatory response to injury is complex, involving not Once it was established that neutrophils were invariably pres-
only the area of injury but also systemic tissues. The four cardi- ent during an acute inflammatory reaction, efforts were
nal signs of acute inflammation—calor (heat), rubor (redness), launched to explore their role in mediating this response. In the
dolor (pain), and tumor (swelling)—were first described by 1960s and 1970s it became clear that during inflammation, neu-
Celsus in the first century A.D. Subsequent descriptions added a trophils actively secreted products that were injurious3,4; these
fifth sign, functio laesa (loss of function). Although the descrip- products included reactive oxygen metabolites (ROMs) as well
tive features of acute inflammation have long been known, there as proteases such as elastase and collagenase.The importance of
is still no single satisfactory definition of this phenomenon. It is the neutrophil in mediating inflammation was further under-
commonly considered to be the reaction of living tissues to all scored by the finding that the increased permeability resulting
forms of injury and involves vascular, neurogenic, humoral, and from an injection of the eicosanoid leukotriene B4 (LTB4), the
cellular responses. complement fragment C5a, or the formylated bacterial peptide
The vascular events associated with inflammation typically formyl-methionyl-leucyl-phenylalanine (fMLP) into the skin
involve changes in blood flow and alterations in permeability. was prevented by previous neutrophil depletion.That neutrophil
In 1924, Lewis first described the so-called triple response, in depletion did not affect the release of serotonin, histamine, and
which drawing a blunt instrument across the skin induced a bradykinin from mast cells in response to these stimuli suggest-
flush, a flare, and a wheal.Thus, directly after injury, local arte- ed that these mast cell–derived agents did not play a major role
riolar dilatation, preceded by a fleeting interval of vasocon- in mediating permeability.5
striction, is the dominant vascular response. Precapillary In the early 1980s, it was shown that neutrophils stimulated
sphincters dilate, leading to increased flow in already active with phorbol myristate acetate damaged endothelial cells via a
capillaries as well as to the opening of inactive capillary beds. mechanism that could be interrupted by catalase, a hydrogen
At the same time, postcapillary venules dilate to cope with the peroxide scavenger.6 Subsequent investigations demonstrated
increased flow of blood. The rise in hydrostatic pressure is fol- that neutrophil-mediated endothelial damage was also caused
lowed by fluid exudation through the vascular bed, leading to by elastase.7 The mechanism of injury required further clarifi-
edema (wheal). As the edema increases, the wheal turns pale as cation, given that normal plasma was known to contain potent
a result of the increased interstitial pressure compressing the protease inhibitors and superoxide dismutase (SOD), which
capillaries. The changes observed in the skin after mechanical inactivate circulating proteases and ROMs, respectively.
trauma are typical of those seen in other organs after various Furthermore, a better understanding of how neutrophils were
injuries. selectively activated was needed, given that indiscriminate
In what follows, we focus on humoral and cellular responses release of proteases and ROMs would clearly be both inefficient
to injury, making use of several types of clinically important and dangerous.
events (e.g., ischemia-reperfusion and sepsis) to illustrate impor- The mechanism by which the neutrophil is currently thought
tant inflammatory pathways. to produce its injurious effects on target cells involves the for-
mation of a protected environment in which there is close con-
tact between cells. This concept, though not proved, has some
Neutrophil evidence to support it. Neutrophils lyse substrate protein only
That leukocytes are involved in inflammation was first when in close contact.8 Phorbol myristate acetate–induced lung
demonstrated in 1887 by Metchnikoff, who found that rose injury occurs only when neutrophils are in direct contact with
thorns inserted into the body of starfish resulted in the local the endothelium.9 When neutrophils are stimulated with fMLP,7
accumulation of leukocytes within hours.1 Metchnikoff also doc- C5a, or endotoxin,10 they must adhere to the endothelium to
umented the existence of different types of leukocytes and the cause vascular injury, which is governed by molecules expressed
ability of these cells to kill and digest bacteria, and he was the on the surfaces of both cell types.
first to suggest that products released by white blood cells could The normal immune action of the neutrophil is to phagocytose
damage vessel walls. In 1960, Marchesi and Florey, using elec- its target cell. This process results in the creation within the neu-
tron microscopy to follow the movement of neutrophils across trophil of a protected microenvironment known as a phagosome,
blood vessel walls in rat mesentery, found that neutrophils pen- which contains the targets for destruction [see Figure 1].When the
etrated vascular endothelium at or near cell junctions and that phagosome has been established, the neutrophil releases large
after their diapedesis, no sign of a passageway could be seen.2 quantities of ROMs and proteases into it. Any plasma protease
Furthermore, they demonstrated that the site at which such dia- inhibitors gaining access to this restricted space are themselves
pedesis occurred was the postcapillary venule. inhibited by the neutrophil secretory products hydrogen peroxide

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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 2
Circulating
energy phosphates; in contrast, such depolarization was limited
NEUTROPHIL during hindlimb reperfusion in neutropenic animals.16
Microbe
Lysosome
Neutrophils have also been implicated as mediators of the
remote lung injury observed to occur as a consequence of skele-
Proteases tal muscle ischemia. Investigators documented leukosequestra-
tion and increased lung permeability during reperfusion after
Phagolysosome
hindlimb tourniquet ischemia.17 Neutropenia, however, was
protective in this setting, maintaining normal microvascular per-
MPO
meability and lung histology.
O2 H2O2
Circulating
Antiproteases Adhesion Molecules
ROMs
To patrol the body effectively for infectious organisms and to
PSGL-1
CD18 Serum repair injuries in disparate regions of the body, neutrophils must
Antioxidants be able both to circulate as nonadherent particles in blood and
lymph and to adhere to and migrate through specific tissues sites
P/E ICAM where they are needed. Evidence from the past 20 years indi-
Selectin Endothelial Damage
cates that inflammatory sites emit signals that activate the adja-
cent endothelium and circulating phagocytes. Inflammatory
events also trigger release of circulating mediators such as
Chemoattractants cytokines, which produce more generalized activation of
Endothelium (Chemokines, C5a, LTB4, PAF) endothelial cells and leukocytes, thereby rendering one or both
cell types more adhesive.18 Circulating phagocytes then adhere
to endothelium; in response to a chemical chemoattractant gra-
Figure 1 Proteases and ROMs are stored in intracellular lyso-
somes. These fuse with microbe-containing phagosomes, forming
dient across the microvessel, they undergo diapedesis between
phagolysosmes that initiate destruction of the microbes. Anti- the junctions of endothelial cells, migrate through the suben-
proteases that gain entry to this area, such as serum α1-anti- dothelial matrix, and participate in the parenchymal inflamma-
trypsin, are inhibited by hydrogen peroxide and myeloperoxidase tory reaction. This phagocyte adherence, though essential to the
secreted by neutrophils. The same neutrophil products that cause immune response and the repair of injury, may induce patho-
microbial destruction may also damage surrounding endothelial logic consequences.
tissue. Tissue damage is somewhat reduced by natural protective For the first few hours of the inflammatory response, the neu-
mechanisms, such as ROM scavenging by SOD, catalase, and α1- trophil is the predominant leukocyte at inflammatory sites.19
antitrypsin. After 12 to 24 hours, mononuclear phagocytes predominate.20
When injury is extensive and the inflammatory response robust,
and myeloperoxidase, either of which is capable of neutralizing this second phase may be accompanied by immunomodulation
α1-antitrypsin, the major protease inhibitor.11,12 Furthermore, and increased susceptibility to infection.
