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which are proteins distinct from their plasma counterparts. LMWK is present in higher concentrations

intracellularly compared with HMWK. Whereas both HMWK and LMWK can be converted to lysylbradykinin by tissue kallikrein, only HMWK is cleaved by plasma kallikrein. Kallidin itself can be

converted to bradykinin by a plasma aminopeptidase. Both kallidin and bradykinin use the same

receptors and perform similar functions, but kallidin is approximately 85% as potent as bradykinin.

Tissue kallikrein is synthesized from a preproenzyme and is converted intracellularly to tissue

Prekallikrein by enzymes that are not yet well-characterized. The secreted Prekallikrein is then

converted to tissue kallikrein extracellularly by plasmin or plasma kallikrein. The only significant

inhibitor of tissue kallikreins is α1

-proteinase inhibitor.39,191

Figure 7-11. Kinin pathway. (Modified from Proud D, Kaplan AP. Kinin formation: mechanisms and role in inflammatory

disorders. Annu Rev Immun 1988;6:49.)

Cellular Kininogenase Activity

Neutrophils, mast cells, and basophils are sources of kininogenase activity. Neutrophils produce

leukokinins by way of cathepsin D. Their role in inflammation is unclear. Bradykinin is metabolized

sequentially to the partially active eight amino acid peptide, des-Arg-bradykinin, by carboxypeptidase

N, and then to inactive five-amino acid and three-amino acid fragments by the angiotensin-converting

enzyme (ACE). ACE is the predominant enzyme to inactivate bradykinin in the pulmonary vasculature.

Arginine released as a byproduct of the carboxypeptidase N reaction may contribute further to the

modulation of inflammation by acting as a substrate for the formation of NO.39,191,195

There are three kinin receptors, of which two are well-characterized.191,193 B1 receptors are expressed

primarily on the vasculature under pathologic conditions such as tissue injury. They bind des-Argbradykinin and des-Arg-kallidin and mediate the hypotension characteristic of sepsis and pain.191,196

Both B1 and the more widely distributed B2 receptors are G protein–coupled receptors. Activation of B2

receptors stimulates IP and PLC, resulting in the accumulation of the second messengers IP3

, DAG, and

calcium. The B2 receptors are more important in mediating the effects of inflammatory kinins, such as

bradykinin and lysyl-bradykinin. These kinins induce arteriolar dilatation and mediate pain. Similar to

histamine, bradykinin increases the gaps between postcapillary venule endothelial cells leading to

increase in vascular permeability. It is a potent constrictor of bronchial, uterine, and gastrointestinal

smooth muscle, and the coronary and pulmonary vasculature.193 Activation of endothelial B2 receptors

stimulates production of NO to further enhance vasodilatation. Kinins have been implicated in

mediating the antihypertensive and cardioprotective effects of ACE inhibitors.197 ACE, in addition to

catalyzing the formation of angiotensin II from angiotensin I, promotes the hydrolysis of bradykinin to

inactive metabolites. In addition, bradykinin can modulate platelet function by stimulating endothelial

cell secretion of PGI2 and thromboxane through activation of PLA2

.191

Neuropeptides

Neuropeptides may provide a neuroendocrine link between psychological stress and inflammatory

diseases such as psoriasis and inflammatory bowel disease. Like cytokines, their actions are pleiotropic

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and redundant. Neuropeptides execute their inflammatory and immunomodulatory effect by binding to

specific G protein–coupled receptors on the surfaces of target cells, and the resultant effect may be

proinflammatory, anti-inflammatory, or both. For example, substance P mediates the hypothalamic

fever response to PGE2

induced by IL-1 and TNFα, whereas ACTH, AVP, and α-melanocyte stimulating

hormone (α-MSH) suppress it. The pituitary peptides prolactin, CRH, and AVP have been shown to

augment immune responses by enhancing TH1 activity.39,198,199

Tachykinins are important proinflammatory neuropeptides that mediate pain and vasodilatation and

promote the classic inflammatory signs of erythema and edema. Substance P stimulates monocyte and

neutrophil influx and neutrophil phagocytosis. Its inflammatory effects appear to be mediated by the

proinflammatory cytokines TNFα and IL-1 from mast cells, monocytes, macrophages, bone marrow, and

endothelial cells. During allergic inflammation, substance P stimulates histamine release from mast

cells. As an effector of immune function, substance P promotes T-cell proliferation and antibody

