672 Journal of Leukocyte Biology Volume 56, December 1994

Neutrophils, host defense, and inflammation: a double-edged

swordJohn A. SmithDivision of Biochemistry & Molecular Biology, School of Life Sciences, Faculty of Science, Australian National University,

Canberra, Australia

Abstract: Neutrophils play important roles in host

defense against all classes of infectious agents but, para-

doxically, they are also involved in the pathology of van-

ous inflammatory conditions. Their microbicidal armory

consists of oxidative and nonoxidative processes that are

activated simultaneously upon phagocytosis. Althoughdestruction of infectious agents occurs intracellulanly,

release of cytotoxic molecules into the extracellulan milieu

can damage body tissues. Neutrophils are heterogeneous.

Subpopulations exist in various stages from dormant toprimed to fully activated. The activities of neutrophils

are regulated locally in microenvironments and systemi-

cally by a plethora of mediators including cytokines,

“classical” neuroendocrine hormones, and bioactive

lipids. The net response depends on a complex balance of

stimulatory and inhibitory pathways that are regulated

by these mediators. Although some effector and regula-

tory pathways are vital, considerable redundancy is alsoevident. Identification of the essential mediators and the

unraveling of any interactions may be the keys to under-standing the neutrophil paradox and developing then-

apeutic strategies that optimize microbial killing and

minimize host tissue damage. Finally, reports that neu-trophils can act as drug delivery vectors and that their

function is influenced by stress and other lifestyle factors

suggest that new homeostatic functions for these cells,

outside their traditional roles in host defense and inflam-mation, remain to be identified: some are speculated onhere.J. Leukoc. Biol. 56: 672-686; 1994.

Key Words: cytokines free radicals hormones . infection. immunoregulation . inflammation . phagocytes

INTRODUCTION

The human immune system consists of a complex network

of interacting cells and humoral factors. The intricate coun-

terregulation and apparent redundancies ofthe immune sys-

tern also apply to the functions of neutnophils, which are also

known as polymorphonuclear leukocytes [1]. Because neu-

trophils are abundant in the circulation, they are readily ac-

cessible to experimental investigation. Indeed, the cytokine

revolution, the emergence of neuroimmunoendocrinology,

and the discovery that phagocytes produce reactive nitrogen

species (RNS) show that although our understanding of neu-

trophil function has increased substantially, we still have a

long way to go. One paradoxical aspect of leukocyte biology

is that while neutrophils are essential for host defense, they

are also involved in the pathology of various inflammatory

conditions. The aim of this brief review is to examine this

neutrophil paradox. Where possible, human studies are exa-

mined. Emphasis is placed on analyzing the microbicidal ar-

mory of these cells and the mechanisms that counterregulate

various neutnophil functions and can potentially contribute

to dysnegulation. The influence of stress and other lifestyle

factors is also mentioned. The review concludes with some

speculation on novel homeostatic functions for neutrophils,

outside their traditional roles in host defense and inflamma-

tion, and suggestions for future work.Neutnophils represent 50 to 60% of the total circulating

leukocytes and constitute the “first line ofdefense” against in-

fectious agents or “nonself” substances that penetrate the

body’s physical barriers. Once an inflammatory response is

initiated, neutrophils are the first cells to be recruited to sites

of infection or injury [2]. Their targets include bacteria,

fungi, protozoa, viruses, virally infected cells, and tumor

cells [3]. Evasion of neutnophil defenses may provide a “win-

dow of opportunity” for local infections to be established un-

less the infectious agent is rendered harmless by interaction

with memory components such as neutralizing antibodies

(e.g., immunoglobulin A or G) already present.

Neutnophil microbicidal processes consist ofthe formation

of a combination of reactive oxygen (and, possibly, nitrogen)

species and various hydrolytic enzymes and antimicrobial

polypeptides. This broad-spectrum arsenal can be

influenced-both positively and negatively-by a wide van-

ety of mediators, which include cytokines, neuroendocrine

factors, and bioactive lipids. Subpopulations of neutrophils

have been identified by various criteria [4]. These cells exist

not only in dormant (nesting) or activated states but also invarious intermediate stages. For example, priming is a

mechanism whereby dormant neutrophils acquire a state of

preactivation that enables a more powerful response to be

generated once microbicidal activity is initiated (activated).

Neutrophils also interact reciprocally with other cells (e.g., T

cells, endothelial cells, and platelets) through either direct

cell-to-cell contact or humoral mediators; they even cooper-ate in the synthesis of eicosaniods [5, 6]. Although neu-

trophils are thought, classically, to be effector cells, they also

synthesize and secrete humoral mediators such as cytokines

that may play a role in regulating the afferent limb of im-

mune or inflammatory responses [7�.

Abbreviations: AIDS, acquired immunodeficiency syndrome; G-CSF,

granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage

colony-stimulating factor; HIV, human immunodeficiency virus; H202,

hydrogen peroxide; HOd, hypochlorous acid; IL, interleukin; MPO, my-

eloperoxidase; NO, nitric oxide; PAF, platelet-activating factor; RNS, reac-

tive nitrogen species; ROS, reactive oxygen species; SERPIN, serine pro-

tease inhibitor; SOD, superoxide dismutase; TNF, tumor necrosis factor.

Reprint requests: John A. Smith, National Quality of Life Foundation,

Department of Physiology and Applied Nutrition, Australian Institute of

Sport, P0 Box 176, Belconnen, ACT, 2616, Australia.

Received July 27, 1994; accepted July 27, 1994.

Smith Neutrophils, host defense, and inflammation 673

BIOLOGY OF NEUTROPHILS

Neutrophils are terminally differentiated cells rich in

cytoplasmic granules and contain a lobulated chromatin-

dense nucleus with no nucleolus. Four types of cytosolic

granules have been characterized; these granules contain

various receptors, enzyme components, and antimicrobial

proteins [8]. Neutrophils mature in the bone marrow before

being released into the circulation, where they spend only 4

to 10 h before marginating and entering tissue pools, where

they survive for 1 to 2 days. Cells of the circulating and mar-

ginated pools can exchange with each other, but little is

known about the size or fate of the individual tissue pools

[9]. Senescent neutrophils are thought to undergo apoptosis

( programmed cell death) prior to removal by macrophages

[10]. This stops disintegration in vivo, which would other-

wise expel their cytotoxic contents into the extracellular

milieu; this process may also play a role in terminating

inflammatory responses [10].

Neutrophils are produced in human bone marrow at the

rate of 10” cells per day [11]. This is controlled by two

colony-stimulating factors (CSFs) [i.e., granulocyte (G-CSF)

and granulocyte-macrophage (GM-CSF)] that direct the

production and differentiation of bone marrow progenitorcells. The rate of neutrophil differentiation can increase as

much as 10-fold during states of stress and infection [11].

CSFs also amplify the activities of various neutrophil func-

tions in vitro [12]. It is unclear why neutrophils are turned

over so rapidly in healthy subjects, but it may be related to

their role in immunosurveillance and/or nonimmunological

roles in maintaining homeostasis.

During the inflammatory response, chemotactic factors

generated by infectious agents themselves, as well as those

released as a result of their initial contact with phagocytes

and other components of the immune system, signal the

recruitment of additional neutrophils to sites of infectionand/or injury. Under normal conditions, neutrophils roll

along microvascular walls via low-affinity interactions of

selectins with specific endothelial carbohydrate ligands. Acti-vation of neutrophil /32-integnins and subsequent high-

affinity binding to intracellular adhesion molecules on the

surfaces ofactivated endothelial cells in postcapillary venules

is the first step in transmigration to sites of infection [13).