myeloperoxidase activates other neutrophil proteases, such as The importance of leukocyte adhesion to the endothelium in
collagenase and gelatinase.4 Thus, ROMs and proteases act in maintaining host defenses against infection is illustrated by the
synergy to digest phagocytosed matter. example of patients with a congenital deficiency of CD11/CD18,
In cases of strong neutrophil activation, the cells may release a family of adhesion-promoting leukocyte surface proteins.21 This
their toxic products in concentrations that overwhelm the body’s condition, known as leukocyte adhesion deficiency I (LAD I),
regulatory defenses, causing substantial damage to surrounding results in a profound defect in phagocyte accumulation because
tissues through excess vascular permeability and parenchymal the leukocytes, though stimulated, cannot adhere and migrate to
injury. extravascular sites.7 Patients with LAD I have little or no pus at
bacterial infection sites and suffer recurrent life-threatening bac-
ROLE IN REPERFUSION INJURY
terial infections.21 On the other hand, unmoderated leukocyte
The importance of neutrophils in the generation of reperfu- adhesion to the endothelium can also have pathologic conse-
sion injury was first demonstrated in the context of myocardial quences, such as lung failure (i.e., the acute respiratory distress
ischemia.13 Histologically, the injury was characterized by an syndrome [ARDS]) or organ transplant rejection.22,23 Thus, a bet-
extensive early accumulation of extravasated neutrophils, thus ter understanding of the mechanism of adhesion may lead to bet-
resembling an acute inflammatory process. Depletion of neu- ter therapy for a number of disease states. Such therapy will have
trophils by means of an antiserum led to a significant reduction to balance the benefit of reducing tissue damage caused by leuko-
in the size of myocardial infarction after temporary occlusion of cyte adhesion against the possible increased risk of infection.
the left circumflex coronary artery.13 Restoration of blood flow Adhesion molecules on endothelial cells and neutrophils may
to ischemic skeletal muscle was also associated with significant be classified into three groups: selectins, integrins, and
leukocyte infiltration,14 whereas leukocyte depletion attenuated immunoglobulins [see Table 1].
the associated increase in microvascular permeability and vascu-
SELECTINS
lar resistance.15
Besides enhancing permeability, neutrophils were shown to The selectins are expressed only on cells within the vascula-
cause skeletal myocyte injury after ischemia, as evidenced by ture or the lymphatic system. To date, three selectins have been
membrane dysfunction. Such dysfunction was indicated by identified: L-selectin, P-selectin, and E-selectin, or endothelial
depolarization of resting skeletal muscle that persisted after leukocyte adhesion molecule–1 (ELAM-1).These molecules are
restoration of blood flow despite repletion of intracellular high- also collectively referred to as lectin cell adhesion molecules

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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 3
Table 1—Adhesion Molecules Involved in INTEGRINS
Adherence of Leukocytes to Endothelium The integrin family comprises a vast number of adhesion mol-
ecules that mediate intercellular recognition and cellular binding
Family Members to the extracellular matrix. These molecules are heterodimeric
transmembrane receptors composed of α and β subunits.The β2
L-selectin (LAM-1, Mel-14, Leu-8, LECCAM-1)
subfamily, collectively known as the leukocyte integrins or leuko-
Selectins E-selectin (ELAM-1)
cyte cell adhesion molecules (LeuCAMs), is expressed exclu-
P-selectin (CD62, GMP-140, PADGEM)
sively by leukocytes. This subfamily comprises three related het-
LFA-1 (CD11a/CD18) erodimers, each made up of a common β subunit (CD18) com-
Integrins Mac-1 (CD11b/CD18, Mo-1, CR3) plexed with a unique α subunit (CD11a, CD11b, or CD11c).
gp 150,95 (CD11c/CD18) CD11a/CD18, also known as lymphocyte function–associat-
ed antigen–1 (LFA-1), is expressed on all leukocytes and medi-
ICAM-1
Immunoglobulins
ates the attachment of unstimulated neutrophils to the vascular
ICAM-2
endothelium through a specific interaction with ICAM-1 and
VCAM-1
ICAM-2. CD11b/CD18, previously referred to as complement
receptor type 3 (CR3) because of its ability to bind the comple-
(LEC-CAMs). In general, the selectins are responsible for the ment fragment iC3b but now more generally known as Mac-1,
earliest interaction of circulating cells, setting the stage for inte- is constitutively expressed on the surface of neutrophils, mono-
grin-mediated firm adherence. All three help regulate the initial cytes, and macrophages. It binds to endothelial ICAM-1 at a site
leukocyte rolling on activated endothelium. Without selectins, distinct from the LFA-1 binding domain.34 In addition to the
leukocytes interact poorly with endothelium. Leukocyte adhe- Mac-1 expressed on the cell surface, an intracellular pool of pre-
sion deficiency II (LAD II), a genetic deficiency of glucosyl- formed Mac-1 is stored within specific granules in neutrophils.
transferase, prevents formation of sialyl Lewisx (sLex), the key Translocation of Mac-1 from intracellular pools to the neu-
selectin counterreceptor, thereby increasing the risk of infection. trophil surface via granule fusion with the plasma membrane is
L-selectin, the smallest member of the selectin family, is pres- stimulated by chemotaxins, including C5a, LTB4, IL-8, and
ent on the surface of neutrophils, monocytes, and lymphocytes platelet-activating factor (PAF).35 Unlike the adhesive interac-
and binds both constitutive and inducible carbohydrate ligands tions between LFA-1 and ICAM-1, which can be demonstrated
on the endothelial cell surface.The migration of neutrophils into in both stimulated and unstimulated cells, Mac-1–mediated
certain inflammatory sites is significantly attenuated by L- adherence to endothelial ICAM-1 requires chemotactic activa-
selectin blockade24; however, other molecules play a greater role tion.36 CD11c/CD18, also referred to as gp150,95, has been
in neutrophil adhesion than L-selectin does. Where L-selectin identified on the surfaces of neutrophils and monocytes and is
plays a dominant role in cell trafficking is in the lymphocyte: L- believed to play its primary role in monocyte adhesion.
selectin–deficient knockout mice have small, poorly formed
lymph nodes and defective T cell function.25 IMMUNOGLOBULINS
P-selectin, the largest member of the family, plays a key part Leukocyte integrins bind to endothelial surface receptors
in leukocyte rolling.26,27 It is constitutively associated with the α- belonging to the immunoglobulin supergene family. Members of
granules of platelets28 and the cytoplasmic Weibel-Palade bodies this superfamily, which includes major histocompatibility com-
of endothelial cells29 and is rapidly translocated to the endothe- plex (MHC) antigens I and II, T cell receptors, ICAM-1, and
lial cell surface after stimulation by histamine or thrombin.29 ICAM-2, share an immunoglobulin domain composed of 90 to
Expression of P-selectin may also be induced by exposure of the 100 amino acids arranged in two sheets of antiparallel β strands
vascular endothelium to free radicals (particularly superoxide),30 stabilized at the center with a disulfide bond. Although constitu-
C5a, the complement membrane attack complex (C5b-C9),31 tively expressed, copies of ICAM-1 are increased after stimula-
and tumor necrosis factor–α (TNF-α).32 The extracellular N- tion by TNF-α, IL-1, LPS, lymphotoxin, or interferon gamma
terminal domain of the molecule binds to its carbohydrate li- (IFN-γ). IL-1–induced upregulation can be observed 30 min-
gand, sLex. An additional P-selectin mechanism of neutrophil- utes after incubation, after which time it increases steadily,
endothelial interaction involves platelets. By adhering to reaching a peak after 24 hours.37 ICAM-1 is the counterreceptor
endothelial surfaces, platelets provide a rich surface of exposed for CD11a and CD11b.
P-selectin to which neutrophils can bind. Platelet-endothelial
binding may occur via fibrinogen attachment to endothelial STUDIES USING ANTIBODIES TO ADHESION MOLECULES
intercellular adhesion molecule–1 (ICAM-1) and to platelet The development of monoclonal antibodies (mAbs) that bind
GPIIb/IIIa. Platelet endothelial cell adhesion molecule–1 to the functional epitopes of adhesion molecules has yielded use-
(PECAM-1) can also mediate attachment. ful data on adhesion mechanisms. In addition, a number of stud-
E-selectin, the third member of the selectin family, is a glyco- ies have aroused clinical interest.
protein found exclusively on the surface of endothelial cells. In two trials, survival was significantly increased in rabbits and
Although not expressed on resting endothelium, E-selectin is baboons treated with a CD18 mAb after hemorrhagic shock.38,39
markedly upregulated in response to the cytokines interleukin-1 In a canine model of myocardial ischemia-reperfusion, treatment
(IL-1) and TNF-α as well as to lipopolysaccharide (LPS). E- with a CD11b mAb led to reduced infarct size and neutrophil
selectin plays an important role in slightly later leukocyte adhe- sequestration.40 In a murine model of liver cell injury induced by
sion to the microvascular endothelium.33 LPS and galactosamine, a CD11b mAb reduced liver cell injury
For all three selectins, P-selectin glycoprotein ligand–1 by more than 90% (as determined by assessment of alanine
(PSGL-1), which is rich in sLex moieties, is the principal coun- aminotransferase levels) but reduced neutrophil sequestration by
terreceptor; it is located on the surface of circulating leukocytes only 40%.41 In this setting, it is likely that neutrophils were
and participates in the earliest inflammatory responses. bound to a large extent by CD11a, which may explain the dis-

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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 4
crepancy between the degree of protection afforded by the anti- Table 2—General Classification of Cytokines
body and the reduction in neutrophil sequestration. The benefi- by Primary Activity
cial effects of CD11b antibodies may well be related more to the
inhibition of neutrophil signaling and function than to seques-
Proinflammatory Cytokines Anti-inflammatory Cytokines
tration per se.
