Inflammatory mediators and intracellular

signalling
A6
Richard Korbut and Tomasz J. Guzik

is mainly produced by phagocytic activity of the

Introduction mesodermic cells, and that it includes “the chemical
action of the blood plasma and tissue fluids…”, thus
Inflammation is a protective response of the introducing the concept of the mediators of inflam-
macroorganism to injury caused by trauma, noxious mation [2]. Further numerous studies since then
chemicals or microbiological toxins.This response is have identified the roles of individual mediators in
intended to inactivate or destroy invading organ- inflammation, and we are beginning to understand
isms, remove irritants, and set the stage for tissue the genetic molecular aspects of the genesis of
repair. The inflammatory response consists of inflammatory process.
immunological and non-immunological reactions.
The latter are triggered by the release from injured
tissues and migrating cells of lipid-derived autacoids, Eicosanoids
such as EICOSANOIDS or “platelet-activating factor”
(PAF), large peptides, such as interleukin-1, small Arachidonic acid (AA) metabolites are formed rap-
peptides, such as bradykinin, and amines, such as idly from lipids of the cellular membrane, following
HISTAMINE or 5-HYDROXYTRYPTAMINE. These constitute activation of cells by numerous chemical and physi-
the chemical network of the inflammatory response cal stimuli (Fig. 1). They exert their effects locally
and result in clinical and pathological manifesta- (autacoids), affecting virtually every step of inflam-
tions of inflammation (Tab. 1). The concept of the mation [3]. EICOSANOIDS encompass cyclic
inflammatory response has been introduced over prostanoid structure, i.e., PROSTAGLANDINS (PGs),
2000 years ago with the description by Cornelius Cel- prostacyclin (PGI2), thromboxane A2 (TXA2) and
sus as “rubor et tumor cum calore et dolore”.Centuries straight chain leukotriene structures (LTs), i.e.,
later, in 19th century this definition was extended by chemotactic LTB4 and pro-inflammatory pepti-
Rudolph Virchow to incude the loss of function dolipids (LTC4, LTD4, LTE4) (Fig. 2). Recently, a new
(“functio lesa”). It was Virchow and his pupils, like J. group of molecules was added to the family of
Cohnheim,who explained the scientific basis for Cel- eicosanoids,namely LIPOXINS (LXA4 and LXB4),which
sus’description of inflammation.They found that red- are products of platelet 12-lipooxygenase metabo-
ness and heat reflected an increased blood flow, lism of neutrophil LTA4 (transcellular biosynthesis).
swelling is related to the exudation of fluid and accu- EICOSANOIDS are synthesized by cyclooxygenation
mulation of cells, while pain follows [1]. The first (prostanoids) or lipooxygenation (LTs) of a 20-car-
understanding of the mechanism of inflammation bon ω-6 polyunsaturated fatty acid (PUFA)- 5,8,11,14-
was introduced by Elie Metchnikoff, who concluded eicosatetraenoic acid (AA) (Fig.1). AA is an impor-
in his book Comparative Pathology of Inflammation tant structural constituent of cellular phospholipids,
published in 1893, that “…inflammation is a local and first must be liberated by acylhydrolases - direct-
reaction, often beneficial, of living tissue against an ly by phospholipase A2 (PLA2) or indirectly by PLC
irritant substance” [2]. This definition stands until before it becomes the substrate for the synthesis of
today.For the first time,he observed that this reaction eicosanoids.
82 Inflammatory mediators and intracellular signalling

oxy to a 15-hydroxy group, in this way yielding PGH2.

TABLE 1. SYMPTOMS OF INFLAMMATION INDUCED BY Eventually, the end-product of PGHS (the complex
INFLAMMATORY MEDIATORS which contains either constitutive COX-1, inducible
COX-2 or recently discovered COX-3) is an unstable
Symptom Mediators cyclic prostaglandin endoperoxide (PGH2), which in
various types of cells is converted by corresponding
Vascular permeability Vasoactive amines isomerases or synthases to stable prostanoids: PGD2,
Bradykinin PGE2, PGF2α, and unstable prostanoids, i.e., PGI2 or
Leukotrienes C4, D4, E4 TXA2. Special biological significance has been
PAF ascribed to PGI2 synthase in vascular endothelial
Complement (C3a and C5a) cells and TXA2 synthase in blood platelets. The tran-
Substance P scellular metabolism providing PGH2 from activated
Nitric oxide platelets to endothelial cells is the main source of
vascular PGI2 [4]. The biological activity of stable
Vasodilation Nitric oxide prostanoids is terminated by catabolic enzymes,
PGI2, PGE1, PGE2, PGD2 such as prostaglandin 15-hydroxy dehydrogenase
Hydrogen peroxide (15-PGDH),D13-reductase or α and ω oxidases which
are present in high concentration in the lungs.These
Vasoconstriction Thromboxane A2 enzymes also break down inactive TXB2 and 6-keto-
Leukotrienes C4, D4, E4 PGF1α.
Superoxide The role of individual CYCLOOXYGENASE enzymes in
the development of inflammation remains unclear.
Chemotaxis and Chemokines The discovery of the inducible form, COX-2, led to the
leukocyte adhesion LTB4, HETE, lipoxins hypothesis that COX-1 is a constitutive enzyme respon-
Complement (C5a) sible for physiological activities of PROSTAGLANDINS
Bacterial antigens while COX-2,which is expressed during inflammation,
produces “bad” PROSTAGLANDINS that generate pain and
Pain Bradykinin fever.This hypothesis quickly turned out to be simplis-
Prostaglandins tic and both enzymes show their activities under phys-
iological and pathological conditions [5]. Moreover
Fever IL-1, TNF, IL-6 COX-2 inhibitory drugs possess fewer analgesic prop-
Prostaglandins erties than non-selective inhibitors. The picture
became even more complicated in 2002, with the dis-
Tissue and endothelial Reactive oxygen species covery of COX-3. This isoenzyme is not a separate
damage Nitric oxide genetic isoform (like COX-2), but a splice variant of
Lyzosomal enzymes COX-1. In fact, COX-1 mRNA gives rise to four different
isoforms including classical COX-1, COX-3 (splice vari-
ant including intron 1) and two partially truncated,
inactive PCOX-1a and 1b. COX-3, due to the presence
Prostanoids of intron 1, which changes its conformational struc-
ture, shows significantly diminished activity (25%)
Prostanoids are produced by the CYCLOOXYGENASE [6].It is expressed mainly in the human brain and the
pathway. Prostaglandin H synthase (PGHS) is a heart. It has been suggested that COX-3 is an isoform
dimeric complex which contains CYCLOOXYGENASE particularly involved in the mechanisms of pain and
(COX) and peroxidase (Px). COX cyclizes AA to an fever during inflammation. Some suggestions exist
unstable cyclic 15-hydroperoxy prostaglandin endo- that this isoform is inhibited by paracetamol, which
peroxide (PGG2) while Px converts the 15-hydroper- could explain its analgesic actions.
 Eicosanoids 83

FIGURE 1
Mediators derived from phospholipids and their actions, the sites of action of anti-inflammatory drugs.

Biosynthesis of prostanoids is initiated by trans- the major prostanoid released by endothelium is

ductional mechanisms in an immediate response to PGI2, while platelets produce TXA2.
activation of various cell membrane receptors or to Prostanoids regulate vascular tone and perme-
various physical and chemical stimuli.These lead to ability in the development of inflammation. They
an increase in the cytoplasmic levels of calcium ions also (TX) induce platelet aggregation and thrombus
Ca2+i and in this way they activate acyl hydrolases, formation. Prostaglandins (in particular PGE2) are
which thereby release free AA for metabolism by also involved in the pathogenesis of pain and fever
PGHS. Alternatively, these enzymes can be induced accompanying inflammation.
by delayed transcriptional mechanisms which are Most actions of prostanoids appear to be brought
usually activated by CYTOKINES or bacterial toxins.The about by activation of the cell surface receptors that
spectrum of prostanoids produced by individual tis- are coupled by G proteins to either adenylate
sues depends on the local expression of individual cyclase (changes in intracellular c-AMP levels) or
enzymes. For example, vascular endothelium pos- PLC (changes in triphosphoinositol – IP3 and diacyl-
sesses prostacyclin synthase and COX-2, but lacks TX glycerol – DAG levels) [7].The diversity of the effects
synthase, present in turn in the platelets.Accordingly, of prostanoids is explained by the existence of a
84 Inflammatory mediators and intracellular signalling

(HPETEs). LOXs differ in their specificity for placing

the hydroperoxy group,and tissues differ in LOXs that
they contain. Platelets have only 12-LOX and synthe-
size 12-HPETE, whereas LEUKOCYTES contain both 5-
LOX and 12-LOX producing both 5-HPETE and 12-
HPETE. HPETEs are unstable intermediates, analo-
gous to PGG2 or PGH2, and are further transformed
by peroxidases or nonenzymatically to their corre-
sponding hydroxy fatty acids (HETEs).12-HPETE can
also undergo catalyzed molecular rearrangement to
epoxy-hydroxyeicosatrienoic acids called hepox-
illins. 15-HPETE may also be converted by lipooxy-
genation of LTA4 to trihydroxylated derivatives called
LIPOXINS (Fig. 1).