Under the influence of a chemotactic gradient, generated lo-

cally and by diffusion of chemoattractants from the infection

site, neutrophils penetrate the endothelial layer and migrate

through connective tissue to sites of infection (diapedesis),

where they finally congregate and adhere to extracellular

matrix components such as laminin and fibronectin [13, 14].

A wide variety of adhesion molecules have been character-

ized on the surface of phagocytic cells [13].

NEUTROPHILS AND HOST DEFENSE

Humans are exposed to thousands of microorganisms and

harbor many potential pathogens on the skin and mucosal

surfaces, but healthy people seldom develop serious infec-

tions. Neutrophils are essential for host defense. These cells

form part of the natural (nonspecific) immune system. Their

major role is to phagocytose and destroy infectious agents

but they also limit the growth of some microbes, thereby

buying time for adaptive (specific) immunological responses

to develop [15]. With many microbes, however, neutrophil

defenses are ineffective in the absence of opsonins and vari-

ous agents that amplify the cytotoxic response. This empha-

sizes the cooperative interaction between natural and adap-tive components of the immune system.

During phagocytosis of most microorganisms, cytosolic

granules fuse with the invaginating plasma membrane to

form a phagolysosome into which they release their contents,

thereby creating a highly toxic microenvironment (Fig. 1).

This normally prevents release of the components of theirmicrobicidal armoury into the extracellular milieu.

However, some targets may be too large to be fully phagocy-

tosed or they avoid engulfment, resulting in “frustrated”

phagocytosis in which no phagosome is formed. These may

be killed extracellularly [16]. However, tissue damage occurs

when neutrophil microbicidal products are released extracel-lulanly to such an extent that host defenses (antioxidant and

antiprotease screens) in the immediate vicinity are over-

whelmed [17].

The importance of neutrophils in host defense is illus-

trated by deficiencies in number and/or function. Neutrope-

nia induced by chemotherapy or rare inherited defects in

neutrophil function such as deficiencies in cytotoxic oxida-

tive metabolism (e.g. , chronic granulomatous disease),

specific granules, and adhesion molecules are substantial risk

factors for developing potentially fatal bacterial and fungal

infections [18]. Gram-negative bacilli and Staphylococcus infec-

tions are the most common bacterial infections in neutro-

penic people [19]. Neutrophils also determine susceptibilityto opportunistic fungi and pathogenesis may be related to

their resistance to the neutrophil’s microbicidal armory [20].

Commensal microorganisms such as Candida albicans become

pathogenic in people with neutrophil dysfunction. Bacterial

virulence can be influenced by the ability of bacteria to resist

phagocytosis (e.g., mucoid cell wall, formation ofcolonies) or

secrete agents that inhibit neutrophil cytotoxicity [21]. Other

immune processes may counter this, but chronic infections

do occur (e.g., Pseudomonas aeruginosa in people with cystic

fibrosis [21]). However, neutrophil killing capacity against

some bacterial, protozoan, and fungal pathogens can be

boosted substantially by cytokines and other humoral media-tons [22-24). This is discussed in greater detail under regula-

tion of neutrophil function. This type of therapy is the sub-

ject of ongoing clinical trials.

Although the importance of neutrophils in fighting bac-

tenial and fungal infections is well recognized, only limited

attention has been paid to their involvement in viral infec-

lions. This is surprising considering that neutrophils are

found in abundance in virally induced lesions [3, 25]. Neu-

trophils appear to be the primary cells responsible for protec-

tion against the influenza virus during the initial stage of in-

fection in mice [26], and they appear to play an important

role in diminishing the severity of vaccinia and herpes infec-tions [27]. Neutrophils bind to opsonized viruses and virally

infected cells via antibody (Fc) and complement (C3b) recep-

tors [3]. The rate of vinion uptake is increased following

cytokine-induced priming [25]. Viruses such as influenza

can be inactivated by neutrophils through damage to viral

proteins (e.g., hemagglutinin and neuraminidase) mediated

by the myeloperoxidase (MPO) released during degranula-

tion [28]. In contrast to these acute diseases, chronic

influenza infections can diminish or exhaust the microbicidal

potency of neutrophils [29). The systemic depression of neu-

trophil activity could lead to secondary bacterial infections

but the presence of priming agents such as cytokines (actingeither locally or systemically) may prevent this [30].

The rapid changes that occur in the antigenic deter-

minants of some viruses such as influenza suggest that

nonspecific defenses (e.g., neutrophils and natural killer

674 Journal of Leukocyte Biology Volume 56, December 1994

NEUTROPHIL

1 MICROBE

PHAGOCYTOSIS

KILLING

Fig. 1. l’hagocvtosis and bacterial killing. This diagram shows that binding of an opsonized microbe to the neutrophil membrane initiates the phagocytic

� lnvagination of the portion of the membrane containing the microbe leads to the formation of the phagosome. Fusion and release of granule con-tents into the phagosome create a highly microbicidal environment (phagolysosome) where killing and degradation takes place via a combination of oxida-

live ansi flOflOXi(lat se processes. See text for full details.

cells) may, out of necessity, play an essential role in combat-

ing these inkctions [29]. The ability ofneutrophils to destroy

human immunodeficiency virus (HIV) [31] may explain why

many HIV-inf#{232}cted people do not develop symptoms of ac-

quired immunodeficiency syndrome (AIDS) lbr many years.

Compared to healthy controls, neutrophil phagocytic and

oxidative burst activities are higher in people with stage 1

(asymptomatic) HIV infection 1321. Suppressed neutrophil

functions are Ibund in patients with AIDS or AIDS-related

complications [33]. Interferon--)’ �34] or GM-CSF/G-CSF

[35] treatment primes the antibody-dependent cytotoxicity

of neutrophils against HIV-infccted lymphocytes in vitro.

NEUTROPHILS AND HOST TISSUE DAMAGE

Although neutrophils arc essential to host defense, they have

also been implicated in the pathology of many chronic

inflammatory conditions 1171 and ischemia-reperfusion in-

jury [361. Hydrolytic enzymes of’ neutrophil origin and ox-idatively inactivated protease inhibitors can be detected in

fluids isolated from inflammatory sites [17]. Under normal

conditions, neutrophils can migrate to sites of’ infection

without damaging host tissues. Some neutrophil secretory

processes are activated during this process (e.g. , to enable the

expression of adherence molecules and to activate signaling

responses to chemoattractants) [8]. However, the same secre-tory responses are also connected with the activation of

microbicidal activity.Host tissue damage may occur through several indepen-

dent mechanisms. These include premature activation dur-

ing migration, extracellular release of microbicidal products

during the killing of some microbes, removal of infected or

damaged host cells and debris as a first step in tissue

remodeling, or failure to terminate acute inflammatory

responses. Ischemia-reperfusion injury is associated with an

influx of neutrophils into the affected tissue and subsequent

activation; this may be triggered by substances released fromdamaged host cells or as a consequence of superoxide gener-

at ion through xanthine oxidase [36, 37]. Superoxide also sig-

nals recruitment of additional neutrophils to the affected site

and ischemia may be sustained by plugging of capillaries

with aggregates of activated neutrophils [36, 37].