In another study, treatment with a mAb to ICAM-1 led to a TNF IL-10
reduction in phorbol myristate acetate–stimulated neutrophil IL-1 IL-4
accumulation in rabbit lungs.42 This mAb also reduced lympho- Chemokines IL-13
cyte sequestration and acute graft rejection after renal transplan- Interferons TGF-β
tation in primates.23 Attempts to modify acute allograft rejection IL-2
in humans have met with little success. It must be kept in mind, CSFs
however, that our understanding of the effects of antibodies to IL-6
adhesion molecules on the immune system is still in its infancy.
This rapidly developing field promises to offer powerful tools
that may well prove applicable to the control of inflammatory discovered in a variety of different settings and consequently
and immunologic reactions. bear several different names associated with specific functions.
For example, TNF, initially named for its ability to cause tumor
necrosis, is also known as cachectin because of its association
Cytokines with cachexia. Current cytokine nomenclature follows a more
The term cytokine refers to a diverse group of polypeptides consistent naming system, in which the term interleukin (IL),
and glycoproteins that are important mediators of inflammation. referring to a substance that acts between leukocytes, is used in
They are produced by numerous cell types but primarily by conjunction with a number (e.g., IL-10); however, many of the
leukocytes. Cytokines are potent intercellular messengers that cytokines are still commonly referred to by their historical (and
exert most of their actions on immune and inflammatory cells. frequently misleading) names.
Taken separately, they have a wide range of effects; considered as The cytokines we address in the following discussion do not
a group, their principal functions are to coordinate the immune constitute an exhaustive list. Such a list would be beyond the
response to foreign antigens by acting as growth factors and cel- scope of this chapter; furthermore, given the rate at which new
lular activators and also to moderate this response via various cytokines are being found and classified, it would be out of date
feedback mechanisms. before it was published. Accordingly, we focus on the better-
Cytokines are not stored intracellularly: upon antigenic stim- known and more thoroughly studied cytokines.
ulus of the cell, they are synthesized rapidly by novel mRNA To facilitate understanding of cytokine function, it may be
transcription, then quickly released. For example, LPS from helpful to classify them on the basis of their primary actions.
bacterial cell walls causes macrophage activation and induces Various classification systems have been proposed; however,
TNF release. The mRNA encoded during this process tends to none have proved entirely satisfactory, because these agents,
be unstable, which ensures that cytokine synthesis after cell being pleiotropic by nature, often have conflicting actions. In our
activation will be brief and self-limiting. Cytokines exert their view, the most useful approach is to categorize the cytokines
effects through very high-affinity binding to various receptors according to whether they exert primarily proinflammatory or
on target cells. Thus, as a rule, very small amounts of cytokines primarily anti-inflammatory effects [see Table 2]. Admittedly, the
suffice to produce their biologic effects. External signals boundary between the two categories is frequently blurred.
increase the number of receptors on a cell, thereby enhancing
PROINFLAMMATORY CYTOKINES
the cellular response to a particular cytokine. For example,
antigenic stimulation of lymphocytes leads to increased The general distinction between proinflammatory and anti-
cytokine receptor expression. The cytokine itself can also stim- inflammatory cytokines is based on the observation that certain
ulate cytokine receptor upregulation via a positive feedback cytokines—principally TNF, IL-1, and IL-8—promote the
mechanism. inflammatory response by upregulating expression of genes
Cytokines usually act on cells locally in an autocrine and encoding phospholipase A2, cyclooxygenase-2 (COX-2), or
paracrine fashion, thereby ensuring that the inflammatory inducible nitric oxide (NO) synthase, thereby increasing pro-
response is evoked at the site of infection or injury. When they duction of the corresponding proinflammatory mediators (PAF,
are produced in very large quantities, they can enter the circula- eicosanoids, and NO, respectively). In addition, proinflamma-
tion and exert significant systemic effects. On one hand, a single tory cytokines mediate neutrophil action, acting as chemoat-
cytokine may act on more than one cell type, causing a number tractants, upregulating expression of leukocyte-endothelial
of different consequences (pleiotropism); on the other, several adhesion molecules, causing cell migration into the tissues, and
different cytokines may produce the same functional effects activating neutrophil degranulation—all of which lead to tissue
(redundancy). This relative nonspecificity of action limits the damage.
clinical usefulness of exogenously administered cytokines: the
likelihood is high that such agents will either have unwanted side Tumor Necrosis Factor and Interleukin-1
effects or be functionally useless as a result of compensation by Although TNF and IL-1 are structurally dissimilar, their func-
other cytokines.43 Another common feature of cytokines is that tions overlap considerably, and they are known to act synergisti-
they often act in a cascade, with one cytokine leading to the pro- cally. Originally discovered as a substance causing ischemic
duction of another. They may also act synergistically or may necrosis of tumors in rabbits treated with endotoxin,TNF is pro-
antagonize the actions of other cytokines. duced principally by monocytes and macrophages but also by
Discussion of cytokines is complicated by the often confusing various other immune and endothelial cells. The most potent
nomenclature. Several of the earliest identified cytokines were stimulus for its production is LPS (endotoxin) from gram-nega-

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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 5
bomodulin; and profound hypoglycemia results from tissue
overutilization of glucose and impaired liver function.43
Table 3 Systemic Actions of TNF
Like TNF, IL-1 is produced primarily by macrophages and to
a lesser extent by other leukocytes and endothelial cells. IL-1 is
Fever
a primary responder in the inflammatory cascade, and its actions
Acute-phase protein synthesis
are very similar to those of TNF. It mediates local inflammatory
Cachexia responses at low concentrations and exerts adverse systemic
Myocardial depression effects at higher concentrations. Unlike TNF, it cannot induce
Disseminated intravascular coagulation (DIC) apoptosis. IL-1 also minimizes the perception of posttraumatic
Hypoglycemia
pain by causing the release of β-endorphins from the pituitary
gland and increasing the number of central opioid receptors.
TNF and IL-1 act synergistically to produce the acute innate
immune response to invading microorganisms, manifested by
tive bacteria, but other stressors (e.g., infection, trauma, neutrophil activation and release of other cytokines that promote
ischemia, and toxins) can also induce its release. further immune activity.