FIGURE 2 Leukotrienes
Typical eicosanoid structures
In activated LEUKOCYTES an increase in Ca2+i binds 5-
LOX to five-lipoxygenase-activating-protein (FLAP),
number of distinct receptors [8].The receptors have and this complex converts AA to 5-HPETE, which in
been divided into five main types, designated DP turn is the substrate for LTA4 synthase. In the course
(PGD), FP (PGF), IP (PGI2),TP (TXA2), and EP (PGE). of transcellular metabolism between LEUKOCYTES and
The EP receptors are subdivided further into EP1 blood cells or endothelial cells, unstable LTA4 is con-
(smooth muscle contraction), EP2 (smooth muscle verted by corresponding enzymes to stable chemo-
relaxation), EP3 and EP4, on the basis of physiologi- tactic LTB4 or to cytotoxic cysteinyl-containing LTs –
cal and molecular cloning information. Subtype- C4, D4, E4 and F4 (also referred to as sulphidopeptide
selective receptor antagonists are under develop- LTs or peptidolipids) [10] (Fig. 1). Note that the tran-
ment. Only one gene for TP receptors has been iden- scellular metabolism of AA can bring about either
tified, but multiple splice variants exist. PGI2 binds to “protection” as is the case during the platelet/endo-
IP receptors and activates adenylate cyclase. PGD2 thelium transfer of PGH2 to make cytoprotective PGI2
interacts with a distinct DP receptor that also stimu- [1] or “damage” as in the case of the leukocyte/
lates adenylate cyclase. PGE1 acts through IP recep- endothelium transfer of LTA4 to make cytotoxic LTC4
tors, PGE2 activates EP receptors but it may also act [6].
on IP and DP receptors. Consecutive splicing of amino acids from the glu-
tathione moiety of LTC4 occurs in the lungs, kidney,
and liver. LTE4 is already substantially deprived of
Products of lipooxygenation of arachidonic most of the biological activities of LTC4 and LTD4.
acid LTC4 may also be inactivated by oxidation of its cys-
teinyl sulphur atom to a sulphoxide group.The prin-
AA can be metabolized to straight chain products by cipal route of inactivation of LTB4 is by ω-oxidation.
lipooxygenases (LOXs) which are a family of cytoso- LTC4 and LTD4 comprise an important endogenous
lic enzymes that catalyze oxygenation of all polyenic bronchoconstrictor, earlier known as “slow-reacting
fatty acids with two cis double bonds separated by a substance of anaphylaxis” (SRS-A) [11].
methylene group to corresponding lipid hydroperox- Three distinct receptors have been identified for
ides [9] (Fig. 1).As in the case of AA, these hydroper- LTs (LTB4, LTC4 and LTD4/LTE4). Stimulation of all of
oxides are called hydroperoxyeicosatetraenoic acids them appears to activate PLC. LTB4, acting on specif-
 Eicosanoids 85

FIGURE 3
Transcellular synthesis of lipoxins and their actions

ic receptors, causes adherence, chemotaxis and acti- expression on endothelium, cause vasodilation and
vation of POLYMORPHONUCLEAR LEUKOCYTES and MONO- attenuate LTC4-induced vasoconstriction by antago-
CYTES, as well as promoting cytokine production in nism of cysLT1 receptor. They also inhibit chemo-
MACROPHAGES and LYMPHOCYTES. Its potency is compa- taxis, adhesion and transmigration, IL-1β and super-
rable with that of various chemotactic peptides and oxide production of POLYMORPHONUCLEAR leukocytes.
PAF. In higher concentrations, LTB4 stimulates the On the other hand, LIPOXINS stimulate monocyte
aggregation of PMNs and promotes DEGRANULATION adhesion and increase IL-4 formation [13, 14].There
and the generation of superoxide. It promotes adhe- is an inverse relationship between the amount of
sion of neutrophils to vascular endothelium and LIPOXIN and LT production, which may indicate that
their transendothelial migration [12]. The cysteinyl- LIPOXINS may be “endogenous regulators of
LTs are strongly cytotoxic, and cause bronchocon- leukotriene actions”. High-affinity G-protein coupled
striction and vasodilation in most vessels except the LIPOXIN receptors (ALXR) have been identified on
coronary vascular bed. numerous cells, including MONOCYTES, PMNs, fibrob-
lasts and endothelial and epithelial cells. Its expres-
sion may be up-regulated by IFN-γ, IL-13 or even IL-
Lipoxins (“lipooxygenase interaction 1β. Activation of this receptor modulates phos-
products”) phatidylinositide 3-kinase (PI3-kinase) activity.
LIPOXINS may also competitively bind and block the
LIPOXINS are formed by sequential transcellular cys-LT1 receptor. There are also suggestions that
metabolism of AA by 15- and 5-, or by 5- and 12-LOX LIPOXINS may also bind within the cell, to ligand-acti-
[13]. Cellular context is critical for the synthesis of vated transcription factors, therefore regulating gene
LIPOXINS (Fig. 3). LIPOXINS have several anti-inflamma- expression in the nucleus.
tory properties as well as concomitant proinflamma- A separate group of LIPOXINS was termed aspirin-
tory actions. LIPOXINS inhibit adhesion molecule triggered LIPOXINS (ATLs), as their synthesis is the
86 Inflammatory mediators and intracellular signalling

result of acetylation of CYCLOOXYGENASE-2, which actions are mediated by PGE2 and PGI2. They are
inhibits endothelial cell prostanoid formation and both naturally occurring vasodilators. PGI2 is the
promotes synthesis of 15(R) HETE. These are then most comprehensive anti-platelet agent which is
converted in PMNs to 15R-enantiomeres: 15-epi LXA4 responsible for the thromboresistance of the vascu-
or 15-epi-LXB4. ATLs share many actions of LIPOXINS, lar wall. PGE2, through a similar adenylate cyclase-
albeit with much greater potency [15]. Due to their dependent mechanism, inhibits the activation of
anti-inflammatory properties, LIPOXIN analogues may leukocytes. PGE2 is also responsible for protection
find an important place in the treatment of inflam- of the gastric mucosa. PGE2 and PGF2α may play a
mation [13, 14]. physiological role in labor and are sometimes used
clinically as abortifacients. Locally generated PGE2
and PGI2 modulate vascular tone and the impor-
Other pathways of arachidonic acid tance of their vascular actions is emphasized by the
metabolism participation of PGE2 and PGI2 in the hypotension
associated with septic shock. These PGs also have
AA can be also metabolized by a NADPH-dependent been implicated in the maintenance of patency of
cytochrome P-450-mediated monooxygenase path- the ductus arteriosus. Various PGs and LTs are
way (MOX). The resulting 19-HETE, 20-HETE and a prominent components released when sensitized
number of epoxyeicosatrienoic and dihydroxye- lung tissue is challenged by the appropriate anti-
icosatrienoic acid isomers show vascular,endocrine, gen. While both bronchodilator (PGE2) and bron-
renal, and ocular effects, the physiological impor- choconstrictor (PGF2α, TXA2, LTC4) substances are
tance of which remains to be elucidated [16]. released, responses to the peptidoleukotrienes
Recently, a non-enzymatic, free radical-mediated probably dominate during allergic constriction of
oxidation of AA, while still embedded in phospho- the airway.The relatively slow metabolism of the LTs
lipids,has been discovered.Subsequently,acyl hydro- in lung tissue contributes to the long-lasting bron-
lases gave rise to a novel series of regioisomers of choconstriction that follows challenge with antigen
ISOPROSTANES.Formed non-enzymatically, ISOPROSTANES and may be a factor in the high bronchial tone that
lack the stereospecificity of prostanoids. Highly toxic is observed in asthmatics in periods between
ISOPROSTANES might contribute to the pathophysiolo- attacks. PGs and LTs contribute importantly to the
gy of inflammatory responses which are insensitive genesis of the signs and symptoms of inflammation.
to currently available steroidal and non-steroidal The peptidoleukotrienes have effects on vascular
anti-inflammatory drugs. The most thoroughly inves- permeability, while LTB4 is a powerful chemoattrac-
tigated regioisomer of ISOPROSTANES is 8-epi-PGF2α. It tant for POLYMORPHONUCLEAR LEUKOCYTES and can
has a potent vasoconstrictor action which is mediat- promote exudation of plasma by mobilizing the
ed by vascular TXA2/PGH2 receptors. source of additional inflammatory mediators. PGs
do not appear to have direct effect on vascular per-
meability; however, PGE2 and PGI2 markedly
Actions and clinical uses of eicosanoids enhance oedema formation and leukocyte infiltra-
tion by promoting blood flow in the inflamed
EICOSANOIDS produce a vast array of biological region. PGEs inhibit the participation of LYMPHO-
effects.TXA2, PGF2α and LTs represent cytotoxic, pro- CYTES in delayed reactions. Bradykinin, CYTOKINES
inflammatory mediators. TXA2 is strongly thrombo- (TNF-α, IL-1, IL-8) appear to liberate PGs and proba-
genic through aggregation of blood platelets. LTC4 bly other mediators that promote hyperalgesia
injures blood vessels and bronchi subsequent to (decreased pain threshold) and the pain of inflam-
activation of leukocytes. On a molecular level, their mation. Large doses of PGE2 or PGF2α, given to
cytotoxicity is frequently mediated by stimulation of women by intramuscular or subcutaneous injection
PLC or inactivation of adenylate cyclase. Cytopro- to induce abortion, cause intense local pain. PGs
tective, but not necessarily anti-inflammatory also can cause headache and vascular pain when
 Eicosanoids 87