Neutrophils have been implicated in the pathology of

many diseases. Chronic Pseudomonas infection in the lung

leads to the destruction of protease inhibitors through oxida-

tive inactivation; the large release of neutrophil proteases(activated by bacterial phenazine pigments) destroys lung

Smith Neutrophils, host defense, and inflammation 675

tissue [38]. Activation of neutrophils by immune complexes

in synovial fluid contributes to the pathology of rheumatoid

arthritis [39]. Chronic activation of neutrophils may also in-

itiate tumor development because some reactive oxygen spe-

cies (ROS) generated by neutrophils damage DNA in vitro[40] and proteases promote tumor cell migration [41]. Chlo-

rinated oxidants, in particular, have been shown to cause tis-

sue damage [17]. Oxidants of neutrophil origin have also

been shown to cause red blood cell damage and destruction

in vivo [42] and to oxidize low-density lipoproteins in vitro;

uptake of these damaged lipoproteins by macrophages isthought to initiate atherosclerosis [43].

A good example ofthe neutrophil paradox is found in peo-ple with the adult respiratory distress syndrome. Neutrophils

have been implicated in the pathology of this condition be-

cause of the large influx of these cells into the lung and the

associated tissue damage caused by oxidants and hydrolyticenzymes released from activated neutrophils. The impair-

ment of neutrophil microbicidal activity that occurs as this

condition worsens may be a protective response on the part

of the host, which is induced locally by inflammatory

products [44]. This “down-regulation” of neutrophil function

may explain why many of these patients eventually die fromoverwhelming pulmonary infections [44]. The acute phase of

thermal injury is also associated with neutrophil activation,

and this is followed by a general impairment in various neu-

trophil functions [45].

NEUTROPHIL SUBPOPULATIONS

Peripheral blood neutrophils have been shown by many

criteria to be heterogeneous. The majority of these so-called

subpopulations have been identified on the basis of cell sur-

face markers, but there are reported examples of functional

heterogeneity within the neutrophil population [4]. The

physiological significance of neutrophil heterogeneity is not

understood, but exercise, infection, and stress alter the distri-

butions of subpopulations in the circulation. Under normal

conditions, blood may contain a mixture of normal, primed,

activated, and spent neutrophils. In fact, not all neutrophilsare phagocytically and oxidatively active. Single-cell assess-

ment of chemiluminescence in neutrophils isolated from

healthy human subjects showed that although 80%

responded to phorbol myristate acetate, an activator of pro-

tein kinase C, only 30% were activated by the particulate

stimulus opsonized zymosan [46]. GM-CSF treatment invitro also increases the percentage of neutrophils responsive

to stimulation with chemotactic N-formylated peptides [47].

A subpopulation of neutrophils with an enhanced oxidative

burst has been detected in the blood of people with an acute

bacterial infection [48] and patients with the adult respira-

tory distress syndrome [49]. Our recent work showed that

moderate exercise increased the percentage of circulating

neutrophils in men that were highly responsive to phorbol

myristate acetate stimulation in vitro U.A. Smith et al., un-

published data). Neutrophils isolated from patients with

blunt trauma show reduced binding of the 31D8 antibody,

which correlates with the increased susceptibility of these

people to infection [50]. These “dull 31D8” neutrophils that

dilute out the circulating population are normally located in

the bone marrow and are probably immature and function-

ally inactive [51]. In patients suffering from severe burns, a

strong correlation has been established between the onset of

bacteremic infection and reductions in the proportion andabsolute numbers of neutrophils positive for antibody and

complement receptors [52].

NEUTROPHIL MICROBICIDAL MECHANISMS

Neutrophil microbicidal mechanisms consist of a combina-

tion of oxidative and enzymatic (oxygen-independent)

processes that appear to be activated simultaneously upon

initiation of phagocytosis (Fig. 1). Phagocytosis is triggered

upon the binding of opsonized microorganisms through op-

sonin receptors (for complement fragments and antibodies)

or through nonspecific glycoslyated receptors that recognize

certain lectins on target microorganisms. Two microbicidal

processes are activated concomitantly with phagocytosis: (1)

the oxidative burst, so called because of the 50- to 100-fold

increase in 02 consumption, which results in the production

of cytotoxic reactive oxygen species (ROS) and, possibly,

through a different mechanism, reactive nitrogen species

(RNS), and (2) degranulation, which corresponds to the

release of contents of azurophilic (primary) and specific

(secondary) granules into the phagosome to form a

phagolysosome. These processes involve rearrangement of

contractile proteins, triggered by Ca2�, which leads to inter-

nalization of the portion of plasma membrane that contains

the receptor-target complex [53]. The following discussion

also illustrates the apparent redundancy of the neutrophil

microbicidal arsenal.

Oxidative mechanisms

Reactive oxygen species

The oxidative or respiratory burst in neutrophils is triggered

upon phagocytosis or when the pathway is activated by an

appropriate synthetic stimulus in vitro. The oxidative burst

results in the sequential production of a variety of microbio-

static and microbicidal ROS (Fig. 1). Superoxide (02) is

formed, initially, by the reduction of molecular oxygen by

single electrons that originate from NADPH generated via

the oxidative segment of the pentose phosphate pathway.

This process is catalyzed by the combined action ofa plasma

membrane NADPH oxidase and cytochrome b558 (with an

redox potential of - 245 mV), which appears to be the termi-

nal electron acceptor of a short electron transport chain thatconveys single electrons from NADPH to oxygen [54]. Two

cytosolic proteins (p47P�i0X, �fi7Phox), a quinone, and an Rac-

related GTP-binding protein are thought to be the other

functional components ofthis electron transport system [55].

The NADPH oxidase system is dissociated and thus inactive

in dormant neutrophils. While some components are mem-

brane bound, others are stored in the cytosolic granules.

Upon activation, the cytosolic components translocate to the

plasma membrane to assemble the active oxidase [54]. The

importance of the oxidative burst in the microbicidal activity

of neutrophils is shown in people with severe impairments in

this pathway (e.g., chronic granulomatous disease). They

suffer from repeated infections that respond poorly to con-

ventional therapy and almost invariably lead to early death;

treatment with interferon-y shows promise [56].

Although 02 may contribute to microbial killing, other

more potent ROS are generated rapidly from this precursor.

Hydrogen peroxide (H202), is formed by spontaneous dis-

mutation and/or the catalytic action of superoxide dismutase(SOD). MPO-dependent oxyhalides such as hypochlorous

acid (HOC1) are generated by the reaction of H202 with the

abundant C1 ions taken up from extracellular fluid; secon-

dary chlorinated amines are generated by the reaction of

HOC1 with nitrogen-containing compounds [17]. The per-

centage of H202 converted to HOC1 varies from 30 to 70%,

depending on the experimental system used 1161. Various

676 Journal of Leukocyte Biology Volume 56, December 1994

groups have claimed that the hydroxyl radical (OH.),

formed by the Fe2�-catalyzed decomposition of H202, and

singlet oxygen (102) are also generated by neutrophils.

There is considerable debate, however, as to whether these

ROS are produced under physiological conditions. Lactofer-rin may, in fact, prevent Fe3� from being used as a Fenton

catalyst and the bulk ofthe H202 may be converted to HOCI

[17]. The importance of MPO oxyhalides in neutrophil

microbicidal activity, however, is poorly understood, because

MPO deficiency has been reported to have little clinical

significance, although some affected people may suffer fromsevere Candida infections [16]. Although neutrophils deficient

in MPO can kill most microbes, they do so at a greater meta-

bolic cost than MPO-rich cells by prolonging H202 produc-

tion, possibly because HOCI is 100 to 1000 times more effec-

tive than H202 [57]. Furthermore, HOC1-induced cell death

occurs very rapidly in comparison to that mediated by H202[58]. However, sustained H202 production could also in-

crease the risk of host tissue damage and perhaps activate

inflammatory cascades, particularly if the generation of

OH . compensates for MPO deficiency. Thus, NADPH oxi-dase is essential for neutrophil microbicidal activity, but the

MPO arm is probably reinforced by other microbicidal

molecules or redundant processes.