In small concentrations, TNF acts in an autocrine and
paracrine fashion, causing increased expression of endothelial E- Chemokines
selectin and leukocyte integrins and promoting leukocyte adhe- The chemokines are a group of cytokines that regulate migra-
sion to the endothelium.TNF causes neutrophils to be attracted tion of leukocytes from blood to other tissues. Chemokines bind
first, followed by monocytes and lymphocytes, thereby helping to heparan sulfate proteoglycans on endothelium, thus present-
to regulate the inflammatory response. It also stimulates ing themselves to white blood cells that have become loosely
endothelial cells to produce a cytokine subset known as the attached to endothelial cells via the action of selectins and that
chemokines (e.g., IL-8), which promote leukocyte migration roll along the endothelium. When a white cell interacts with a
into the tissues, and induces mononuclear phagocytes to pro- chemokine, its shape changes rapidly, becoming flatter and more
duce IL-1. Finally, TNF can cause apoptosis in some cell types, motile. At the same time, surface integrins are exposed, allowing
an event that is of unknown physiologic significance.43 firmer attachment to the endothelium. Chemokines also induce
In large concentrations, TNF mediates a number of physio- rhythmic contraction of cellular actin filaments, causing cellular
logic effects that are well-recognized components of infection. It migration from the blood vessel at an interendothelial cell junc-
acts on the hypothalamus via prostaglandin synthesis to cause tion across a chemokine concentration gradient to the region of
fever [see Table 3]. It also induces hepatocyte production of some tissue injury [see Figure 2].
of the acute-phase proteins, the best known of which are C-reac- For the purposes of this discussion, chemokines may be clas-
tive protein, fibrinogen, serum amyloid A, and α1-antitrypsin. sified into two main subsets, CC and CXC, depending on
Prolonged TNF production also leads to cachexia, probably by whether their two terminal cysteine residues are adjacent (CC)
reducing appetite. Massive doses of TNF produce the classic or separated (CXC). The CXC group, which includes IL-8
symptoms of septic shock. Hypotension results from reduced (probably the best known of the chemokines), acts mainly on
vascular smooth muscle tone and myocardial depression; dis- neutrophils, whereas the CC chemokines act on monocytes,
seminated intravascular coagulation (DIC) results from eosinophils, and lymphocytes. Both subsets are produced by
endothelial expression of tissue factor and inhibition of throm- leukocytes, endothelial cells, and fibroblasts in response to
Leukocyte Endothelium
Ligand (e.g., sLeX) Leukocyte
Chemokine Displayed Integrin
on Endothelium
Loose
Binding Diapedesis
Leukocyte
Firm Adherence
Rolling
Endothelial Integrin Ligand Chemokines
Selectin (P or E) (ICAM-1/VCAM-1) in Tissues
Figure 2 Leukocytes bind loosely to the endothelium via low-affinity interaction between selectins and their li-
gands. Once loosely bound, they begin rolling slowly along the endothelium. Chemokines cause a conformational
change in the leukocyte, exposing the leukocyte integrins that bind to endothelial ICAM-1 and VCAM-1 and thus
inducing firm adherence. The leukocyte then undergoes diapedesis into tissues along the chemokine gradient.

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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 6
Naive Interferon Gamma
Antigen-
Presenting Helper T Cell
IFN-γ is produced by T cells and NK cells in response to anti-
Cell
gen, an event greatly enhanced by IL-12. It activates macro-
phages, inducing them to kill microbes by activating enzymes
that trigger synthesis of ROMs and NO. In addition, by increas-
ing IL-12 release from the macrophages it activates, IFN-γ
increases differentiation of T helper cells into the Th1 cell subset
that produces it while inhibiting proliferation of the Th2 subset,
which produces anti-inflammatory cytokines [see Figure 3].
IL-12 IL-4 Interleukin-6
It is probably most accurate to think of IL-6 as a mixed proin-
Activated flammatory/anti-inflammatory cytokine. IL-6 is produced by
Macrophage mononuclear phagocytes as well as by endothelial cells and
fibroblasts. It acts in a proinflammatory manner by providing a
potent stimulus for hepatocyte synthesis of acute-phase proteins.
Th1 Th2 It is also involved in promoting T cell differentiation and matu-
Cell Cell ration and immunoglobulin production and in enhancing NK
cell activity.44 However, IL-6 also has important anti-inflamma-
tory effects. It has been shown to inhibit production of TNF and
IL-1 by mononuclear cells and to reduce release of TNF sec-
Positive Positive ondary to endotoxin challenge.45 Furthermore, IL-6 leads to
Cytokine Production
Feedback Feedback reduced plasma concentrations of soluble TNF receptors and
IL-1 receptor antagonist (IL-1Ra), which are naturally occurring
anticytokines.46
Proinflammatory Anti-inflammatory
Cytokines Cytokines Interferon Alfa and Interferon Beta (Type I Interferons)
TNF IL-4, IL-5, IFN-α and IFN-β are two functionally similar cytokines that
IL-2 IL-10, IL-13 are often jointly referred to as the type I interferons; they are also
IFN-γ known as the antiviral interferons because they are produced by
Figure 3 The function of T helper cells is to secrete cytokines in antigen-stimulated T cells in response to viral infection.They act
response to antigenic stimulation; these cytokines then act to reg- in a paracrine fashion to inhibit viral replication, preventing
ulate an appropriate immune response by triggering proliferation infection of neighboring cells and putting cells into an antiviral
and stimulation of T cells, B cells, and other leukocytes. There are state. IFN-α and IFN-β also increase the efficiency of cytotoxic
two distinct subsets of T helper cells—Th1 and Th2—which are T cell–mediated cell lysis by upregulating expression of MHC
descended from the same precursors, namely, naive CD4+ T cells. class 1 molecules on infected cells. They are frequently used in
The development of Th1 and Th2 subsets occurs in response to the clinical setting: IFN-α seems to be effective therapy for viral
naïve T cell stimulation by different cytokines. In turn, each sub- hepatitis, and INF-β has been used, with some success, to treat
set secretes a distinct set of cytokines. IL-12 is the major stimu-
multiple sclerosis.
lant for Th1 subset development. This subset assists in phagocyte-
mediated responses, with IL-12 being produced by phagocytes in Interleukin-2
response to a variety of antigens (most of which are intracellu-
lar). Th1 cells secrete the proinflammatory cytokines, mainly Unlike the cytokines already mentioned, which exert most of
IFN-γ, IL-2, and TNF. IL-2 acts in an autocrine fashion, stimulat- their effects via the innate immune system, IL-2 mediates
ing further growth of this subset. Th2 cell growth is stimulated by acquired immunity—that is, the proliferation and differentiation
the anti-inflammatory cytokine IL-4. This subset produces the of lymphocytes after antigen recognition. Originally known as T
anti-inflammatory cytokines IL-4, Il-5, IL-13, and IL-10, with IL- cell growth factor, IL-2 acts in an autocrine manner on the T
4 acting as an autocrine growth factor through a positive feed- cells that produce it (i.e., T helper cells), upregulating IL-2
back mechanism. These cytokines also mediate the immune receptors and thereby causing preferential proliferation of T cells
response to helminths and allergens.
specific for the stimulating antigen.43 It also promotes growth
and differentiation of NK cells.
stimulation by either antigens or other cytokines (e.g., TNF IL-2 has an immunomodulatory function as well, in that it
and IL-1). Some are also produced constitutionally by lym- potentiates apoptosis of antigen-stimulated T cells. This action
phocytes to regulate the flow of these cells through the lym- appears to limit immune responses. It is noteworthy that IL-2
phatic system. knockout mice experience overproduction of lymphocytes with
hyperplasia of lymphoid organs and the development of autoim-
Interleukin-12 mune diseases (most strikingly, autoimmune hemolytic anemia
When activated by intracellular microbes (e.g., viruses, Liste- and inflammatory bowel disease). These events are probably
ria, and mycobacteria), mononuclear phagocytes and dendritic attributable to loss of the regulatory apoptosis function.
cells (professional antigen-presenting cells [APCs] located in
lymph nodes) produce IL-12, the essential action of which is to Hematopoietic Cytokines
mediate the early immune response. The most important single The hematopoietic cytokines—including the colony-stimulat-
action of IL-12 is to stimulate synthesis and release of IFN-γ by ing factors (CSFs), IL-7, and IL-3—are, strictly speaking, proin-
T cells and natural killer (NK) cells (see below). flammatory, in that they induce the growth and differentiation of

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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 7
bone marrow progenitor cells; however, this is a normal compo- knockout mice acquire multifocal inflammatory disease, partic-
nent of bone marrow hematopoiesis. CSFs are produced by ularly involving the heart and the lungs.51
bone marrow stromal cells and act to stimulate the formation of
THERAPEUTIC IMPLICATIONS
bone marrow cell colonies. There are several different varieties,
named according to the type of cell line they promote (e.g., gran- Interleukin-1 Receptor Antagonist
ulocytes [G-CSF], monocytes [M-CSF], or both [GM-CSF]).