infused intravenously. The capacity of PGs to sensi- Aspirin selectively inhibits COX-1, explaining its
tize pain receptors to mechanical and chemical inhibitory effect on the biosynthesis of TXA2 in
stimulation appears to result from a lowering of the platelets (causing reduced thrombotic tendency), of
threshold of the polymodal nociceptors of C fibers. PGI2 in endothelial cells and of PGE2 in gastric
Hyperalgesia also is produced by LTB4. PGE2, when mucosa (leading to gastric damage). This action of
infused into the cerebral ventricles or when inject- aspirin is more pronounced than that on the biosyn-
ed into the hypothalamus, produces fever. The thesis of prostanoids at the site of inflammation,
mechanism of fever involves the enhanced forma- where inducible COX-2 is most active. Consequently,
tion of CYTOKINES that increase the synthesis of PGE2 aspirin at low doses seems to be a better anti-throm-
in circumventricular organs in and near to the pre- botic than anti-inflammatory drug. Aspirin irre-
optic hypothalamic area, and PGE2, via increases in versibly acetylates the active centre of COX-1. Unlike
c-AMP, triggers the hypothalamus to elevate body endothelial cells, platelets lack the machinery
temperature by promoting increases in heat gener- required for de novo synthesis of COX-1, and, accord-
ation and decreases in heat loss. ingly, aspirin-induced inhibition of TXA2 synthesis in
Synthetic PGE1, acting through IP and EP recep- platelets is essentially permanent (until new platelets
tors, is given by infusion to maintain the patency of are formed), in contrast to the easily reversible inhi-
the ductus arteriosus in infants with transposition of bition of PGI2 synthesis in vascular endothelium.The
large vessels until surgical correction can be under- net effect of aspirin is, therefore, a long-lasting anti-
taken. PGI2 (epoprostenol) is occasionally used to thrombotic action. Unfortunately, most NSAIDs are
prevent platelet aggregation in dialysis machines more effective inhibitors of COX-1 than of COX-2.
through inhibition of the thrombocytopenic action Meloxicam was the first clinically available drug
of heparin [17].PGI2 is also used for the treatment of which is claimed to be a selective COX-2 inhibitor –
primary and secondary pulmonary hypertension an anti-inflammatory drug with few side-effects on
[18]. Stable analogues of PGI2 (e.g., iloprost), as well the gastro-intestinal tract, which causes no bleeding.
as of PGE1,are used in selected patients with periph- However, population studies have verified that while
eral vascular disease [17].The PGE1 analogue, miso- protective for gastric mucosa, high doses of COX-2
prostol, is approved in the USA for the prevention of selective inhibitors may induce cardiovascular (due
peptic ulcers, especially in patients who are to inhibition of endothelial COX-2) or renal side-
required to take high doses of non-steroidal anti- effects [19]. NSAIDs usually are classified as mild
inflammatory drugs (NSAID) for treatment of their analgesics and they are particularly effective in set-
arthritis. tings in which inflammation has caused sensitization
of pain receptors to normally painless mechanical or
chemical stimuli. NSAIDs do not inhibit fever caused
Pharmacological interference with eicosanoid by direct administration of PGs, but they do inhibit
synthesis and actions fever caused by agents that enhance the synthesis of
IL-1 and other CYTOKINES, which presumably cause
PLA2 and COX are inhibited by drugs which are the fever at least in part by inducing the endogenous
mainstays in the treatment of inflammation. We dis- synthesis of PGs.
covered that glucocorticosteroids (hydrocortisone,
dexamethasone) inhibit the generation of prostan-
oids in vivo through prevention of the release of AA Platelet-activating factor (PAF)
from phospholipids [10]. This effect is mediated by
intracellular steroid receptors which,when activated, PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)
increase expression of lipocortins which inhibit is a specialized phospholipid with an alkyl group
phospholipases. At present, many other actions of (12-18C) attached by an ether bond at position 1 of
glucocorticosteroids on AA metabolism are known, glycerol and acetylated at position 2. PAF is not
one of them being inhibition of COX-2 transcription. stored in cells but it is synthesized from 1-O-alkyl-2-
88 Inflammatory mediators and intracellular signalling

FIGURE 4
The synthesis and metabolism of
platelet-activating factor (PAF)

acyl-glycerophosphocholine as required (Fig. 4) choline by an acyltransferase.This latter step is inhib-

[20].Initially,PLA2 converts the precursor to the inac- ited by Ca2+.
tive 1-O-alkyl-2-lysoglycerophosphocholine (lyso- PAF is synthesized by platelets, neutrophils,
PAF) with concomitant release of AA. Incidentally, in MONOCYTES, basophils and mast cells, EOSINOPHILS,
GRANULOCYTES, AA produced in this way represents a renal mesangial cells, renal medullary cells, and vas-
major source for the synthesis of PGs and LTA4. In a cular endothelial cells.In most instances,stimulation
second step, lyso-PAF is acetylated by acetyl coen- of the synthesis of PAF results in the release of PAF
zyme A in a reaction catalyzed by lyso-PAF acetyl- and lyso-PAF from the cell. However, in some cells
transferase.This is the rate-limiting step.The synthesis (e.g., endothelial cells) PAF is not released and
of PAF in different cells is stimulated during antigen- appears to exert its effects intracellularly.
ANTIBODY reactions or by chemotactic peptides (e.g., PAF exerts its actions by stimulating a single G
f-MLP), CYTOKINES, thrombin, collagen, and autacoids. protein-coupled, cell-surface receptor [21]. High-
PAF can also stimulate its own formation. Both PLA2 affinity binding sites have been detected in the plas-
and lyso-PAF acetyltransferase are calcium-depend- ma membranes of a number of cell types.Stimulation
ent enzymes, and PAF synthesis is regulated by the of these receptors triggers activation of phospholipas-
availability of Ca2+. The anti-inflammatory action of es C, D, and A2. and mobilization of Ca2+i. Massive
glucocorticosteroids is at least partially dependent direct and indirect release of AA occurs with its sub-
on inhibition of the synthesis of PAF by virtue of the sequent conversion to PGs, TXA2, or LTs. EICOSANOIDS
inhibitory effect of lipocortin on the activity of PLA2. seem to function as extracellular representatives of
Inactivation of PAF also occurs in two steps (Fig. the PAF message. As its name suggests, PAF unmasks
4) [20]. Initially, the acetyl group of PAF is removed fibrinogen receptors on platelets, leading directly to
by PAF acetylhydrolase to form lyso-PAF; this enzyme platelet aggregation.In endothelial cells the synthesis
is present in both cells and plasma. Lyso-PAF is then of PAF may be stimulated by a variety of factors, but
converted to a 1-O-alkyl-2-acyl-glycerophospho- PAF is not released extracellularly. Accumulation of
 Toll-like receptors 89