Although critical to neutrophil antimicrobial function, the

exact mechanisms by which different oxidants contribute to

microbial killing have not yet been elucidated [59]. Bacterial

growth may be arrested by the inhibition of DNA synthesis

[59], but killing may involve multiple hits on essential

microbial constituents with unprotected functional groups

and/or fatal disruption of metabolic homeostasis [16]. The

exact targets, which include proteins, lipids, and nucleic

acids, may vary considerably between different species of

microorganisms. The nonoxidative processes also contribute

to varying degrees (see nonoxid#{225}tive mechanisms). Chiori-

nated oxidants are thought to be an important component of

defense against protozoa, fungi [23], and some viruses [28].

Microbial virulence may be related to the capacity to detox-

ify neutrophil ROS. Many microorganisms can catalytically

detoxify 02 and H2O2 but not HOCI [57].

Neutrophil-generated ROS also influence some cellular

functions. Superoxide and H2O2 may augment phagocytosis

independently of the MPO-halide system, whereas chlori-nated oxidants may limit this process by oxidatively mac-

tivating opsonin receptors [60]. Oxidants also promote the

margination of neutrophils by triggering the expression of

adhesion molecules on endothelial cells [61]. The mechan-

isms by which the oxidative burst is terminated are not

known, but inactivation of the oxidase may occur when acti-vation factors are consumed or when the enzyme is macti-

vated by oxidants and hydrolytic enzymes released by the

neutrophil or as a result of specific dephosphorylation reac-

tions [62].

Neutrophils contain large reserves of endogenous antioxi-

dants such as glutathione and ascorbate [57, 63]. Their abil-

ity to maintain these antioxidants in the reduced state during

phagocytosis [63] may prevent death from oxidative suicide.A substantial proportion of activated neutrophils are not des-

troyed during the killing process, but a significant refractory

period must elapse before these cells can be reactivated with

a secondary stimulus [64]. The antioxidant protection of

host tissues and fluids also plays a role in preventing

neutrophil-mediated destruction.

Reactive nitrogen species

Like macrophages, neutrophils appear to produce reactive

nitrogen species (RNS). The pathway is an oxidative process

in which short-lived nitric oxide (NO ) is derived from the

guanidino nitrogen in the conversion of L-arginine to L-

citrulline. This reaction is catalysed by N0 synthase and-

like the oxidative burst-it involves 02 uptake [65]. The con-

stitutive form of N0 synthase has been purified from hu-

man neutrophils [66]. Caution is needed in interpreting

many of these studies, as the measurements may not reflect

RNS generation through the N0 synthase pathway. Most

reports of phagocyte production of RNS involve indirect or

nonspecific methods for measuring the presence of N0

such as nitrite and L-citrulline accumulation or

chemiluminescence. Nitrosylation reactions with thiol-

containing compounds and proteins, particularly

glutathione, or heme iron also consume N0 [67]. Further-

more, doubt has been cast on the authenticity of RNS

production assessed by arginine analogues because these in-

hibitors of NO synthase have also been reported to block the

activities of heme-containing enzymes [68]. Nonspecificityhas also been a problem in the measurement of ROS in neu-

trophils [69]. In both cases, controversy may be avoided if

two or more independent techniques are used.

Independent pathways are involved in the synthesis of

ROS and RNS [65]. Dormant neutrophils incubated at

37#{176}Cproduce N0 continuously but activation arrests this

pathway in favor of the oxidative burst [70]. Neutrophil

migration is mediated by N0 because N0 synthase inhi-

bitors attenuate neutrophil responses to chemotactic gra-

dients in vitro [71], and it also interferes with assembly of

NADPH oxidase in activated neutrophils [72]. Thus,

although the ROS and RNS pathways are independent, they

may compete for common substrates such as NADPH and

02 and exert other modulating effects on each other. Thesteady-state production of these species may dictate the anti-

/proinflammatory balance. This may be controlled by the

level of neutrophil degranulation because heme enzymes

such as MPO scavenge N0 [68]. Various pathways deter-

mine the ultimate fate of N0 , which appears to be anti-

inflammatory itself.

Nitric oxide may contribute to the microbicidal activity of

neutrophils by reacting with ROS to form secondary cyto-

toxic species such as peroxynitrite (O0N02) [73]. For ex-ample, RNS appear to contribute to the killing of

Staphylococcus aureus by neutrophil cytoplasts, but not intact

cells, in vitro because the process is attenuated by M�-monomethyl arginine; this was reversed by adding L-

arginine [74]. At acidic pH, HNO3/NO2 either alone or

combined with H202, or H202 and MPO, in vitro is toxic

to Escherichia coli [75]. Therefore, microbial killing appears to

ROS dependent in normal neutrophils but RNS may play arole in cells with deficiencies in the NADPH oxidase/MPO

pathways. It would be interesting to determine whether RNS

production is greater in neutrophils isolated from people

with chronic granulomatous disease or MPO deficiency.

The main role of neutrophil-derived N0 may be to facili-

tate the migration of neutrophils from blood vessels to sur-

rounding tissues by causing vasodilation [69]. N0 facili-

tates relaxation of vascular smooth muscle, and ROS initiate

vasoconstriction through the production of O2, which re-

moves N0. Activated neutrophils have been implicated in

the development of inflammatory injury to the microvascula-

ture through the release of ROS and hydrolytic enzymes but

a shift in the balance in favor of RNS production may pre-

vent this. For example, N0 inhibits neutrophil adhesion to

vascular endothelium and this may prevent inflammatory

and ischemia-reperfusion injuries [76].

Hypertensive patients have circulating neutrophils that

are more oxidatively active than those of their normotensive

Smith Neutrophils, host defense, and inflammation 677

counterparts [77]. This suggests that some types of hyperten-

sion may be mediated by the collective failure of neutrophils

and endothelial cells to produce sufficient RNS to maintain

optimal vascular tone. Further support for this hypothesis

comes from a report suggesting that excessive formation ofNO may mediate the hypotensive effects of tumor necrosis

factor (TNF) [78]. Much more work, however, is required to

unravel the interactions between these pathways and the

physiological consequences of their cooperative and recipro-

cal activities.

Non-oxidative mechanismsNeutrophils contain an abundance of hydrolytic enzymes

and antimicrobial polypeptides. Acid hydrolases and an-

timicrobial defensins are contained within the intracellular

granules [8]. The azurophilic granules contain many proteo-

lytic and saccharolytic enzymes capable of digesting

microbial structural proteins and mucopolysaccharides,

whereas the contents of the specific granules include binding

proteins such as lactoferrin, which deprive microorganisms

of essential nutrients, and lysozyme and collagenase, which

destroy cell envelope components. Most of these proteins arepositively charged, which enhances their binding to cell surfaces.

Neutrophil hydrolytic enzymes augment microbial damage

initiated by ROS and participate in the digestion ofdead mi-

crobes and damaged host cells. Serine proteases such as

elastase and cathepsin G hydrolyze proteins in bacterial cell

envelopes and lysozyme degrades the polysaccharide compo-nents. The enzymes may also limit the spread of inflamma-

tion within local microenvironments by degrading priming

agents such as TNF-a and lymphotoxin [79]. Cathepsin G

also contributes to bacterial killing through a mechanism in-

dependent of its enzyme activity [80]. Neutrophils also acti-

vate platelets through cathepsin G [6]. Bactericidal/permeability-

increasing protein, a factor that is highly toxic to gram-

negative bacteria but not to gram-positive bacteria or fungi,

can also neutralize endotoxin, the toxic lipopolysaccharide

component of gram-negative bacterial cell walls [81].