production. Substance P released locally by nerve terminals is important in mediating the perception of

pain.39,191

CRH induces the release of IL-1, IL-6, and superoxide anion from macrophages and negatively

regulates its own proinflammatory effects through cortisol release. Cortisol downregulates production

of proinflammatory cytokines such as IL-1, TNFα, and IL-2, metalloproteinases, and iNOS, and through

this negative feedback loop, inhibits the production of CRH, ACTH, and AVP. AVP, growth hormone,

and prolactin are other important proinflammatory neuropeptides.39,198,199

Vasoactive intestinal peptide (VIP) and its homologues display both proinflammatory and antiinflammatory effects. VIP is widely distributed throughout the central and peripheral nervous systems

and serves as a chemoattractant for macrophages, neutrophils, and T cells, and may play an important

role in granulomatous reactions. VIP stimulates the release of histamine and IL-5 and is a potent

vasodilator. It inhibits IL-6, TNFα, and IL-12 release and iNOS expression in activated macrophages. VIP

has also been shown to stimulate the production of the anti-inflammatory cytokine IL-10 by

macrophages and to inhibit T lymphocyte proliferation and the production IL-2 and IFN-γ.200

Somatostatin and α-MSH are primarily anti-inflammatory in action. Somatostatin, which colocalizes

with substance P in sensory nerves, inhibits IgE formation and NK cell activity, whereas α-MSH inhibits

leukocyte chemotaxis, IFN-γ production, and downregulates TH1 activity. ACTH, calcitonin, and β

endorphin are other neuropeptides with predominantly anti-inflammatory properties.39

Calcitonin gene-related peptide (CGRP) is an immunomodulator that inhibits the activity of T cells

and macrophages, in part through the induction of IL-10. It also is an inhibitor of antigen presentation.

CGRP promotes vasodilatation and neutrophil influx, and synergizes with bradykinin and histamine to

promote edema formation.

Nitric Oxide

Endogenous NO was first discovered in 1987, and NO was the first gaseous molecule shown to be

synthesized for the purpose of cell signaling.201 NO is a weakly reactive radical that diffuses short

distances from cell to cell independent of membrane channels or receptors. Its half-life is short because

of its rapid inactivation by hemoglobin and other endogenous substances; thus, it functions primarily in

a paracrine and autocrine fashion. The enzyme NO synthase (NOS) catalyzes the formation of NO and

citrulline from the substrates L-arginine and oxygen.53,202–204 NOS contains prosthetic groups for flavinadenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, iron protoporphyrin IX, and zinc.

Three isoforms of NOS have been identified. The calcium-dependent constitutive isoforms, neuronal

NOS (nNOS) and endothelial NOS (eNOS), generate the small amounts of NO necessary for those

processes maintaining physiologic homeostasis, such as neurotransmission and endothelial regulation of

vascular tone. The expression of inducible NOS (iNOS), however, requires stimulation and produces

larger, sustained amounts of NO that possess both cytoprotective and cytotoxic properties. This

distinction is not absolute, as certain cell populations express low basal levels of iNOS, and constitutive

NOS transcripts can be enhanced by certain stimuli such as shear stress and hypoxia.53,202–204

Many of the physiologic effects of NO are mediated by the activation of soluble guanylate cyclase.

Increased levels of intracellular cyclic guanosine monophosphate trigger a reduction in calcium

concentration and promote vascular smooth muscle relaxation and the inhibition of platelet aggregation

and adhesion. The cellular response to NO also likely involves multiple signal transduction mechanisms,

including the MAPK pathway.

Inflammation secondary to endotoxemia, hemorrhagic shock, and ischemia/reperfusion are associated

with increased NO production by iNOS. First described in macrophages, iNOS can be expressed in

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essentially any cell type in response to immunologic stimuli. Unlike nNOS and eNOS, iNOS does not

depend on elevations in intracellular calcium levels for its activity.53,202–204 Important inducers of iNOS

upregulation include LPS, IL-1, TNFα, and IFN-γ. Expression is primarily transcriptionally regulated,

although stabilization of iNOS mRNA also appears to play a role. IFN-γ stabilizes iNOS mRNA, whereas

TGFβ can destabilize it. Transcription of the iNOS gene is controlled by NFκB, IFNγ-responsive element,

and TNF-responsive element. Induction of iNOS can be inhibited by glucocorticoids, thrombin,

macrophage deactivation factor, PDGF, IL-4, IL-8, IL-10, and IL-13.53,202–204 Dexamethasone may inhibit

iNOS induction by impairing the DNA binding capacity of NFκB and by increasing levels of IκB.205,206