PAF intracellularly is associated with the adhesion of PAF receptor antagonists include PAF structural
neutrophils to the surface of the endothelial cells and analogues, natural products (e.g., ginkgoloids from
their diapedesis, apparently because it promotes the Ginkgo biloba), and interestingly, triazolobenzodi-
expression or exposure of surface proteins that recog- azepines (e.g., triazolam). The development of PAF
nize and bind neutrophils.Activated endothelial cells receptor antagonists is currently at an early stage of
play a key role in “targeting” circulating cells to clinical development, still leaving the hope that such
inflammatory sites. Expression of the various adhe- antagonists may find future therapeutic application
sion molecules varies among different cell types in inflammation and sepsis.
involved in the inflammatory response. For example,
expression of E-selectin is restricted primarily to
endothelial cells and is enhanced at sites of inflam- Toll-like receptors
mation. P-selectin is expressed predominantly on
platelets and on endothelial cells. L-SELECTIN is The TOLL-LIKE RECEPTORS (TLR1-10) are a part of the
expressed on LEUKOCYTES and is shed when these cells innate immune defence, recognizing conserved pat-
are activated. Cell adhesion appears to occur by terns only on microorganisms, but not mammalian
recognition of cell surface glycoprotein and carbohy- proteins [22].TLRs and their signalling pathways are
drates on circulating cells by the adhesion molecules present in mammals, fruit flies, and plants.Ten mem-
whose expression has been enhanced on resident bers of the TLR family have been identified in
cells. Endothelial activation results in adhesion of humans, and several of them appear to recognize
LEUKOCYTES by their interaction with newly expressed specific microbial products, including lipopolysac-
L-SELECTIN and P-selectin, whereas endothelial- charide (LPS), bacterial lipoproteins, peptidoglycan,
expressed E-selectin interacts with glycoproteins on bacterial DNA and viral RNA. Signals initiated by the
the leukocyte surface, and endothelial ICAM-1 inter- interaction of TLRs with specific microbial patterns
acts with leucocyte integrins. direct the subsequent inflammatory response,
PAF also very strongly increases vascular perme- including mononuclear phagocytic cell cytokine
ability. As with substances such as HISTAMINE and production. Thus, TLR signalling represents a key
bradykinin, the increase in permeability is due to component of the innate immune response to micro-
contraction of venular endothelial cells, but PAF is bial infection [22]. These mechanisms are further
1000–10000-fold more potent than HISTAMINE or discussed in Chapter 4.
bradykinin.
Intradermal injection of PAF duplicates many of
the signs and symptoms of inflammation, including Cytokines
vasodilation, increased vascular permeability, hyper-
algesia, oedema, and infiltration of neutrophils. CYTOKINES are peptides produced by immune cells,
Inhaled PAF induces bronchoconstriction, promotes which play key roles in regulating virtually all mech-
local oedema and accumulation of EOSINOPHILS, and anisms of inflammation, including INNATE IMMUNITY,
stimulates secretion of mucus. In anaphylactic antigen presentation, cellular differentiation, activa-
shock, the plasma concentration of PAF is high and tion and recruitment, as well as repair processes.
the administration of PAF reproduces many of the They are produced primarily by MACROPHAGES and
signs and symptoms of experimental anaphylactic LYMPHOCYTES, but also by other leukocytes, endothe-
shock. PAF receptor antagonists prevent the develop- lial cells and fibroblasts. Substances considered as
ment of pulmonary hypertension in experimental CYTOKINES include INTERLEUKINS (IL)-1 to -25, INTERFER-
septic shock. Despite the broad implications of these ONS (IFNs), TUMOUR NECROSIS FACTORS (TNFs),platelet-
experimental observations,the clinical effects of PAF derived growth factor (PDGF), transforming growth
antagonists in the treatment of bronchial asthma, factor- (TGF-)β, CHEMOKINES (which will be discussed
septic shock and other inflammatory responses have separately) and the COLONY-STIMULATING FACTORS.The
been rather modest. cytokine production profile in response to immune
90 Inflammatory mediators and intracellular signalling

insult determines the nature of immune response Production of IL-1ra alleviates potentially deleterious
(cell-mediated, humoral, cytotoxic or allergic) [23, effects of IL-1 in the natural course of the disease.
24]. IL-18,although structurally close to the IL-1 family,
INTERLEUKIN-1 is the term given to a family of four exerts actions more related to IL-12. It was originally
CYTOKINES consisting of two active agonists: IL-1α, IL- derived from liver, but is produced by numerous cell
1β, an endogenous IL-1-receptor antagonist (IL-1ra) types (including lung, kidney and smooth muscle
and recently cloned cytokine IL-18, which is struc- cells) apart from LYMPHOCYTES. In contrast to other
turally related to IL-1. Both IL-1α, IL-1β, as well as a CYTOKINES, IL-18 procytokine is constitutively
related protein IL-18, are synthesized as a less active expressed and therefore its activity is regulated pri-
precursor.Their secretion in response to various stim- marily by caspase-1. It plays a critical role in cellular
uli (antigens, endotoxin, CYTOKINES or microorgan- adhesion, being the final common pathway leading
isms) depends on the cleavage of the pro-cytokines to ICAM-1 expression in response to IL-1, TNF-α and
to their active forms by IL-1 converting enzyme (ICE other CYTOKINES. It also synergises with IL-12 in stimu-
or caspase 1). IL-1α remains cell-associated and is lating IFN-γ production. Soluble IL-18 receptor may
active mainly during cell-to-cell contact, while the be particularly interesting from an immunopharma-
soluble IL-1β is a form predominant in biological flu- cological point of view as it has lost its signalling
ids. IL-1 is an important inflammatory mediator and domain and may therefore serve as a potent anti-
it is believed to be implicated in several acute (e.g., inflammatory molecule.
systemic inflammatory response syndrome (SIRS) in
sepsis) or chronic (e.g., rheumatoid arthritis) inflam-
matory diseases. IL-1 is also important in immune Tumour necrosis factor-α and -β
responses, facilitating interaction of both B or T cells (TNF-α and -β)
with antigen.
One of the principal actions of IL-1 is activation of These CYTOKINES are produced primarily in mononu-
T LYMPHOCYTES and B cells by enhancing the produc- clear phagocytes (TNF-α) and in LYMPHOCYTES (TNF-
tion of IL-2 and expression of IL-2 receptors. In IL-1 β), but also by numerous other cells. Activation of
knockout animals, diminished immune responses or TLRs (TLR2 and TLR4) by LPS is the most commonly
state of tolerance is observed.In vascular endothelial recognised intracellular pathway leading to produc-
cells, IL-1 increases the synthesis of leukocyte adhe- tion of TNF. TNF-α and -β bind with similar affinity to
sion molecules (VCAM-1, ICAM-1 and E-selectin), the same cell surface receptors – TNFR 1 (p55) and
stimulates nitric oxide (NO) production, releases TNFR 2 (p75), therefore their activities are very close
PDGF, and activates PLA2, thus inducing the synthesis to each other. Their name is based on tumour cyto-
of prostanoids and PAF. It stimulates fibroblasts to toxic effects; however, their pharmacological use in
proliferate, to synthesize collagen and to generate the treatment of tumours is limited by severe side-
collagenase. It regulates the systemic inflammatory effects.TNF is responsible for severe cachexia during
response by stimulating synthesis of acute-phase pro- chronic infections and cancer.
teins (C-reactive protein, amyloid and complement), In endothelial cells these CYTOKINES induce:
producing neutrophilia, and causing fever by alter- expression of adhesion molecules (ICAM-1 and
ing a set-point of temperature in the hypothalamus. VCAM-1), synthesis of prostacyclin and of CYTOKINES.
IL-1 also induces the generation of other CYTOKINES TNFs act as chemoattractants, as well as potent acti-
such as the IFNs, IL-3, IL-6, and, in BONE MARROW, the vators for neutrophils and MACROPHAGES.TNF-α caus-
COLONY-STIMULATING FACTORS. It synergises with TNF-α es fever and releases acute-phase proteins. TNF and
in many of its actions, and its synthesis is stimulated IL-1 produce many of the same proinflammatory
by TNF-α.The therapeutic effects of GLUCOCORTICOIDS responses which include induction of CYCLOOXYGE-
in rheumatoid arthritis and other chronic inflamma- NASE and lipooxygenase enzymes as well as the acti-
tory and autoimmune diseases may well involve vation of B cells and T cells. It is finally important to
inhibition of both IL-1 production and IL-1 activity. point out that TNF is the primary mediator of haemo-
 Cytokines 91

dynamic changes during septic shock through its haematopoiesis and are chemotactic for neutrophils,
negative inotropic effects as well increase in vascular as well as activating neutrophils and MACROPHAGES.
permeability.
Anti-inflammatory cytokines. It is important to point
TGF-α (transforming growth factor-α) is a trophic out that, apart from pro-inflammatory actions, some
regulator of cell proliferation and differentiation CYTOKINES may inhibit inflammatory processes.These
which is important in repair processes; it is involved include IL-1ra mentioned above, as well as TGF-β or
in angiogenesis and in the organization of the extra- the IL-10 family (includes IL10, 19, 20, 22 and 24).
cellular matrix, and it is chemotactic for MONOCYTES.