Azurocidin is also active against gram-negative bacteria and,

to a lesser extent, gram-positive bacteria and fungi; its

microbicidal mechanism is unknown but proteolysis is not

involved [82]. Lactoferrin sequesters free iron, thereby

preventing the growth of ingested microorganisms that sur-

vive the killing process [83]. Lactoferrin also increases bac-

terial permeability to lysozyme [84]. The candidastatic ac-

tivity of neutrophils may be due to binding of Zn2� and Ca2�by the calprotectin complex of proteins [85]. Thus neu-

trophil microbiostatic and microbicidal mechanisms may be

independent to some extent.

Defensins, which constitute 30-50% of azurophilic gran-

ule protein, are small (molecular weight < 4000) potent an-

timicrobial peptides that are cytotoxic to a broad range of

bacteria, fungi, and some viruses. Their toxicity may be due

to membrane permeabilization of the target cell [86].

Limited attention has been paid to microbial DNA damage

by neutrophils, but endonuclease activity in human neu-

trophils has been reported [87]. Although neutrophils do not

degrade the DNA of phagocytosed E. colt, monocytes, in con-trast, degrade chromosomal DNA but not plasmid DNA;

this could have important implications in antibiotic

resistance [88].

Interactions between oxygen-dependent and oxygen-independent mechanisms

The oxidative and non-oxidative processes may kill some mi-

crobes independently, but the combination of the two in-

creases the microbial killing potential ofneutrophils substan-

daily. Unfortunately, the potential for host tissue damage is

also increased. In fact, cooperative interaction is vital in

many cases. Gram-negative bacteria, for example, are resis-

tant to lysozyme unless they are subjected simultaneously to

oxidants and/or complement factors [86]. Defensins and ox-

idants interact synergistically to lyse tumor cells in vitro[89]. Chlorinated oxidants also sustain the activity of some

proteases [17]. Serine proteases such as elastase constitute

part of a regulatory circuit that modulates the oxidative and

phagocytic functions of neutrophils, the activation of lym-

phocytes, and the activities of complement factors [90].

These proteases can prime the responsiveness of NADPH

oxidase to N-formylated peptides and phorbol esters by

modifying proteins present on the outer surface of the

plasma membrane, thereby increasing the lateral mobility ofmembrane lipids [90].

Hydrolytic damage to host tissue and therefore chronic

inflammatory conditions may occur only when antioxidant

and antiprotease screens are overwhelmed. Antiprotease

deficiency is thought to be responsible for the pathology ofemphysema [91]. Many antiproteases are members of the

serine protease inhibitor (SERPIN) family. Although the cir-

culation is rich in antiproteases, these large proteins may be

selectively excluded at sites of inflammation because neu-

trophils adhere tightly to their targets. Oxidative stress may

initiate tissue damage by reducing the concentration of ex-

tracellular antiproteases to below the level required to inhibit

released proteases [17]. Chlorinated oxidants and H2O2 can

inactivate antiproteases such as cr3-protease inhibitor and

a2-macroglobulin (which are endogenous inhibitors of

elastase) but, surprisingly, simultaneously activate latent

metalloproteases such as collagenases and gelatinase [17, 92],which contribute to the further inactivation of antiproteases

[93]. Many antiproteases are susceptible to oxidative attack,

probably because of exposed thiol groups. These destructive

processes may prolong the inflammatory response by trans-

forming the immediate nonspecific effects mediated by ROS

into more prolonged effects that depend on the activation ofa complex array of endogenous, and perhaps more specific,

proteolytic activities. These reactions may be regulated by

negative feedback because SERPINs such as a1-protease in-

hibitor, for example, block O2 production by activated neu-

trophils [94], possibly by inhibiting the synthesis of cytokines

and bioactive lipids [95] and/or by inhibiting NADPH oxi-dase activity by mechanisms not linked directly to their an-

tiprotease activities [94].

Priming and activation

Neutrophils exist in various states of activation, which varyfrom dormant to primed to fully activated. While activation

triggers the immediate expression of neutrophil microbicidal

activity, priming stimuli amplify the magnitude of the

response when it is activated subsequently and/or switch

cells from a nonresponsive to a responsive state. Althoughpriming and activation appear to be distinct processes, they

are biochemically integrated and require at least two events

(initiation and prolongation), which may be mediated by

separate mechanisms [96]. For example, subactivating con-

centrations of stimuli such as N-formylated peptides and

phorbol esters induce priming of the oxidative burst [90].

Stimuli at priming concentrations also regulate other neu-trophil activities; for example, N-formylated peptides at

nanomolar concentrations activate chemotaxis, whereas

micromolar concentrations arrest cell movement and trigger

microbicidal activity directly [97]. Although the distinct

678 Journal of Leukocyte Biology Volume 56, December 1994

multistep mechanisms responsible fbr the induction of’ prim-

ing and activation (and the interaction between them) arepoorly understood, several levels of involvement have been

identified. The transmembrane signaling processes involved

share salient features with those that occur in other cell

types. They begin with the binding of soluble or particulate

mediators to a complementary cell surface receptor and the

transduction of this initial signal to the effector machinery.The signal transduction processes involved in coupling

receptor-ligand binding vary according to the nature of the

stimulus.

Cell surface markers

Priming and activation are associated with changes in the ex-

pression of a variety of molecules on the cell surface. Up-

regulation of complement receptors occurs in vitro in

response to many neutrophil priming and activating agents

including cytokines, eicosanoids, and bacterial chemoattrac-

tants [98, 99] as well as in vivo in patients with thermal in-jury [45] and in response to exercise U.A. Smith et al., un-

published data). Dormant neutrophils do not express Fc�yRI

under normal conditions, but its expression can be induced

by treatment with interferon-’y [1]. In contrast, Fc�yRII and

Fc�yRIII expression decreases by 50 to 80% in activated neu-

trophils [99] and this may coincide with an increase in cir-culating forms of these receptors [100]. Selectins are also

shed from the surface of activated neutrophils [13].

Several novel neutrophil activation markers have been

described. The lymphocyte activation marker CD69 has

been reported to be expressed on the surface of the plasma

membrane of activated (but not dormant) neutrophils [101].Furthermore, CD66 and CD67, which are stored in the

specific granules, and CD63, a marker of azurophilic

degranulation, have also been reported to be neutrophil acti-

vation markers; the functions ofthese proteins are not known

[102, 103]. No marker that is exclusive to primed neutrophils

has been described yet, although expression of CD1O andbinding of the monoclonal antibody 7D5 have been reported

to be early activation markers in circulating neutrophils [104].

Signal transduction

Multiple signal transduction processes are involved in prim-

ing and activation of neutrophil microbicidal activity. Theactivation of NADPH oxidase activity has received the most

experimental attention [105]. This subject is discussed only

briefly here. At present priming and activation appear to be

biochemically inseparable. For example, primed neutrophil

oxygenation activity may be the result of an alteration to one

or more of the components of the transduction system. Thiscould include fluxes of free cations (Nat, K� and Ca2�),

changes in membrane potential, activation of intracellular

proteases, changes in arachidonic acid and phospholipid

metabolism, phosphorylation of specific proteins (i.e., oxi-

dase components), and changes in the intracellular concen-

trations of cyclic nucleotides. Small changes may triggerpriming and large changes lead to full activation including

degranulation. Furthermore, these signaling processes may

be involved in other neutrophil functions such as expression

of adhesion molecules and chemotaxis inter alia. Thus it is

difficult to characterize signaling mechanisms of a single

neutrophil function in intact cells.At present, two distinct signaling pathways of NADPH ox-

idase activation have been identified: one is Ca2� dependent

and leads to the activation of the Ca2�/phospholipid-

dependent protein kinase C; the other is Ca2� independent

and does not involve phospholipase C or protein kinase C;

both pathways must be functional, however, for activation ofthe oxidative burst [106]. Distinct pathways of activation of

a common NADPH oxidase appear to be triggered by differ-

ent classes of intracellular messengers, suggesting that the

activation pathways converge at a common intermediate

[107]. Interactions between protein kinase C, cAMP- and

cGMP-dependent protein kinases may combine to regulate

NADPH oxidase activity [107]. Some of the proximal steps

of the Ca2�-protein kinase C pathways may be bypassed in

vitro by using Ca� ionophores and/or protein kinase C activators.