A naturally occurring cytokine inhibitor is the endogenously
IL-7 is synthesized by fibroblasts and bone marrow cells and
secreted protein IL-1Ra, which competitively binds the IL-1
induces the differentiation of B and T cells. IL-3 is also known
receptor without signaling. IL-1Ra is secreted by mononuclear
as multilineage colony-stimulating factor (multi-CSF) because
phagocytes and has nearly the same affinity for the IL-1 recep-
its actions promote the differentiation of bone marrow precur-
tor as IL-1 itself does.52 IL-1Ra is present in high concentrations
sors into all known mature cell types.
in the serum of patients with a variety of diseases, often in much
ANTI-INFLAMMATORY CYTOKINES higher concentrations than IL-1.53 In patients with sepsis, juve-
nile rheumatoid arthritis, and inflammatory bowel disease, IL-
The anti-inflammatory cytokines exert their effects by inhibit-
1Ra levels have been found to correlate with disease severity;
ing the production of proinflammatory cytokines, countering
presumably, IL-1Ra acts as a natural negative feedback mecha-
their actions, or both. In so doing, they reduce gene expression
nism in inflammatory states. Unfortunately, the results of clini-
of agents such as TNF, IL-1, and the chemokines, thereby miti-
cal trials evaluating recombinant IL-1Ra in the setting of septic
gating or preventing their subsequent inflammatory effects.
shock have been largely disappointing.54
Interleukin-10 TNF Antagonists
IL-10 is important in the homeostatic control of both innate There are two cell surface receptors for TNF, p55 and p75.
and acquired immunity. It is produced by activated macro- These are normally shed after activation and are found in the cir-
phages and is a potent inhibitor of these cells, regulating them culation. In the presence of disease and in response to endotox-
via negative feedback; it is also produced by Th2 cells and B in challenge, they circulate in much higher concentrations.55 It is
cells. IL-10 antagonizes a broad range of immune functions: it thought that p55 and p75 act as carriers for TNF, thereby reduc-
inhibits IL-12 production by activated macrophages, thereby ing the amount of TNF available to act on cells. TNF itself
reducing IFN-γ release and, consequently, decreasing macro- induces the production of its soluble receptors. Elevated levels of
phage activation. It also inhibits Th1 cells, thereby reducing pro- TNF receptors have been recorded in cancer patients, and these
duction of proinflammatory cytokines (e.g., TNF, IFN-γ, and levels correlate well with tumor burden and the extent of metas-
IL-2). However, IL-10 also has proinflammatory effects, stimu- tasis.56 Elevated TNF receptor levels in blood or joint fluid have
lating B cell function and enhancing the development of cyto- also been recorded in patients with a variety of autoimmune and
toxic T cells.47 inflammatory diseases.
The importance of IL-10 in normal regulation of the immune In response to such observations, commercial anti-TNF drugs
response is demonstrated by studies of IL-10 knockout mice, began to be developed. The first such drug to be approved was
which experience fatal inflammatory bowel disease and show etanercerpt, in 1998.This agent, a recombinant TNF p75 recep-
markedly increased levels of TNF in response to endotoxin chal- tor, has proved to be effective in treating rheumatoid arthritis
lenge.48 Administration of recombinant IL-10 before lethal doses and spondyloarthropathies. Infliximab, approved in 1999, is a
of endotoxin reduces TNF release and prevents death.49 In genetically engineered anti-TNF mAb that is capable of binding
humans, it prevents fever, proinflammatory cytokine release, and to both cell-bound and soluble TNF. It causes lysis of TNF-pro-
clotting cascade activation during endotoxin challenge. ducing cells via complement- and antibody-dependant cytotox-
Interleukin-4 and Interleukin-13 icity.57 Infliximab is approved for use in patients with Crohn dis-
ease, in whom it can induce and maintain remissions, bring
IL-4 and IL-13 are structurally similar, and both are produced about endoscopically and histologically demonstrable mucosal
by Th2 cells as well as by mast cells and basophils.They are con- healing, reduce the number of perianal fistulae, and decrease
sidered anti-inflammatory cytokines by virtue of their ability to inflammation in ileoanal pouches.58 Infliximab also shows great
antagonize INF-γ and thereby reduce macrophage activation. promise in the treatment of rheumatoid arthritis, ankylosing
Because INF-γ is the major promoter of Th1 cells, these two spondylitis, and psoriatic arthritis.
agents indirectly suppress cytokine production by Th1 cells in
much the same way that IL-10 does. Modulation of Cytokine Activity in Sepsis, SIRS, and CARS
IL-4 is the principal growth factor for Th2 cells, which, in addi- Key terms and concepts associated with the systemic activa-
tion to their immune suppressor functions, mediate the immune tion of the body’s inflammatory mechanisms—systemic inflam-
response to helminths and are responsible for allergic reactions. It matory response syndrome (SIRS), sepsis, septic shock, and
is also the major stimulus for production of IgE by B cells. multiple organ dysfunction syndrome (MODS)—are defined
and discussed more fully elsewhere [see VI:10 Clinical and
Transforming Growth Factor–β
Laboratory Diagnosis of Infection,VI:11 Blood Cultures and Infection
Transforming growth factor–β (TGF-β) is produced by a in the Patient with the Septic Response, and VII:7 Multiple Organ
large variety of cells, including activated T cells, phagocytes, and Dysfunction Syndrome].59 The general understanding is that sys-
endothelial cells. It has potent anti-inflammatory effects, inhibit- temic inflammatory activity (i.e., SIRS), which may occur in
ing T cell differentiation and proliferation and macrophage acti- response to either infectious or noninfectious stimuli, is the fun-
vation. It also counteracts the effects of proinflammatory damental phenomenon; in this view, sepsis may be understood
cytokines, causing downregulation of endothelial intercellular as SIRS occurring specifically in response to microbial invasion.
adhesion molecules,50 downregulation of TNF endothelial This whole-body inflammatory response can lead ultimately to
receptor expression, and inhibition of chemokine action. TGF-β MODS, which is associated with mortalities higher than 50%.

8. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice
8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 8
The role of cytokines in the pathophysiology of SIRS and sep- Vascular Endothelium
sis was first appreciated when experimental studies demonstrat-
ed that antibodies to TNF reduced mortality in animals to which
endotoxin had been administered.60,61 Further research revealed Selectin-
that the proinflammatory cytokines TNF and IL-1 were both Mediated
able to evoke a clinical picture mimicking that of sepsis (i.e., Rolling
Clotting
hypotension and fever). In addition, TNF activated the coagula- Cascade
tion cascade.54 Accordingly, it was postulated that SIRS and sep- VIIIa
sis were attributable to an overwhelming proinflammatory
Neutrophil
immune response mediated by TNF and other cytokines. A great
deal of research on sepsis has now been carried out in animal
Activated
and clinical trials, mostly using TNF mAbs and soluble TNF Protein C
Va
receptors. The initial results were positive, in that TNF amelio-
ration led to increased survival in endotoxin-sensitized animals.
Subsequently, however, these findings were countered by the
Thrombin
observation that TNF had no significant effect in animal sepsis
models of cecal ligation and puncture (which would be more rel-
evant to clinical application in humans). Large-scale clinical tri- Monocyte Cytokine Production
als employing TNF mAbs and receptors or IL-1 ablation with
IL-1Ra have met with little success.62 IL-1, TNF, IL-6
The disappointing clinical results from proinflammatory
cytokine ablation have brought into question the thesis that sep- Figure 4 Activated protein C has both anti-inflammatory and
antithrombotic actions that make it a valuable antisepsis drug. It
sis is entirely understandable in terms of persistent and uncon-
reduces cytokine release by mononuclear phagocytes, reduces
trolled inflammation. Another view—which is not incompatible leukocyte adhesion to the vascular endothelium, and inhibits
with the basic concept of sepsis as a systemic inflammatory thrombin formation.