PDGFs cause proliferation of fibroblasts, vascular Intracellular signalling by cytokine receptors

endothelial cells and smooth muscle; they are impli- [23, 24]
cated in angiogenesis, atherosclerosis and possibly
in chronic asthma. Binding CYTOKINES to their receptors leads to the acti-
vation of cytoplasmic tyrosine kinases. Janus kinases
IFNs constitute a group of inducible CYTOKINES which (JAKs), a recently described family of four related
are synthesized in response to viral and other stim- cytoplasmic protein tyrosine kinases, function in
uli.There are three classes of IFNs, termed IFN-α, IFN- cytokine signalling. There are four JAKs: JAK1, JAK2,
β and IFN-γ.IFN-α is not a single substance but a fam- JAK3, and TYK2, which transduct signals from
ily of 15 proteins with similar activities. The three cytokine receptors to effector mechanisms. On bind-
IFNs (α, β and γ) have antiviral activity and IFN-γ has ing of the cytokine, JAKs bind to the receptor and
a significant immunoregulatory function and only mediate tyrosine kinase activity. and phosphoryla-
modest anti-viral activity. Their anti-viral effects are tion of the receptor and of receptor-associated JAKs
achieved by inhibition of viral replication within (Fig. 5).The next step in signal transduction involves
infected cells as well as by stimulation of cytotoxic tyrosine phosphorylation of signal transducers and
LYMPHOCYTES and NK cells. All IFNs can be induced activators of transcription (STATs) in the cytoplasm.
by other CYTOKINES such as IL-1,IL-2,TNF and COLONY- Upon activation, STATs become phosphorylated,
STIMULATING FACTORS.IFN-α and IFN-β are produced in form homodimers,and migrate to the nucleus,where
many cell types – MACROPHAGES, fibroblasts, endothe- they bind to regulatory sequences in the promoters
lial cells, osteoblasts, etc.; they can be strongly of cytokine-responsive genes, e.g., ICAM-1 or other
induced by viruses, and less strongly by other cytokine genes. Cytokine signalling is based on a rel-
microorganisms and bacterial products. IFNs induce atively small number of redundant tyrosine kinases.
the expression of the major histocompatibility mole- For instance, JAK-1 and JAK-3 transduct signals from
cules (MHC I and II) that are involved in antigen gc CYTOKINES (i.e.,IL-2 or IL-4),while JAK-2 is involved
presentation to T cells.IFNs also stimulate the expres- in IL-3, IL-6 and GM-CSF signalling. Similarly, the num-
sion of Fc receptors on GRANULOCYTES, promote the ber of STATs is low when compared to the number of
differentiation of myeloid cells and modulate the CYTOKINES. Therefore one can conclude that some
synthesis of CYTOKINES. IFN-γ is primarily made by T additional mechanisms will guide different respons-
LYMPHOCYTES (T helper type 1), and which may sug- es to various CYTOKINES. An additional pathway used
gest that it is more of an IL than an IFN. Indeed, it by many cytokine receptors includes Ras-dependent
functions as an inhibitor of IL-4-dependent expres- cascades. In this signal transduction cascade, Ras,
sion of low-affinity IgE receptors, therefore inhibiting Raf-1, Map/Erk kinase kinase (MEKK) and finally
IgE synthesis. mitogen-activated protein kinases (MAPK) are
sequentially activated and lead to regulation of cel-
Colony-stimulating factors. These include IL-3 and lular proliferation by growth factors and responses to
GM-CSF (granulocyte macrophage colony-stimulat- IL-2 or IL-3. The activation of other signalling path-
ing factor) and several other CYTOKINES.They regulate ways, like insulin receptor substrates (IRS-1, IRS-2),
92 Inflammatory mediators and intracellular signalling

trophils, and other GRANULOCYTES, as well as vascular

smooth muscle cells and a variety of other cells [25].
There are 47 chemokines,sharing 30–60% homology.
CHEMOKINES are characterised by the presence of 3-4
conserved cysteine residues. Recently, a new classifi-
cation of CHEMOKINES has been proposed based on
the positioning of the N-terminal cysteine residues
(Tab. 2). CHEMOKINES are usually secreted proteins
except for fractalkine (CX3CL1), which is the only
membrane-bound chemokine. Most CHEMOKINES play
roles in recruiting and activating immune cells to
and at the site of inflammation while others are
important in maintaining homeostasis within the
immune system (housekeeping chemokines: CCL5,
CCL17-19, 21, 22, 25, 27, 28, CXCL13, CXCL14). Homeo-
static CHEMOKINES are expressed in an organ-specific
manner while inflammatory CHEMOKINES can be pro-
duced by multiple cell types.
Their activities are achieved through interaction
with chemokine receptors. There are 18 chemokine
receptors currently known; therefore some receptors
may bind several ligands,which leads to overlapping
functions of known chemokines. Moreover, a single
cell may express several chemokine receptors. One
of the key features of chemokine receptors, owed to
their heptahelical transmembrane structure, is their
ability to signal through different intracellular sig-
nalling pathways. Binding of the chemokine to the
FIGURE 5 receptor leads to activation of Gα protein and bind-
Cytokine-induced intracellular signalling ing of GTP. The Gα subunit activates Src kinases and
subsequently MAPKs and protein kinase B (PKB).
During activation of Gα protein,a Gβγ complex is lib-
erated and may independently lead to activation of
can also mediate some other biological activities of PKB and MAPKs (via PI3), PKC activation via phos-
CYTOKINES, including proliferation and regulation of pholipase C (PLC) and finally through Pyk-2 [25].
apoptosis. In conclusion, it becomes apparent that These pathways lead to up-regulation of membrane
combination of the signalling mechanisms INTEGRINS and initiate rolling and adhesion of cells as
described above will lead to many distinct respons- well as their conformational changes. Some of these
es to different CYTOKINES. intracellular pathways (in particular PLC activation)
may then lead to an increase in intracellular calcium
and its consequences, including DEGRANULATION, NOS
Chemokines and their intracellular activation, etc., within the target cells.
It is difficult to accurately describe the relative
signalling importance of individual chemokines.The largest
number of studies were conducted looking at
CHEMOKINESare a family of 8–12 kD molecules,which actions of IL-8 as the most important chemoattrac-
induce chemotaxis of MONOCYTES, LYMPHOCYTES, neu- tant for POLYMORPHONUCLEAR leukocytes, although it
 Neuropeptides / Kinins 93

TABLE 2. CLASSES OF CHEMOKINES

Subfamily Chemokines Characteristics

C-X-C CXCL 1-16, includes IL-8 (CXCL8) First two cysteines separated by a variable amino-acid.
C-C CCL1-28 (include MIP-1 MCP and RANTES) First two cysteines are adjacent to each other.
C XCL 1 (lymphotactin)-and XCL 2 Lacks first and third cysteine residue
CX3CL1 CX3CL1 (Fractalkine) Two N-terminal cysteine residues separated by 3
variable amino acids.

appears late during the inflammatory response. also called substance K, and neurokinin B (NKB).
Other well-investigated members of this family They occur mainly in the nervous system, particu-
include CCL3 (MIP-1a) or RANTES (CCL5). larly in nociceptive sensory neurons and in enteric
Apart from effects on chemotaxis, CHEMOKINES neurons. They are released as neurotransmitters,
have direct and indirect effects on T-cell differentia- often in combination with other mediators. SP and
tion into T helper 1 or 2 subclasses, therefore regulat- NKA are encoded by the same gene and they have
ing the nature of immune responses [24, 25]. a similar distribution. Three distinct types of
Due to the critical role of CHEMOKINES in inflam- tachykinin receptor are known: NK1, NK2, and NK3.
mation,interest has focused on potential therapeutic They are selective for three endogenous tachykinins
effects of inhibiting their activity. Both peptide antag- with the following affinity: SP> NKA> NKB for NK1,
onists as well as gene transfer approaches have been NKA> NKB> SP for NK2 and NKB> NKA> SP for NK3
successfully used to inhibit inflammation in various receptor. Receptor cloning has shown that
animal models (e.g.,allergic inflammation models or tachykinin receptors belong to a family of G-pro-
ApoE-knockout atherosclerosis-prone mice). tein-coupled receptors. Several potent antagonists
of NK1 and NK2 and NK3-receptors have been dis-
covered [26], and novel therapeutic agents for vari-
Neuropeptides ous disease states (e.g., pain, asthma, arthritis,
headache) may be developed.
Neuropeptides are released from sensory neurons CGRP differs from other tachykinins. It is coded
and in some tissues they contribute to inflammatory for by the calcitonin gene which also codes for cal-
reactions. For example, substance P and other citonin itself. Differential splicing allows cells to pro-
tachykinins produce smooth muscle contraction, duce either procalcitonin (expressed in thyroid
mucus secretion, cause vasodilation and increase cells) or pro-CGRP (expressed in neurons) from the
vascular permeability. “Calcitonin gene-related pep- same gene. CGRP is found in non-myelinated senso-
tide”(CGRP) is a potent vasodilator, acting on CGRP- ry neurons and it is a potent inducer of neurogenic
receptors leading to activation of adenylate cyclase. inflammation.
The overall pattern of effects of tachykinins is similar,
though not identical, to the pattern seen with kinins.
Kinins
Tachykinins Kinins are polypeptides with vasodilator/hypoten-
sive, thrombolytic, pro-inflammatory and algesic
The mammalian tachykinins comprise three related actions. The two best known kinins are bradykinin
peptides: substance P (SP), neurokinin A (NKA), and kallidin and they are referred to as plasma
94 Inflammatory mediators and intracellular signalling