Physiological examples of neutrophil priming andactivation

Various infectious and inflammatory conditions trigger the

priming response or neutrophil activation. Neutrophils iso-

lated from patients with acute bacterial infections show

primed oxidative responsiveness [48] and enhanced

antibody-mediated phagocytosis [108]. Primed neutrophils

have been found in people with essential hypertension [77],Hodgkin’s disease [109], inflammatory bowel disease [110],

psoriasis [111], sarcoidosis [112], and septicemia, where prim-

ing correlates with high concentrations of circulating TNF-a

I113]. In most cases, the mechanisms involved are not known.

Our group has shown that moderate exercise also triggers the

priming response in both trained and untrained men [114].In contrast to priming, activation is indicated by the in-

creased concentration of neutrophil granule contents found

in plasma, for example, from people with septicemia or bac-

terial meningitis [115], or after strenuous exercise [116, 117].

Elevated plasma concentrations of autoantibodies against

some neutrophil cytoplasmic components are associated withsome autoimmune diseases; as these antibodies can activate

neutrophils, they may play a role in the pathology [118],

although some may, possibly, neutralize cytotoxic neutrophil

components.

REGULATION OF NEUTROPHIL FUNCTION

The activities of immune cells such as neutrophils are or-

chestrated by a complex balance of counterregulatory (and

somewhat redundant) pathways. The immune system is not

autonomous: cellular and humoral immune activities areinfluenced by a plethora of soluble mediators secreted from

the endocrine, nervous, and cardiovascular systems as well as

by those produced by other immune cells. These mediators

include cytokines, hormones, and bioactive lipids, many of

which are secreted in response to stress. As immune cells

synthesize and release small amounts of most of these fac-tors, they have the potential to function in autocrine and

paracrine amplification networks [119, 120]. A large number

of mediators have been reported to modulate neutrophil

functions in vitro and some in vivo (Fig. 2). This list is ex-

panding rapidly. Activation of the complement cascade also

generates fragments that enhance phagocytosis (C3b),

chemotaxis, and microbicidal activity (C5a) [98, 105].

Cytokines

Many cytokines including hematopoietic growth factors and

pyrogens have been shown to be potent neutrophil primingagents in vitro [12]. Cytokines are immunoregulatory pro-

teins produced and secreted in different combinations, and

at different rates, by most immune cells, including macro-

phages, neutrophils, and lymphocytes [7, 121]. They have

powerful and multiple overlapping (pleiotropic) actions on

target cells; they can, in a concentration-dependent manner,

PAF(+) A�ATP(+)

Adenosine (-)

1�

GM-CSF (+)

G-CSF (+)

r

Interleukins 4 (+1-), 6 (+), 10 (-)

Interferon-i (+)

GM-CSF (+)

Growth Hormone (+)

TNF(+)

PAF (+)

PGE2 �

TNF (+)

PAF (+)

GM-CSF (+)

Leukotriene B4 (+) Interleukins 1,6,8 (+)

Adrenalin (-)

<SGlucocorticoids (-)

IAdenosine () Prostacyclin (-)

PAF(+)�11 GM-CSF(+)

Leukotriene B4 (+) Interleukins 6,8 (+)

Atrial Natriuretic Peptide (+)

Substance-P (+)

Interleukins 1,6,8 (+)

Smith Neutrophils, host defense, and inflammation 679

Fig. 2. Regulation of neutrophil function. This diagram illustrates the plethora of mediators that modulate various neutrophil functions and the potential

cellular sources (compiled from refs. 2, 6, 7, 12, 121, 189, 190). See text for full explanation.

amplify or diminish all responses of the immune system.

Some cytokines also interact to produce additive or synergis-

tic amplification. Neutrophils also synthesize and secrete

small amounts ofsome cytokines including interleukins (ILs)

1, 6, and 8, TNF-a, and GM-CSF; they may act in an auto-

crine or paracrine manner [7]. In fact, under normal condi-

tions, most cytokines act locally in microenvironments. They

are virtually undetectable in the circulation except under

pathological conditions.

The pyrogenic cytokines, IL-i [122], TNF-a [123], and

IL-6 [124] all prime various pathways that contribute to the

activation of NADPH oxidase. TNF-a also primes HOC1

production in these cells both in vitro and in vivo [125].

MPO release is also primed by IL-1j3 [122]. High concentra-

tions of circulating TNF-a have been detected in patients

with bacterial infections, cancer, and thermal injury [126].

IL-8, which is also known as neutrophil-activating factor, is

a potent chemoattractant and activating agent; it synergizes

with interferon-y, TNF-a, GM-CSF, and G-CSF to amplifyvarious neutrophil cytotoxic functions in vitro [127].

Cytokines also increase the microbiostatic and killing ca-

pacities of neutrophils against bacteria [22], protozoa [23],

and fungi [24]. G-CSF and interferon-rny prime the neu-

trophil oxidative burst to soluble stimuli and ingestible and

noningestible fungal forms [24]. Administration of G-CSF tohuman subjects increases neutrophil complement receptor

expression and enhances superoxide generation in stimu-

lated cells in vitro [128]. Interferon-’y and GM-CSF indepen-

dently amplify neutrophil antibody-dependent cytotoxicity

[33, 35]. These observations have physiological implications

because priming of neutrophils by nontoxic doses of TNF-aand IL-i appears to be responsible for the increased

resistance ofmice to bacterial infection [129] and for the en-

hanced candidicidal activity of human neutrophils that ac-

companies the priming of the oxidative burst [130]. Further-

more, administration of IL-i to neutropenic mice protects

them from lethal Candida albicans infection [131].The effect of the anti-inflammatory (type II) cytokines,

IL-4 and IL-lO, on neutrophils has received little experimen-

tal attention. Although IL-4 diminishes #{176}2 production in

monocytes [132], one group has reported that it increases the

neutrophil oxidative burst and bacterial killing in vitro [133].

However, IL-4 inhibits the production of IL-8 in lipopoly-saccharide-stimulated neutrophils [134] and blocks the ac-

tion of IL-i by increasing the expression of apparently non-

functional IL-i (type II) receptors [135] on the cell surface.

IL-lO inhibits the release of TNF-a, IL-1f3, and IL-8 [136]

and blocks IL-1/3 transcription [137] from lipopolysaccharide-

stimulated neutrophils.Like neutrophils, cytokines have been implicated in the

pathology of inflammatory disease. Synovial inflammation

and other manifestations of rheumatoid arthritis may be

caused by cytokines [i38]. However, although plasma TNF

is elevated in the majority of people with the adult respira-

tory distress syndrome, a direct cause-and-effect relationshipwas not established [49]. Furthermore, some cytokines

prolong neutrophil survival [139]. The acute inflammatory

response may be terminated by the secretion of macrophage

inflammatory protein-la from neutrophils; this protein may

signal mononuclear cell recruitment and clear neutrophils

from the affected tissue site [140]. Cytokine effects can be

blocked by endogenous inhibitors or carrier proteins; this in-

cludes soluble receptors shed from neutrophils and cytokine

autoantibodies that have been isolated in plasma 1141, 142].