response—is that the body also mounts a countering anti-
inflammatory reaction to an insult, which then can lead to what
is referred to as the compensatory anti-inflammatory response by presumably combat CARS. In one, administration of G-CSF
syndrome (CARS).63 According to this hypothesis, when the to patients with community-acquired pneumonia resulted in
proinflammatory response to an insult predominates, SIRS and reduced rates of systemic complications and sepsis.62 INF-γ was
shock result, and when the anti-inflammatory response predom- employed in a small clinical study of patients with sepsis and
inates, CARS, immunosupression, and increased susceptibility later in a multicenter study of burn patients,66 the rationale being
to infection ensue. Homeostasis and return to health reflect a that monocyte function had been found to be depressed in
state in which the two responses are in balance. It is likely that in severely ill patients. In these settings, the goal of restoring mono-
cases of trauma or infection, the body’s first response is proin- cyte activity, as defined by HLA-DR expression, was achieved,
flammatory, and the resulting SIRS is responsible for early mor- though no clinical benefit was noted with regard to mortality or
tality. This initial response is followed by an anti-inflammatory incidence of infectious complications.
response, and the resulting CARS is responsible for late mortal- It is clear that the role of cytokines in sepsis is a complex one:
ity secondary to immunodepression.62 both hypoinflammation and hyperinflammation occur, and
Numerous studies have measured cytokine concentrations in both are major determinants of clinical outcome. To date, no
the peripheral vasculature in an effort to identify markers of sep- therapy has been found that reliably regulates cytokine activity
sis, predictors of mortality, and possible clinical interventions. and reduces sepsis-related mortality. It may be that future treat-
Perhaps surprisingly, levels of the proinflammatory cytokines ments will be chosen and given on a patient-to-patient basis,
(TNF, IL-1, IL-12, and IFN-γ) are not consistently correlated with markers of immune competence (e.g., HLA-DR, IL-6, and
with either disease severity or mortality in patients with septic TNF) indicating whether a proinflammatory approach or an
shock. This finding provides some evidence for the CARS anti-inflammatory approach is preferable in a specific instance.
hypothesis. It must be remembered, however, that systemic
blood sampling is not indicative of tissue cytokine dynamics. In
patients with pneumonia, for example, concentrations of TNF, Activated Protein C
IL-1, and IL-12 were found to be markedly higher in bronchial It has long been recognized that the clotting system plays a
lavage samples than in serum.64 The cytokine for which periph- major role in inflammation and in the response to invading organ-
eral blood levels seem to correlate best with disease severity is isms; DIC is one of the cardinal signs of sepsis. The proinflam-
IL-6; it is unclear why this is so, given that IL-6 certainly does matory cytokines TNF, IL-1, and IL-6 are known to trigger acti-
not produce shock. A 1994 study found that circulating levels of vation of the clotting cascade by stimulating the release of tissue
the anti-inflammatory cytokine IL-10, which are normally factor from monocytes and endothelium, leading to thrombin for-
unmeasurable, were detectable in more than 80% of patients mation and a fibrin clot.These cytokines also inhibit endogenous
with severe sepsis.65 A so-called refractory immunologic state fibrinolytic mechanisms by stimulating the release of plasmino-
developing after infection or trauma has also been described,47 gen-activator inhibitor–1 (PAI-1) from platelets and endothelium.
characterized by reduced proinflammatory cytokine production At the same time, thrombin stimulates many inflammatory path-
by mononuclear cells but increased synthesis of IL-1Ra (which ways and suppresses natural anticoagulant response by activating
correlates with CARS). thrombin-activatable fibrinolysis inhibitor (TAFI).67 This proco-
Several studies have examined therapies aimed at promoting agulant response leads to microvascular thrombosis and is impli-
cytokines that enhance the proinflammatory response and there- cated in the multiple organ failure associated with sepsis.

9. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice
8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 9
Protein C is an endogenous anticoagulant that, when activat- Table 4—Reactive Oxygen Metabolites
ed by thrombin bound to thrombomodulin, reduces the genera- and Other Free Radicals
tion of thrombin by inactivating clotting factors Va and VIIIa. It
also exerts an anti-inflammatory effect by inhibiting monocyte
Chemical
production of TNF, IL-1, and IL-6 and limiting leukocyte adhe- Radicals and Metabolites
Symbol
Comments
sion to endothelium by binding selectins68 [see Figure 4].
Activation of protein C is impaired during the inflammatory Oxygen-centered free
response because endothelial injury results in reduced levels of radicals
thrombomodulin. Decreased circulating levels of activated pro- Molecular oxygen
3
tein C are usually present in sepsis and are associated with Ground state (triplet) O2 Constitutes 20% of the
oxygen atmosphere
increased mortality.69 1
Singlet oxygen O2 Much more reactive
These observations have led to the investigation of activated than 3O2
protein C as a potential treatment for severe sepsis. A multicen- Superoxide radical O2•
–
Initial product of
ter, placebo-controlled phase III clinical trial comprising 1,690 respiratory burst
patients documented a 6.1% reduction in total mortality in of neutrophils
patients treated with drotrecogin alpha (the generic name for Hydroxyl radical OH• Highly unstable;
extremely toxic
recombinant human activated protein C).67 The only major side Alkoxy radical RO• Important toxic
effect noted with this agent was an increased risk of serious bleed- intermediate of free
ing: 3.5%, compared with 2% in the placebo group. Drotrecogin radical chain reactions
is thus the first therapy to have shown a real clinical benefit in Peroxy radical ROO• Important mediator of
DNA damage
sepsis trials, and high hopes are held for its future therapeutic use.
Lipoxy radical LOO• Important toxic
The United States Food and Drug Administration has recently metabolite of lipid
approved this agent for treament of sepsis, and further clinical tri- peroxidation
als are planned. It is marketed under the brand name Xigris.
Non–oxygen-centered
free radicals
Reactive Oxygen Metabolites Nitrogen centered
Nitric oxide NO• Neurotransmitter
Oxygen and its metabolites [see Table 4] are often very toxic,
Peroxynitrite OONO• Important endogenous
and this toxicity seems to have influenced the early evolution of toxin
Carbon centered
metabolic pathways to detoxify oxygen and make life possible in
Lipid radical L• Important toxic
an aerobic environment.70 Although aerobic organisms are capa- metabolite of lipid
ble of exploiting oxygen’s high reactivity for cellular energy, this peroxidation
reactivity also poses a continual assault on living tissues. Sulfur centered
Whenever the rate of endogenous oxidant generation exceeds Thiyl R-S• Metabolites of cysteine
the body’s endogenous antioxidant capacity, tissue injury ensues. and D-penicillamine
This occurrence is known as oxidative stress. Hydrogen centered
Free radicals and other ROMs have been implicated in a Hydrogen atom H• —
diverse array of human disease processes, including reperfusion Iron centered
injury, ischemic hepatitis and pancreatitis, inflammatory bowel Perferryl radical Fe3+-O2-Fe2+ Important final mediator
of tissue injury
disease, and ARDS. The common pathway for the tissue injury (along with OH•)
in these illnesses is the action of free radicals.
Non–free radical reactive
CHEMICAL STRUCTURE metabolites
Ozone O3 Major air pollutant
The high reactivity of free radicals is a consequence of their
Hydroperoxides
chemical structure. Free radicals are molecules that contain one
Hydrogen peroxide H2O2 Important endogenous
or more unpaired electrons in their outer orbits. As a result, they tissue toxin
possess higher energy levels than molecules with full outer orbits Lipid peroxide LOOH Intermediate lipid
and, accordingly, are less stable and more reactive. peroxidation
The most abundant radical in the biologic system is molecular Hypochlorous acid HOCl Important mediator of
oxygen itself (O2), which has two unpaired electrons in its outer neutrophil toxicity
orbit. In this ground (i.e., low-energy) state, the two electrons have Chloramines R′RNCl Important final mediator
of neutrophil toxicity
parallel spins, rendering the molecule relatively unreactive. When
the electrons have antiparallel spins, the radical—termed singlet
oxygen (1O2)—is much more reactive.Various oxygen-centered free
radicals and ROMs are believed to play important roles in tissue The hydroperoxides constitute an important class of toxic
–
injury.The superoxide anion (O2• ) and the hydroxyl radical (OH•) ROMs, with hydrogen peroxide (H2O2) playing a particularly
each have one unpaired electron in the outer orbit.The latter is par- important part. Much of the superoxide-initiated tissue damage
ticularly unstable: it reacts within 10-6 seconds of its formation and caused by neutrophils and macrophages can be attributed to
within a distance of 15 nm. Consequently, the hydroxyl radical is halogenated oxidants (e.g., hypochlorous acid [HOCl]) and
not only highly toxic but also very difficult to scavenge effectively. chloramines, which are formed by the secondary reaction of
Some ROMs, technically speaking, are not true radicals but halogenated radical species with nitrogen compounds. This pro-
are still quite toxic. For example, much of superoxide’s tissue duction of toxic ROMs as redox products substantially amplifies
toxicity is actually mediated by its oxidant reduction products. the toxicity of free radicals.