FIGURE 6
The formation and metabolism of
kinins

kinins. Since 1980, when Regoli and Barabe divided bradykinin occurs in plasma due to the activity of
the kinin receptors into B1 and B2 classes, first- and plasma aminopeptidases.
second-generation kinin receptor antagonists have The half-life of kinins in plasma is about 15 sec-
been developed, leading to a much better under- onds and concentrations of kinins found in the cir-
standing of the actions of kinins. culation are within the picomolar range. Bradykinin
Bradykinin is a nonapeptide, kallidin is a deca- is inactivated by a group of enzymes known as kini-
peptide and has an additional lysine residue at the nases. The major catabolizing enzyme in the lung
amino-terminal position. These two peptides are and in other vascular beds is kininase II, which is
formed from a class of α-2 globulins known as kinin- identical to peptidyl dipeptidase – known as
ogens (Fig. 6).There are two kininogens: high molec- angiotensin-converting enzyme (ACE). Kininase II is
ular weight (HMW) and low molecular weight inhibited by captopril, resulting in an increased con-
(LMW) kininogen, which are products of a single centration of circulating bradykinin, which con-
gene that arises by alternative processing of mRNA. tributes substantially to the antihypertensive effect of
The highly specific proteases that release bradykinin captopril. On the other hand, kininase I is arginine
and kallidin from the kininogens are termed carboxypeptidase and it has a slower action than
kallikreins. Two distinct kallikreins, formed by differ- kininase II. It removes the carboxyl-terminal arginine
ent activation mechanisms from inactive prekalli- residue producing des-Arg9-bradykinin or des-Arg10-
kreins, act on the kininogens. One of these is plasma kallidin, which are themselves potent B1-kinin recep-
kallikrein and the other is tissue kallikrein. LMW tor agonists.
kininogen is a substrate only for the tissue kallikrein There are at least two distinct receptors for kinins:
and the product is kallidin, while HMW kininogen is B1 and B2. The classical, constitutive bradykinin
cleaved by plasma and tissue kallikrein to yield receptor, now designated the B2 receptor, selectively
bradykinin and kallidin, respectively. binds bradykinin and kallidin and mediates a major-
Kallidin is similar in activity to bradykinin and ity of the effects of bradykinin and kallidin in the
need not be converted to the latter to exert its absence of inflammation, such as the release of PGI2
effects. However, some conversion of kallidin to and NO from endothelial cells. On the other hand,
 Nitric oxide 95

inducible B1 receptors are upregulated by inflamma- pancreatitis-induced hypotension,bronchial asthma,

tion. They bind des-Arg metabolites of bradykinin rhinovirus-induced symptoms and in fighting pain.
and kallidin.In contrast to B1 receptors,the signalling
mechanism of B2 receptors has been well character-
ized.The B2 receptor is coupled to G protein and acti- Nitric oxide
vates both PLA2 and PLC.While stimulation of the for-
mer liberates AA from phospholipids, with its subse- In animal tissues, nitric oxide (NO) is generated
quent oxidation to a variety of pro-inflammatory enzymatically by synthases (NOS). The three NOS
eicosanoids, the activation of PLC through IP3 and isoenzymes (neuronal, endothelial and inducible)
DAG leads directly to pro-inflammatory effects. are flavoproteins which contain tetrahydrobiopterin
During the last decade the existence of other and haeme and they are homologous with
types of kinin receptors (B3, B4, B5) has been suggest- cytochrome p 450 reductase [30]. Isoenzymes of
ed. However, recent studies indicate that some of NOS act as dioxygenases using molecular oxygen
them may actually represent functions of the B2 and NADPH to transform L-arginine to L-citrulline
receptor [18]. and NO (Fig. 7). NO formed by endothelial constitu-
Kinins are among the most potent vasodilators tive NOS (eNOS) is responsible for maintaining low
known, acting on arteriolar beds of the heart, liver, vascular tone and preventing LEUKOCYTES and
skeletal muscle, kidney, intestines, and ovaries. They platelets from adhering to the vascular wall. eNOS is
are claimed to play a minor role in the regulation of also found in renal mesangial cells. NO formed by
blood pressure in healthy individuals,but they play a neuronal constitutive NOS (nNOS) acts as a neuro-
major vasodepressor regulatory role, most likely modulator or neuromediator in some central neu-
mediated by arterial endothelium, in hypertensive rons and in peripheral “non-adrenergic non-choliner-
patients [27]. Indeed, kinins contract veins and non- gic” (NANC) nerve endings. NO formed by inducible
vascular smooth muscle,such as gastrointestinal and NOS (iNOS) in MACROPHAGES and other cells plays a
bronchial muscle. Bradykinin and kallidin have simi- role in the inflammatory response.
lar contracting properties.At the level of the capillary NO was discovered by Furchgott and Zawadzki as
circulation, kinins increase permeability and pro- “endothelium-derived relaxing factor”(EDRF) [31].It
duce oedema. Stimulation of B1 receptors on inflam- soon became obvious that EDRF, like nitroglycerine,
matory cells such as MACROPHAGES can elicit the pro- activates soluble guanylate cyclase in vascular
duction of the inflammatory mediators such as IL-1 smooth muscle by binding to its active haem centre.
and TNF-α [28]. Kinins are also potent pain-inducing The rise in cyclic GMP achieved is responsible for
agents in both the viscera and skin. In acute pain, B2 vasodilation and for other physiological regulatory
receptors mediate bradykinin algesia. The pain of functions of NO.
chronic inflammation appears to involve an The activities of constitutive nNOS and eNOS are
increased expression of B1 receptors. controlled by intracellular calcium/calmodulin lev-
As in the case of other autacoids, the therapeutic els.For instance,nNOS in central neurons is activated
interest in kinins has focused particularly on by glutamate binding to NMDA receptors with a sub-
attempts to modulate their formation or metabolism sequent rise in Ca2+i due to opening of voltage calci-
in vivo [29]. Blockade of kinin formation with a um channels, whereas eNOS is activated by blood
kallikrein inhibitor, aprotynin (Trasylol), has been shear stress or stimulation of endothelial muscarinic,
used with some success to treat acute pancreatitis, purinergic, kinin, substance P or thrombin receptors.
carcinoid syndrome or Crohn disease. Experimen- This triggers an increase in Ca2+i at the expense of
tally, progress has been made in the development of the release of Ca2+ from endoplasmic reticulum.
selective antagonists of kinins. Currently, they are not Calcium ionophores (e.g., A23187) and polyca-
available for clinical use; however, recent studies tions (e.g., poly-L-lysine) cause a rise in Ca2+i and
indicate that kinin receptor antagonists might be use- activate eNOS, thereby bypassing the receptor mech-
ful for the treatment of patients with septic shock, anisms.
96 Inflammatory mediators and intracellular signalling

triggers a chain of protein phosphorylation which

eventually leads to the activation of the major tran-
scription protein NF-k-B. This is responsible for tran-
scription of the message: to make iNOS. In cells
which lack m-CD14, the induction of iNOS is com-
pleted by a complex of soluble s-CD14 with LBP and
LPS itself. In a similar manner, LPS can also induce
COX-2. Although NO fulfils more paracrine than
autoendocrine functions, yet in the case of iNOS,
large amounts of locally formed NO may inhibit
iNOS itself as well as COX-2, in a negative feedback
reaction. Glucocorticosteroids and some CYTOKINES,
such as TGF-β, IL-4 or IL-10, inhibit the induction of
iNOS.