680 Journal of Leukocyte Biology Volume 56, December i994

Pyrogenic cytokines also mediate septic shock; clinical trials

are in progress to examine the therapeutic potential of

cytokine neutralizing antibodies and soluble receptors [143].Although most neutrophil priming studies in vitro have in-

volved cell suspensions, some cytokines can stimulate the ox-

idative burst directly in adherent cells but not in cell suspen-

sions [144]. Neutrophils must adhere to matrix proteins for

cytokines to activate the oxidative burst directly; this may

prevent premature or “accidental” activation of circulating

neutrophils by cytokines [144] but without preventin.g prim-

ing. Adherence may also lower the steady-state concentration

of cytosolic cAMP, which could prepare neutrophils for the

onset of the oxidative burst by driving the concentration of

this potent inhibitor below its effective threshold [14]. Bac-

terial endotoxin (lipopolysaccharide), a potent stimulus ofpyrogenic cytokine secretion, may prime neutrophils directly

in vitro. This does not occur with cells in suspension, possi-

bly because additional serum factors are required [145]. The

stimulatory effect of lipopolysaccharide is enhanced when it

is complexed to the lipopolysaccharide-binding protein

found in the plasma of healthy humans in trace concentra-

tions [146].

Bioactive lipids

Arachidonic acid and its metabolites also modulate various

neutrophil functions. Neutrophil membranes are rich in

arachidonic acid and neutrophils are potent producers of bi-

oactive lipids. Leukotriene B4 is a strong neutrophil

chemoattractant that may play a role in the priming process

[76]. Vasoactive leukotrienes produced by neutrophils and

platelets (C4, D4, E4) increase microvascular permeability

and may contribute to ischemia-reperfusion injury [36, 76].

In contrast to leukotrienes, prostaglandins suppress most

neutrophil functions, possibly through their ability to elevate

intracellular cAMP [147].

Platelet-activating factor (PAF) is the most extensively

studied neutrophil lipid mediator. In many inflammatory

conditions, the levels of PAF rise in the affected tissues, but

injury can be attenuated by PAF antagonists [76]. PAFdirectly primes superoxide generation and elastase release

[i48]. The mechanisms by which cytokines prime neu-

trophils are not known, but PAF appears to mimic many of

the roles ascribed to TNF-a and IL-i [149, 150]. It may be

a direct second messenger for some cytokines because they

induce PAF accumulation in neutrophils and this increasesfurther upon activation [151]. The synthesis and release of

IL-I are triggered by PAF in a positive feedback manner; this

may be an important factor in the amplification of some im-

mune and inflammatory responses [149, 150]. GM-CSF also

primes PAF, arachidonic acid, and leukotriene B4 synthesis

in response to secondary stimuli [152, 153].

Neuroendocrine hormones

The major “stress hormones” are involved in immunoregula-

tion at both the systemic and, perhaps, local levels. The bi-

directional interactions of cytokines and neu rotransmitters

with neurons and immune cells, respectively, provide ameans of indirect chemical communication between the neu-

roendocrine and immune systems. The plasma concentra-

tions of these hormones fluctuate throughout the day be-

cause of pulsatile secretion and rapid metabolic clearance,

both of which are partially regulated by negative feedback

[154]. This may explain diurnal variations in various T cellsubset numbers in the circulation [155]. It is not known

whether neutrophil activities fluctuate throughout the day

under normal conditions. This may be an important variable

in many studies.

Growth hormone deficiency, which reduces the potency of

virtually all immune mechanisms, increases vulnerability to

infection [156]. Growth hormone also primes the oxidative

burst ofhuman neutrophils [157]. This is initiated by growth

hormone binding to the prolactin (and not the growth hor-

mone) receptor on neutrophils in a zinc-dependent process

[157]. The mechanisms underlying growth hormone-induced

priming have not been identified, but protein synthesis ap-

pears to be involved [157]. The growth-promoting effects of

growth hormone are mediated through insulin-like growth

factor 1, which is also a strong neutrophil-priming agent

[157]. Growth hormone synergizes with interferon-’y to re-

store the suppressed neutrophil oxidative burst and bacteri-

cidal activity in cells isolated from senescent rats; interferon-,y alone did not have any priming effect [158].

Other neuroendocrine factors have been reported to be

potent neutrophil priming agents. Prolactin-which shares

considerable functional and structural similarities with

growth hormone - is also a strong immunopotentiating

agent [i56]. Prolactin primes the oxidative burst of macro-

phages and neutrophils to the same intensity as that induced

by growth hormone [157]. Atrial natriuretic peptide has also

been reported to be a potent neutrophil-priming agent [159].

This hormone may play a role in neutrophil activation in

heart tissue during ischemia-reperfusion injury [159].

Although glucocorticoids and opioids may enhance someimmune responses at very low concentrations, they are

generally considered to be immunosuppressive [154]. These

contrasting responses may be controlled by the presence of

multiple receptors for the same mediator that are coupled to

stimulatory and inhibitory pathways. Saturation of the

stimulatory receptor may eventually trigger expression and

activity of the inhibitory receptor-linked pathway. In fact,

containment of the stress response may be the principal role

of glucocorticoids [160]. Glucocorticoids severely impair the

phagocytic and cytotoxic activities of neutrophils [161] and

their capacity to produce ROS and secrete lysozyme in

response to stimulation with chemotactic peptides in vitro[162]. Treatment of cattle with glucocorticoids inhibits the

oxidative burst, chemotaxis, and antibody-dependent

cytotoxicity [163]. Some cytokines protect against

glucocorticoid-induced impairment of neutrophil responses

to certain fungi [24]. However, glucocorticoids increase the

expression of the nonfunctional type II receptor for IL-i,

which may down-regulate the effect of this cytokine on neu-

trophils [135]. The physiological relevance of many studies is

questionable, however, because of the very high concentra-

tions of glucocorticoids used [2].

Catecholamines and opioids also suppress a variety of im-

mune activities. Epinephrine treatment of isolated cells invitro inhibits the oxidative burst of macrophages and neu-

trophils [164] and the tumoricidal and antiviral activities of

macrophages [165]. Opioid addiction increases susceptibility

to a variety of infections. This may be due to suppressed T

cell proliferation and neutrophil microbicidal activity [166].

Oxidant production by neutrophils is also inhibited by f3-

endorphin activity mediated via nonopioid receptors [167].

Other mediators

Histamine, [168], lipoxins A4 and B4 [169], and some

unidentified products from platelet lysates [170] are potent

inhibitors of neutrophil microbicidal activity. However,

platelets are immunomodulatory because activated cells can

bind to neutrophils and stimulate the oxidative burst [171],

Smith Neutrophils, host defense, and inflammation 681

possibly through the release of ATP [i72]. The interactions

between platelets and neutrophils are essential for platelets

to synthesize vasoconstrictive leukotrienes [6]. Likeprostaglandins, many immunosuppressive mediators use

cAMP as a second messenger [173]. Increased intracellular

cAMP in neutrophils is associated with decreases in a num-

ber of microbicidal functions [53]. Phagocyte priming and

activation may, in fact, be controlled by shifts in the intracel-

lular ratio ofcGMP to cAMP [173], since cGMP is stimula-

tory [174].