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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 10
Mitochondrion
ELECTRON TRANSPORT CHAIN
Cytochrome Cytochrome
Cytochrome Complex
Complex Complex
Ubiquinone Cyt.C
2 NADH
4 e- 4 e-
4 e-
4 e-
2 ATP 2 ATP 2 ATP
2 NAD+
Oxygen
Water
e-
Hydroxyl
e- H2O
Superoxide Radical
O2 Hydroperoxyl Hydrogen
Anion e-
Radical e- Peroxide
OH•
O2•
Catalase/
HO2• H2O2
Peroxidase
Superoxide
Dismutase
UNIVALENT LEAK
Figure 5 The mitochondrial cytochrome oxidase complex reduces molecular oxygen by the essentially simulta-
neous addition of four electrons, producing water and generating adenosine triphosphate (ATP) without the pro-
duction of free reactive oxygen intermediates. However, 1% to 5% of oxygen leaks from this pathway and under-
goes stepwise, univalent reduction. The one-electron reduction of molecular oxygen generates the superoxide free
radical (superoxide anion). This can subsequently be reduced to hydrogen peroxide (H2O2), most commonly in a
dismutation reaction catalyzed by SOD. Hydrogen peroxide can be directly reduced to water (by a catalase- or
peroxidase-catalyzed reduction), or it can be reduced to the hydroxyl radical (OH·) (usually in an iron-catalyzed
reaction). The highly reactive hydroxyl radical can subsequently abstract a hydrogen atom to reduce itself to
water. Normally, protective biochemical mechanisms detoxify the toxic intermediates generated by this so-called
univalent leak. When increased concentrations of oxygen generate excessive amounts of reactive oxygen metabo-
lites, however, protective mechanisms may be overwhelmed and tissue injury may result.
FORMATION
and peroxidases) can generate ROMs.71 The arachidonic acid cas-
All cells continuously generate ROMs as normal by-products cade, which produces eicosanoids [see Eicosanoids, below], also
of metabolism. During oxidative phosphorylation, mitochondrial generates peroxy compounds and hydroxyl radicals as by-products.
cytochromes couple the production of adenosine triphosphate In inflammatory cells, ROMs are the primary product of a
(ATP) to the tetravalent reduction of molecular oxygen to water highly specialized NADPH-dependent oxidant system. When
[see Figure 5]. In the course of this process, all four electrons are activated, they cause reduction of oxygen to the superoxide
effectively added to the oxygen molecule at once. No intermedi- anion.This process, known as the respiratory burst, may account
aries are formed, and thus no free radicals are generated. for more than 90% of the oxygen consumption of an activated
However, about 1% to 5% of the oxygen reduced to water with- neutrophil [see Figure 6].72 It is followed by formation of the
in the mitochondria leaks from this pathway and undergoes a major toxic product, HOCl, catalyzed by an enzyme (myeloper-
stepwise, noncatalyzed reduction—the so-called univalent leak— oxidase [MPO]) that is unique to phagocytes.
that produces a series of toxic intermediaries.These ROMs must
then be detoxified by endogenous antioxidants to prevent tissue MECHANISM OF TISSUE DAMAGE
injury. All biochemical cell components are potentially subject to
ROMs are also formed in many other circumstances. Certain attack by free radicals; however, lipids, proteins, and nucleic
cellular oxidases (e.g., cytochrome P-450, mixed-function oxidase, acids are particularly vulnerable. It should also be noted that the

11. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice
8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 11
toxic and mutagenic effects of ionizing radiation and those of 2. Protein denaturation. Both structural and enzymatic proteins
many toxic chemicals (e.g., carbon tetrachloride [CCl4]) are in are vulnerable to free radical–mediated denaturation. This
fact mediated by the formation of ROMs. ROMs exert their process can lead to loss of membrane fluidity and structural
inflammatory effects in five major ways. disintegration, which can cause the destruction of organelles
or even entire cells. Calcium adenosine triphosphatase
1. Lipid peroxidation. Because ROMs are so reactive, they gen- (ATPase) is particularly susceptible to oxidative damage;
erally react with the first molecule encountered, which is denaturation of this enzyme results in increased intracellular
often a cell membrane lipid. By removing a hydrogen atom calcium concentration with subsequent swelling and injury.
from the carbon chain of a polyunsaturated fatty acid, a ROM 3. Nucleic acid denaturation. Free radicals can attack nucleic
can form a fatty acid radical (LOO•), which is then able to acids directly, causing base hydroxylation, cross-linking, or
react with neighboring lipids, proteins, or nucleic acids, in a scission. Such attacks may be facilitated by the presence of
chain reaction.Thus, a single free radical can cause the break- DNA-bound iron acting as a catalyst. Depending on the
down of several polyunsaturated fatty acids, generating dam- extent of DNA damage, the effect may be mutagenic or
aged and reactive products along its path. The reaction prod- lethal. Ionizing radiation exerts most of its damaging effect by
ucts include aldehydes, hydrocarbon gases (e.g., pentane, this mechanism.
methane, and ethane) and various chemical residues; these 4. Cell matrix injury. Free radicals also attack extracellular pro-
agents cause cell edema, influence vascular permeability, give teins and glycoproteins (e.g., collagen and hyaluronic acid).
rise to inflammation and chemotaxis, and alter the activity of When these molecules lose their structural integrity, damage
cell enzymes, including phospholipase. This destructive to the connective tissue basement membrane and the extra-
sequence is terminated only when two of these radicals react cellular matrix may ensue, which can affect vascular perme-
together or when the radicals are scavenged by an electron ability and epithelial membrane integrity and can even result
acceptor. in petechiae.73 This process may be the cause of certain con-
HOCl
Cl-
H2O2
O2
H2O
-
•
O2 O2
Myeloperoxidase
e-
Plasma Membrane
NADPH Oxidase
Neutrophil
Cytoplasm
NADP NADPH
Lysosome
Hexose Monophosphate Shunt
Figure 6 Neutrophils and other phagocytes generate the superoxide radical via the membrane-integrated
enzyme NADPH oxidase. On stimulation, more than 90% of the oxygen used by the neutrophil is consumed in
this respiratory burst. Although the superoxide radical itself is toxic to many invading microorganisms, the sec-
ondary generation of HOCl from hydrogen peroxide and chloride ions, catalyzed by the neutrophil-generated
enzyme myeloperoxidase, substantially increases the bactericidal potential of the neutrophil.

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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 12
ATP XANTHINE DEHYDROGENASE
ADP Proteolysis Oxidation
Ca2+
ISCHEMIA
AMP
Adenosine
Inosine
Hypoxanthine Xanthine Oxidase Xanthine Oxidase
Xanthine Urate
SOD
O2 O2 • H2O2
REPERFUSION
Fe2+
NH3
OONO – NO2 Neutrophil RCINO3
OH • HOCl
Peroxynitrite Nitric Chloramines
Oxide
Endothelial Cells
Reactive Adhesion/
Oxidants Activation TISSUE
INJURY
Neutrophils
Figure 7 Xanthine oxidase may serve as the initial source of free radical generation in postischemic reperfusion
injury. With the onset of ischemia, ATP is degraded to its purine bases (e.g., hypoxanthine), the concentration of
which increases. Simultaneously, xanthine dehydrogenase is converted by ischemia to xanthine oxidase. Although
the concentrations of substrate and enzyme are high during ischemia, the absence of oxygen prevents purine oxi-
dation until reperfusion. At reperfusion, oxygen becomes available, suddenly and in excess, and the oxidation of
hypoxanthine (and xanthine) proceeds rapidly, generating a burst of superoxide radicals as a by-product, proba-
bly at or near the endothelial cell surface.
nective tissue diseases (e.g., osteoarthritis and rheumatoid can occur immediately after formation—a critical point, in view
arthritis), with inflammatory cells serving as the source of of the short radii of the antioxidants’ activity. For example, SOD
ROMs. is located almost exclusively in the mitochondrial matrix, where
5. Apoptosis. Apoptosis, a genetic mechanism that regulates cell it scavenges superoxide radicals formed via the univalent leak,
numbers, is characterized by cell shrinkage (pyknosis), in and vitamins A and E are found in cell membranes, where they
contrast with the cell swelling and rupture typical of necrosis. inhibit lipid peroxidation.