Nitric oxide as an effector of inflammation

Kinetics of nitric oxide production by iNOS differ

greatly from production by eNOS or nNOS (Fig. 8)
[32]. Inducible NOS produces very large, toxic
amounts of NO in a sustained manner, whereas con-
sititutive NOS isoforms produce NO within seconds
and its activities are direct and short acting.There are
multiple intracellular mechanisms through which
NO may act as an inflammatory mediator [33]. Low
levels of NO produced by constitutive synthases pri-
marily interact directly with positively charged metal
ions of guanylate cyclase,cytochrome p450 and NOS
itself. Activation of guanylate cyclase leads to an
FIGURE 7 increase in intracellular cyclic guanosine mono-
The synthesis and metabolism of nitric oxide (NO) phosphate (cGMP), which in turn activates cGMP-
dependent protein kinases which mediate NO
actions including vasorelaxation, increase of vascu-
In contrast to the constitutive isoforms of NOS, lar permeability, as well as anti-proliferative, anti-
iNOS does not require a rise in Ca2+i to initiate its platelet and anti-oxidant effects of NO. Recent data
activity. In MACROPHAGES, MONOCYTES and other cells, have also indicated that NO produced by constitu-
the induction of iNOS and the presence of L-arginine tive NOS enzymes may be involved in immune regu-
are sufficient to initiate the generation of NO. lation of T helper cell proliferation and cytokine pro-
Induction of iNOS can be initiated by IFN-γ,TNF−α or duction. During the course of an inflammatory
IL-1. However, the best recognized inducer is LPS or response, the large amounts of NO formed by iNOS
endotoxin from Escherichia coli, which is known to surpass the physiological amounts of NO which are
be responsible for the development of SIRS in the usually made by nNOS or eNOS. The functions of
course of sepsis due to gram-negative bacteria. iNOS-derived NO are also different. In immunologi-
Myeloid cells have a receptor for LPS on their cell cally or chemically activated MACROPHAGES, NO kills
membrane, m-CD14 protein. LPS, using an “LPS bind- microorganisms and destroys macromolecules. NO
ing protein” (LBP), is anchored to m-CD14 and then formed by constitutive isoforms of NOS is stored as a
 Nitric oxide 97

FIGURE 8
Differences between kinetics of nitric oxide generation by eNOS and iNOS

nitrosothiol in albumin and acts physiologically as N- NO formed by eNOS seems to be mostly cytoprotec-
nitrosoglutathione and N-nitrosocysteine. Eventually, tive, possibly due to its unusual redox properties.
within a few seconds, NO is oxidized to nitrites or Large amounts of NO and ONOO– may target
nitrates. Large amounts of “inflammatory NO” from numerous proteins and enzymes critical for cell sur-
myeloid cells are usually generated side by side with vival and signalling. These include signalling mole-
large amounts of superoxide anion (O2–).These two cules involved in cytokine signalling like JAK or STAT
can form peroxynitrite (ONOO–) which mediates the proteins, NK-κB/IκB pathway as well as MAPK, some
cytotoxic effects of NO, such as DNA damage, LDL G proteins and transcription factors. Nitration of cys-
oxidation, isoprostane formation, tyrosine nitration, teines in these proteins may lead to their activation
inhibition of aconitase and mitochondrial respira- or inactivation.
tion.The discovery of this reaction opens new possi- NO is scavenged by haemoglobin, methylene
bilities for the therapeutic use of superoxide dismu- blue and pyocyanin from Pseudomonas coereleus.
tase (SOD). Indeed superoxide dismutase mimetics These last two are also claimed to be inhibitors of
have been successfully used to limit the extent of guanylate cyclase. GLUCOCORTICOIDS selectively inhib-
inflammation. Interestingly, over-stimulation of NMDA it the expression of iNOS.Arginine analogues,such as
receptors by glutamate may activate nNOS to such L-NG-monomethyl arginine (L-NMMA) and L-NG-nitro-
an extent that NO itself exerts neurotoxic properties. arginine methyl ester (L-NAME) inhibit inducible
98 Inflammatory mediators and intracellular signalling

and constitutive NOS isoforms non-selectively. respond similarly to NO.NO-induced changes in lym-
Selective iNOS inhibitors (e.g., alkylisothioureas or phocyte proliferation seem to be dependent more
aminoguanidines) are being intensively investigated on the effects on the cell cycle proteins than on
in the hope that selective inhibition of iNOS may pre- changes in the cytokine profile [32].
vent development of SIRS or MODS (multiple organ It is also important to discuss mechanisms
dysfunction syndrome). Indeed, over-production of through which cells that produce NO protect them-
NO by iNOS during septicaemia is claimed to be selves against its toxic actions [35]. Recent studies
responsible for irreversible arterial hypotension, show that GSH-GSSG anti-oxidative systems protect
vasoplegia (loss of responses to noradrenaline), lac- MACROPHAGES against iNOS-generated large amounts
tic acidosis, suffocation of tissues, their necrosis and of NO. Similarly, endothelial cells are not the primary
apoptosis. However, it is important to remember that responder to eNOS-produced NO. This is possibly
NO made by iNOS is of benefit to the host defence due to the fact that increases of intracellular calcium
reaction by contributing to microbial killing. which mediate eNOS activation are also able to
Moreover, NO generated by eNOS is essential to inhibit guanylate cyclase activity.
maintain tissue perfusion with blood, to offer cyto-
protection in the pulmonary and coronary circula-
tion against toxic lipids which are released by LPS Reactive oxygen species
and to preserve red cell deformability which
becomes reduced in septicaemia [34]. Preliminary REACTIVE OXYGEN SPECIES (ROS) production plays an
clinical experience with L-NMMA has been reason- important role in modulation of inflammatory reac-
ably encouraging, as long as a low dose of the NOS tions. Major ROS produced within the cell are super-
inhibitor is used. In animal models of endotoxic oxide anion, hydrogen peroxide and hydroxyl radi-
shock, non-selective NOS inhibitors were reported to cal [33]. Extracellular release of large amounts of
decrease cardiac output, to increase pulmonary superoxide anion produced by the RESPIRATORY BURST
pressure, to decrease nutritional flow to organs, to in LEUKOCYTES is an important mechanism of
damage gastric mucosa and to increase mortality pathogen killing and also leads to endothelial dam-
rate. On the other hand, inhalation of NO gas (10 age, resulting in an increased vascular permeability
ppm) in septic patients has been found to prevent as well as cellular death. However, vast evidence has
the mismatch of the ventilation/perfusion ratio in recently implicated intracellular ROS production in
their lung. The exact role of NO in various stages of playing a key role in modulation of release of other
sepsis,SIRS and MODS still awaits further elucidation mediators of inflammation. This is related mainly to
and evaluation. the constitutive expression of NAD(P)H oxidases
(termed NOXs- non-phagocytic oxidases) in various
tissues [32].ROS produced by this family of enzymes
Nitric oxide in immune regulation can regulate adhesion molecule expression on
endothelium and inflammatory cells, thus regulating
The exact role of NO in immune regulation is also cellular recruitment to the sites of inflammation.
unclear. Initial mouse studies suggested that antigen- They also increase chemokine and cytokine expres-
presenting cell-derived NO may inhibit T cell prolifer- sion.At least part of these effects results from the abil-
ation, particularly of the Th1 subset of T helper cells. ity of ROS (in particular H2O2) to stimulate MAPK
Mouse Th1 cells were also shown to produce NO,sug- activity which leads to activation of several transcrip-
gesting that the above mechanism is a part of nega- tion factors. It is possible that intracellular ROS may
tive feedback. In this way NO would inhibit Th1 and act as second messengers in inflammatory signal
therefore promote Th2-type cytokine responses lead- transduction [32].
ing to humoral and allergic responses. Subsequent Inflammatory CYTOKINES (like TNF-α) may in turn
studies, however, indicate that both Th1 and Th2 pro- increase NAD(P)H oxidase activity and expression
duce similar amounts of NO, and both subsets which closes the vicious circle of inflammation.
 Amines 99