The endothelial dysfunction and vascular hyporeactivity that characterizes septic shock is

consequential, in part, to iNOS production of NO.207 NO has been shown to be the effective mediator of

the negative myocardial inotropy of TNFα, IL-6, and IL-2 and the TNFα-induced vasodilatation in the

systemic and microcirculations.208 NO may indirectly increase prostaglandin production by increasing

the catalytic activity of cyclooxygenase and decrease LT production by inhibiting 5-LO.209 NO plays an

autoregulatory role in the TH1 subset of TH cells by limiting their own proliferation.210

NO can mediate tissue injury in inflammation by modulating organ perfusion, mediating interactions

with neutrophils, contributing to proinflammatory signaling, and by regulating apoptosis.211 Whereas

eNOS primarily regulates perfusion during homeostasis, both eNOS and iNOS modulate organ flow in

pathophysiologic states. Basal NO production from eNOS prevents the adherence of neutrophils to the

endothelium and inhibits chemotaxis under physiologic conditions. Animal studies have demonstrated

that pharmacologic inhibition of iNOS or genetic deletion of iNOS attenuates neutrophil accumulation in

organs after ischemia/reperfusion injury.212 Conversely, similar experiments in endotoxemia implicate

an antiadhesive role for iNOS, suggesting that the effect of induced NO on neutrophil accumulation is

insult specific.211 Activated neutrophils can be stimulated by fMLP, PAF, LTB4

to produce NO. NO

produced by neutrophils at sites of inflammation can combine with superoxide to form peroxynitrite as

another means of effecting toxicity.39,213

The reaction of NO with superoxide is the only reaction that outcompetes the reaction of superoxide

with superoxide dismutase. Small amounts of peroxynitrite are produced under basal conditions from

constitutively produced NO and superoxide from mitochondria and other cellular sources. However,

endogenous antioxidants such as GSH, vitamins E and C, and superoxide dismutase likely limit its

toxicity. A low concentration of peroxynitrite has been shown to inhibit neutrophil adhesion. Higher

concentrations of peroxynitrite can initiate a wide range of toxic oxidative reactions through a

peroxynitrous acid intermediate. These include the initiation of tyrosine nitration, lipid peroxidation,

and direct inhibition of mitochondrial respiratory enzymes. The balance between superoxide and NO

determines the reactivity of peroxynitrite; excess NO reduces the oxidation elicited by peroxynitrite. In

addition, peroxynitrite may contribute to cytotoxicity by a more indirect pathway. Peroxynitriteinduced single strand breaks in DNA activate the nuclear enzyme poly (ADP-ribose) synthetase, leading

eventually to irreversible energy depletion of the cells and necrotic-type cell death.39,41

Inducible NOS plays a key role in host defense, with NO or peroxynitrite exhibiting potent

antimicrobial activity against a number of pathogens including viruses, fungi, and bacteria. Although

microbicidal susceptibility to NO-mediated killing can vary considerably between species, essential roles

have been identified in tuberculosis and bacterial peritonitis.214 Induced NO has been shown to be

essential for the upregulation of the inflammatory response in hemorrhage shock and other

inflammatory processes. NO produced by iNOS leads to the activation of NFκB.215 This is followed by

the induction of proinflammatory cytokines and increased leukocyte recruitment and activation.

NO possesses both proapoptotic and antiapoptotic effects depending upon the circumstances. NO

derived from eNOS may inhibit apoptosis.216 Proapoptotic effects appear to be associated with

pathophysiologic conditions in which iNOS is upregulated. Low concentrations of peroxynitrite have

also been shown to induce apoptosis, whereas higher concentrations promote cell necrosis in vitro. The

role of NO-mediated apoptosis in the regulation of the inflammatory response is yet to be more clearly

defined.

In summary, NO mediates tissue injury both directly through the formation of peroxynitrite, as well

as, indirectly through the amplification of the inflammatory process. Like many mediators, NO has dual

regulatory functions, and it is therefore difficult to characterize NO as distinctly proinflammatory or

anti-inflammatory. In general, basal levels of NO produced by constitutive NOS may confer antiinflammatory effects, whereas induced NO may tend to promote the upregulation of the inflammatory

response. It is likely that an optimal level of NO is necessary in host defense; too little NO may be as

harmful as too much.

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