Pyknotic cells are cleared by macrophages with minimal
ROLE IN REPERFUSION INJURY
inflammation. Apoptosis is induced by ROMs, acting via
diverse mechanisms. Postischemic reperfusion disease provides the classic example
of free radical–mediated tissue injury. This phenomenon was
ANTIOXIDANT DEFENSE MECHANISMS
first demonstrated in 1981 in the feline small intestine, where
Various endogenous mechanisms have evolved to limit or administration of SOD near the end of the ischemic period but
counteract the tissue damage caused by ROMs. An example is before reperfusion largely prevented the microvascular and
the production and release of enzymatic and nonenzymatic epithelial injury seen after periods of partial ischemia.74 Since
antioxidants such as vitamin E, vitamin C, provitamin A, glu- that initial study, SOD has been shown to attenuate reperfusion
tathione, superoxide dismutase (SOD), and glutathione peroxi- injury in many organs, including skeletal muscle75 and the
dase, which act by scavenging ROMs. The enzymatic antioxi- heart.76 In similar models, injury was ameliorated by (1) scav-
dants require trace metals as cofactors for maximal effect: sele- enging of hydrogen peroxide by catalase,77 (2) scavenging of
nium for glutathione peroxidase; copper, zinc, or manganese for hydroxyl radicals by dimethyl sulfoxide or mannitol,78 or (3) pre-
SOD; and iron for catalase. vention of radical formation by chelating iron with transferrin or
These endogenous antioxidants are generally present at sites deferoxamine.79 Equivalent protection was provided by inhibit-
where free radicals are produced, which ensures that scavenging ing generation of superoxide by xanthine oxidase through

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8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 13
administration of allopurinol, pterin aldehyde, or tungsten.80 dizing agent. Experimental studies using NO• donors and antag-
These studies suggest that xanthine oxidase, which is known to onists have not yielded sufficiently consistent results to permit a
be present in high concentrations in the intestinal mucosa, is the clear definition of the role of NO• in mediating or moderating
initial trigger of free radical generation at reperfusion. inflammatory events.
In nonischemic tissue, the dehydrogenase form of xanthine
oxidoreductase predominates. Ischemia appears to mediate the
conversion of xanthine dehydrogenase to xanthine oxidase via a Complement
mechanism that remains to be defined. At the same time, catab- Complement was discovered in 1889 by Bordet while he was
olism of ATP during ischemia leads to an increased concentra- studying the bactericidal action of serum.86 Bordet demonstrat-
tion of the purine metabolites, xanthine and hypoxanthine. At ed that if fresh serum containing an antibacterial antibody was
reperfusion, oxygen is suddenly reintroduced in excess.The sub- added to the bacteria at physiologic temperature, the bacteria
sequent generation of superoxide by xanthine oxidase triggers a were lysed, but if the serum was heated to a temperature of 56º
free radical chain reaction and subsequent tissue injury [see C or higher, the bacteria were not lysed. Reasoning that the
Figure 7]. That either scavenging the hydroxyl radical or block- serum’s loss of lytic capacity could not have been caused by
ing its generation by an iron-catalyzed reaction has the same decay of antibody activity—because antibodies were known to
protective effect as more proximal blockage of the cascade sug- be heat stable, and even heated serum remained capable of
gests that the hydroxyl radical, or one of its metabolites, is the agglutinating the bacteria—Bordet concluded that serum must
final agent of injury.77 contain another heat-labile component that assisted or comple-
Reperfusion injury is also known to be mediated by neu- mented the lytic function of antibodies.
trophils, which would seem to suggest that the injury is entirely
COMPLEMENT CASCADE
accounted for by neutrophil superoxide generation.This thesis is
unlikely to be true, however: a more plausible thesis is that neu- The complement cascade consists of the classical and alter-
trophils act in concert with ROMs. Thus, in some models of native pathways, each of which comprises a series of plasma pro-
injury, neutrophil depletion fails to ameliorate injuries that are teins that are known to mediate several aspects of the inflamma-
blocked by SOD and catalase. It has been observed that free rad- tory response.This cascade has four principal biologic functions:
ical scavengers prevent neutrophil infiltration into postischemic
tissue,81 which has led to the hypothesis that ROMs act primar- 1. Osmotic cell lysis, effective in the killing of foreign organisms.
ily as neutrophil chemoattractants. 2. Binding of complement proteins to the surfaces of foreign
organisms or particles. These opsonins bind to specific coun-
Clinical Trials terreceptors on macrophages and leukocytes, facilitating
In view of the success achieved in moderating reperfusion phagocytosis.
injury with antioxidants in animal models, it is perhaps surpris- 3. Augmentation, potentiation, and promotion of the inflam-
ing that similar approaches have not met with comparable suc- matory response. Complement fragments bring about
cess in humans. A 1989 study using human recombinant SOD chemotaxis of inflammatory cells. In addition, C3a and C5a
to treat myocardial ischemia-reperfusion injury after angioplasty stimulate a neutrophil oxidative burst and mast cell degranu-
and renal ischemia failed to demonstrate any benefit.82 lation, which potentiate the inflammatory response.
However, a renal transplant study in which SOD was infused 4. Clearance of immune complexes from the circulation.
immediately before reperfusion reported reduced rates of acute
renal failure in graft recipients who underwent cold ischemia for The classical pathway and the alternative pathway have differ-
longer than 30 hours83; graft survival was longer as well. ent activating mechanisms; however, both result in the formation
In general, ROM scavengers are partially effective at best. of the membrane attack complex C5b-C9, which leads to the
Despite the enormous amount of data that has been collected, creation of pores in the cell membrane and consequently to
the precise relationship between ROM generation and tissue abnormal ion transport, impaired signaling, and potential lysis,
injury remains unclear. particularly of nonnucleated cells. Activation of either pathway
leads to production of the biologically active anaphylatoxins C3a
and C5a. These peptides are generated via cleavage of native C3
Nitric Oxide and C5 complement components by C3 convertase and C5 con-
NO• is produced in a variety of tissues (e.g., endothelial cells vertase, which are formed at intermediate steps in the process of
and platelets) by either a constitutive or an inducible form of complement activation. C3a and C5a exert a variety of physio-
NO• synthase. In the setting of inflammation, the inducible logic effects, including vasodilatation and increased vascular per-
enzyme is activated by cytokines such as TNF. NO• is a potent meability. The effects on neutrophils include chemotaxis, dia-
inhibitor of leukocyte and platelet adhesion, exerting its effects pedesis, degranulation, and free radical production as well as
via activation of soluble guanylcyclase and generation of cyclic translocation of intracellular complement receptors to the neu-
guanosine monophosphate (cGMP).84 With regard to platelets, trophil-plasma membrane. In addition, the complement frag-
NO• has been shown to inhibit the binding of fibrinogen to ments C3b and C4b serve as opsonins, thus facilitating phago-
GPIIb/IIIa and to limit the expression of P-selectin.85 Another cytosis of particles to which they have attached. Inactive C3b
important anti-inflammatory role of NO• is its ability to scav- (iC3b) is a counterreceptor for the neutrophil CD11b/CD18
enge superoxide at least as fast as SOD does. Finally, inhaled (Mac-1) receptor and thus has the ability to enhance neutrophil
NO• moderately reduces the pulmonary hypertension seen with binding and signaling in response to complement-coated mole-
ARDS; however, it does not increase cardiac output or reduce cules, thereby facilitating their phagocytosis.
mortality in these patients. The complement cascade has specific features that make it an
Moreover, NO• acts as a source of potent free radicals when important effector mechanism in the inflammatory response.
it interacts with superoxide to form peroxynitrite, a strong oxi- Many of its components, once activated, possess enzymatic