While loss of NAD(P)H oxidase activity in cells leads secretion (H2), contracts most of the smooth muscle
to diminished inflammation in the vascular wall, sev- other than that of blood vessels (H1), causes vasodi-
eral humoral factors may affect constitutive lation (H1), and increases vascular permeability by
NAD(P)H oxidase expression in the vascular wall acting on the post-capillary venules [37]. Injected
and therefore intracellular ROS production. These intradermally, HISTAMINE causes the triple response:
include angiotensin II, endothelins, high glucose or local vasodilation and wheal by a direct action on
high cholesterol levels.Their effects on baseline ROS blood vessels and the surrounding flare which is due
production may therefore mediate modulatory to vasodilation resulting from an axon reflex in sen-
effects of these factors on inflammation which tradi- sory nerves, thereby releasing a peptide mediator
tionally were not associated with inflammation. [37].Of the many functions of HISTAMINE, stimulation
Accordingly, attempts have been undertaken to of gastric acid secretion, and mediation of type 1
inhibit intracellular ROS production in order to limit hypersensitivity, such as urinary and hay fever, are
inflammatory responses.Apocynin, an NAD(P)H oxi- among the most important.The full physiological sig-
dase activation inhibitor, has been successfully used nificance of the H3-receptor has yet to be established
in limiting inflammation in animal models of [38]. HISTAMINE may also be involved in T helper cell
rheumatoid arthritis, while decoy peptides prevent- immune regulation (extensively reviewed in [39]).
ing association of NAD(P)H oxidase subunits were
shown to be effective in inflammation related to ath- 5-HYDROXYTRYPTAMINE (5-HT, SEROTONIN) was original-
erosclerosis. ly isolated and characterized as a vasoconstrictor
released from platelets in clotting blood. 5-HT
occurs in chromaffin cells and enteric neurons of
Amines the gastrointestinal tract, in platelets and in the cen-
tral nervous system (CNS). It is often stored together
HISTAMINE, 2-(4-imidazolyl)-ethyl-amine, is an essen- with various peptide hormones, such as somato-
tial biological amine in inflammation and allergy.It is statin, substance P or “vasoactive intestinal polypep-
found mostly in the lung, skin and gastro-intestinal tide”(VIP).The biosynthesis and metabolism of 5-HT
tract. It is stored together with macroheparin in gran- closely parallels that of CATECHOLAMINES, except the
ules of mastocytes or basophils (0.01–0.2 pmoles per precursor for decarboxylase of aromatic amino
cell), from which it is released when complement acids is 5-hydroxytryptophan instead of tyrosine
components C3a and C5a interact with specific (Fig. 9). 5-HT is inactivated mainly by the mono-
receptors, or when antigen interacts with cell-fixed amine oxidases A or B (MAO A or B) to 5-hydroxyin-
IgE.These trigger a secretory process that is initiated doleacetic acid (5-HIAA) which is excreted in the
by a rise in cytoplasmic Ca2+ from intracellular urine. Some 5-HT is methylated to 5-methoxytrypta-
stores. Morphine and tubocurarine release HISTAMINE mine, which is claimed to be involved in the patho-
by a non-receptor action. Agents which increase genesis of affective disorders.
cAMP formation inhibit HISTAMINE secretion, so it is The actions of 5-HT are numerous and complex,
postulated that, in these cells, c-AMP-dependent pro- showing considerable variation between species
tein kinase is an intracellular restraining mechanism. [40]. For instance, in the inflammatory response 5-
Replenishment of the HISTAMINE content of mast cell HT seems to be more important in rats than in
or basophil after secretion is a slow process,whereas humans. 5-HT is known to increase gastrointestinal
turnover of HISTAMINE in the gastric histaminocyte is motility, to contract bronchi, uterus and arteries,
very rapid. although 5-HT may also act as a vasodilator through
HISTAMINE is synthesized from histidine by a spe- endothelial release of NO. In some species, 5-HT
cific decarboxylase and metabolized by histaminas- stimulates platelet aggregation, increases microvas-
es and/or by imidazole N-methyltransferase. HIS- cular permeability and stimulates peripheral noci-
TAMINE exerts its effects by acting on H1-, H2- or H3- ceptive nerve endings.A plethora of pathophysiolog-
receptors on target cells [36]. It stimulates gastric ical functions proposed for 5-HT includes control of
100 Inflammatory mediators and intracellular signalling

1, 2 and 4 are G-protein-coupled receptors,type 3 is a

ligand-gated cation channel. 5-HT1 receptors occur
mainly in the CNS (all subtypes) and in blood ves-
sels (5-HT1D subtype). 5-HT1B and 5-HT1D receptors
appear to be involved, at least in part, in the modula-
tion of neurogenically induced (following electrical,
chemical or mechanical depolarization of sensory
nerves) vascular inflammation. 5-HT2 receptors (5-
HT2A subtype being functionally the most important)
are more distributed in the periphery than in the
CNS and they are linked to phospholipase C which
catalyses phosphatidylinositol hydrolysis.The role of
5-HT2 receptors in normal physiological processes is
probably a minor one, but it becomes more promi-
nent in pathological conditions, such as asthma,
inflammation or vascular thrombosis. 5-HT3 recep-
tors occur particularly on nociceptive sensory neu-
rons and on autonomic and enteric neurons, on
which 5-HT exerts an excitatory effect and evokes
pain when injected locally.

CATECHOLAMINES. It has become increasingly recog-

nized that the release of CATECHOLAMINES at autonom-
ic nerve endings and from the adrenal medulla may
modulate the function of immunocompetent cells.
The major LYMPHOID ORGANS (spleen, lymph nodes,
thymus, and intestinal Peyer’s patches) are extensive-
ly supplied by noradrenergic sympathetic nerve
FIGURE 9 fibres. Sympathetic nervous system innervation of
The synthesis and breakdown of 5-HT these LYMPHOID ORGANS, as well as the presence of
adrenergic and dopamine receptors on immune
cells, provide the channels for noradrenergic sig-
peristalsis, vomiting, haemostasis, inflammation and nalling to LYMPHOCYTES and MACROPHAGES by sympa-
sensitization of nociceptors by peripheral mecha- thetic nerves [42].CATECHOLAMINES have a wide range
nisms or control of appetite,sleep,mood,stereotyped of direct effects on immune cells, particularly on
behaviour and pain perception by central mecha- MACROPHAGES and LYMPHOCYTES. Stimulation of β-
nisms. Clinically, disturbances in the 5-HT regulation adrenergic receptors on LPS-pretreated MACROPHAGES
system have been proposed in migraine, carcinoid prevents the expression and release of pro-inflamma-
syndrome, mood disorders and anxiety [40]. tory TNF-α and IL-1, while the release of anti-inflam-
These diverse actions of 5-HT are not mediated matory IL-10 is augmented. On the other hand, α-
through one type of receptor. The amino acid adrenergic stimulation augments phagocytic and
sequence for many 5-HT receptor subtypes has tumoricidal activity of MACROPHAGES. CATECHOLAMINES
been determined by cloning, and the transduction acting through β-adrenergic and dopaminergic
mechanisms to which these receptors are coupled receptors, which are linked to adenylate cyclase
have been explained. The basic four types of recep- through cyclic-AMP, modulate the function of
tors are 5-HT1–4. 5-HT1 and 5-HT2 receptors are fur- immune cells. An increase in intracellular cyclic-
ther subdivided into A, B and C subtypes [41].Types AMP inhibits lymphocyte proliferation and produc-
 Summary 101

tion of proinflammatory CYTOKINES. The demonstra- and INTERFERONs. TOLL-LIKE RECEPTORS are also briefly
tion of the presence of α2-, β-adrenergic, D1 and D2 discussed.
receptors on various immune cells has recently pro- Apart from the mechanisms mentioned above,
vided the basis for regulation of cytokine produc- NITRIC OXIDE and REACTIVE OXYGEN SPECIES production
tion, specifically ILs and TNF, by these receptors in and interaction appear to play an important role in
response to LPS [42].Vasopressor and inotropic CAT- inflammation.These not only act as important effec-
ECHOLAMINES seem to have potent immunomodulat- tors, damaging to invading microorganisms (NO
ing properties which,as yet,have not been adequate- from iNOS or superoxide anion) but may also be
ly explored and may contribute to the therapeutic very important in immune regulation in part by the
effects of dobutamine or dopexamine in the treat- redox-sensitive gene regulation. The differences in
ment of septic shock and SIRS. the biology of these radicals reflected by their con-
trasting functions are presented. Summarising all of
the above mechanisms is very important in the regu-
Summary lation of inflammation. Therefore coordinated phar-
macological interventions, which would modify dif-
Inflammation is a protective response of the ferent parallel pathways in the inflammatory cas-
macroorganism to injury caused by trauma, noxious cade, are needed to treat inflammatory diseases.
chemicals or microbiological toxins.This response is
intended to inactivate or destroy invading organ-
isms, remove irritants, and set the stage for tissue Selected readings
repair. The inflammatory response consists of
immunological and non-immunological reactions. Cirino G, Fiorucci S, Sessa WC (2003) Endothelial nitric
The latter are triggered by the release from injured oxide synthase: the Cinderella of inflammation?
tissues and migrating cells of lipid-derived autacoids, Trends Pharmacol Sci 24: 91–95
such as EICOSANOIDS or platelet-activating factor, large Coleman JW (2001) Nitric oxide in immunity and inflam-
peptides, such as INTERLEUKIN-1 and CYTOKINES, small mation. Int Immunopharmacol 1: 1397–1406
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HISTAMINE or 5-HYDROXYTRYPTAMINE. These constitute and superoxide in inflammation and immune regula-
the chemical network of the inflammatory response tion. J Physiol Pharmacol 54: 469–487
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in inflamation are discussed in this chapter.
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