Adenosine provides an interesting example ofhow a single

mediator may play dual roles. Adenosine, a vasodilator, is a

potent anti-inflammatory agent released from damaged host

cells. Neutrophil chemotaxis is activated by adenosine oc-

cupancy ofA1 receptors and inhibition ofthe oxidative burst

triggered through A2 receptors [175]. Adenosine suppresses

the oxidative burst only if it is added before the triggering

agent [176], but it has no effect on the initiation or progress

ofdegranulation [175]. Circulating adenosine, at physiologi-

cal concentrations, may prevent the premature activation of

peripheral blood neutrophils by cytokines without prevent-

ing priming [176].

This discussion highlights the complexity and apparent

redundancy of the humoral mediators that regulate neu-

trophil function. Identification of people with deficiencies

will verify which mediators serve crucial roles that cannot be

replaced by substitutes. The plethora of humoral mediatorsthat influence neutrophil function means that therapeutic

strategies that are employed to correct neutrophil deficien-

cies must be carefully examined in animal models before

proceeding to human trials.

Interactions between the immune and neuroendocrinesystems

The communication between the neuroendocrine and im-

mune systems and its mediation by hormones and cytokines

is one the most active areas of current biological research

[119, 120]. Pyrogenic cytokines stimulate the hypothalamus

to secrete corticotropin-releasing factor which, in turn, acti-

yates the immunosuppressive arm of the pituitary-adrenal

axis by stimulating the secretion of adrenocorticotropic hor-

mone [119, 120, 154]. Immune cells also synthesize and

release hypothalamic regulatory factors such as somatosta-

tin, which may modulate hormone secretion from immune

cells in a manner analogous to the regulation of pituitary

hormone secretion by hypothalamic hormones [177]. The

physiological significance of cell communication at this level

is not known, but hormone secretion by immune cells may

participate in the paracrine amplification of afferent

pituitary signals in microenvironments such as infection

sites. Immune cells in these regions may secrete cytokines

and other mediators that feed back to the pituitary gland and

the brain [178]. In contrast to hormones of neuroendocrine

origin, hormones secreted by leukocytes are not stored and

must be synthesized de novo; the quantities produced by leu-kocytes are much smaller than those produced by neuroen-

docrine cells, but immune cells are mobile and can concen-

trate the hormone at distant targets [119]. Although antigenic

stimuli are not recognized directly by the central nervous or

endocrine systems, leukocytes may convey information deli-

vered by antigens, via humoral mediators, to these systems [178].

STRESS AND OTHER LIFESTYLE FACTORS

Cells of the immune system, including neutrophils, are

influenced by age, diet, psychological stress, health status,

and physical activity. Advancing age is associated with im-

paired cell-mediated immunity, but studies of neutrophils

have been conflicting. Aging, when combined with malnutri-

tion, does produce significant impairments in neutrophil

function [179]. Dietary deficiency or excessive intake of some

vitamins and minerals may impair immune responses [180].

Low neutrophil oxidative activity is associated with a poor

prognosis in some cancer patients [181]. Neutrophils isolated

from cancer patients not receiving any therapy have defec-

tive cytotoxic responses to antibody-coated tumor cells in

vitro [182]. Psychological stress has been shown by many

groups to suppress cell-mediated immunity and increase sus-

ceptibility to infection [183], but neutrophil responses have

not been examined.Exercise has intensity-dependent effects on the immune

system [116, 184]. We have shown that the neutrophil oxida-tive burst is deficient in athletes compared with untrained

men; in contrast, moderate episodes of exercise prime these

cells [114]. Differential secretion of neuroendocrine hor-

mones into the circulation may be responsible for theintensity-dependent effects ofexercise on the immune system

[184]. Priming of neutrophil microbicidal activity may cx-

plain, partially, why moderate exercise programs appear to

reduce the susceptibility of humans to infection and some

cancers, while overtraining is a high-risk factor for infectious

disease [185, 186]. In fact, the immunological and endocrineresponses to psychological stress and intensive physical train-

ing are remarkably similar [i84]. Further work is required to

link the immunological and clinical evidence directly and to

investigate the regulatory mechanisms involved. Circadian

and seasonal variation must also be taken into account.

NOVEL ROLES FOR NEUTROPHILS

As well as their functions in immunity and inflammation,

neutrophils may play other roles in the maintenance ofhomeostasis. This may explain why neutrophil function is

affected by stress and other lifestyle factors and why neu-

trophils are turned over so rapidly in healthy people. Neu-

trophils scavenge and degrade toxic molecules [81] and assist

in platelet removal [6]. Because of their motility and ability

to gain access to most body tissues, neutrophils are ideal car-

riers ofchemical signaling molecules and enzymes inter alia.

Containment and transport of regulatory factors in neu-

trophils may prevent their degradation or nonspecific inter-

actions during transit. For example, under normal condi-

tions, neutrophils may act as “vectors” to deliver essential

molecules such as proteases directly to sites of tissueremodeling (e.g., muscle adaptation to physical training).

This would prevent indiscriminate activation of proteolytic

inflammatory cascades by circulating free proteases or inhi-

bition by circulating antiproteases. Free elastase can activate

some proteolytic cascades including those of the comple-

ment, coagulation, and fibrinolytic systems [i87]. Because of

their accumulation in sites of infection and inflammation,neutrophils cells provide a convenient vehicle for delivering

drugs and reducing the potential for debilitating side effects

[188]. Packaging of cytotoxic molecules in cytosolic granules

with differential sensitivity to neutrophil activating agents

confers additional safety [17]. However, these safety meas-

ures break down during sustained inflammatory responses.

FUTURE DIRECTIONS

The greatest challenge to workers in this field is to determine

how optimal neutrophil function can be maintained and

682 Journal of Leukocyte Biology Volume 56, December 1994

what the essential regulatory factors are, given the apparent

high level of redundancy. The key to solving the neutrophil

paradox at the cellular level may lie in unraveling the

balance between secretory responses involved in the

chemotactic and cytotoxic pathways, and priming and acti-vation. The significance of neutrophil heterogeneity has still

not been solved [4] but this may provide further clues. For

example, ifneutrophils do produce RNS, are there subpopu-

lations that can be divided into ROS and RNS dominant?

Mutants or transgenic manipulation of animal cells may

solve some of these issues.Although many functional assays of neutrophil activity

have been described, we still do not know what constitutes a

normal response. For example, there is great variability in

the magnitude of the oxidative burst in neutrophils isolated

from healthy human subjects and stimulated in vitro [69]. As

a starting point, a reference range for the major neutrophil

functions should be determined in healthy subjects under

standard conditions. This information would aid therapeutic

strategies aimed at boosting specific neutrophil functions in

immunocompromised people or preventing inflammatory

conditions mediated by activated neutrophils.

CONCLUSIONS

Neutrophils are highly destructive cells that are essential for

host defense but also contribute to various inflammatory dis-

eases. This brief review has highlighted the complex path-

ways involved in regulating the diverse and somewhat redun-

dant array of neutrophil functions at the cellular and

humoral levels. Uncoupling of any essential part of the se-

quence may lead to neutrophil dysfunction. This balance is

controlled by the interaction of multiple regulatory cascades.

Unraveling these interactions is one of the greatest

challenges in leukocyte biology, but it is probably the key to

resolving the neutrophil paradox.

ACKNOWLEDGEMENTS

I am grateful to Dr. Maurice Weidemann and Dr. David

Pyne of the Division of Biochemistry and Molecular Biology,

Australian National University for their critical evaluation of

the manuscript. I also thank Prof. Chris Bryant of the Divi-

sion of Biochemistry and Molecular Biology, Australian Na-

tional University for continued support. My work has been

supported by an Australian Postgraduate Research Award,

grants from the Faculties Research Fund of the Australian

National University, and the National Quality of Life Foun-

dation.

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