Clinical Science (1998) 94.461471 (Printed in G r e a t Britain)
461
Editorial Review
Neutrophil priming: pathophysiological consequences
and underlying mechanisms
A. M. CONDLIFFE, E. KITCHEN and E. R. CHILVERS
Respiratory Medicine Unit, Department of Medicine (RIE), Rayne Laboratory, University of Edinburgh Medical School.
Teviot Place. Edinburgh EH8 9AG. U.K.
rapidly to sites of inflammation. These cells respond to
injurious agents by phagocytosis, the release of preformed granular enzymes and proteins, and by the de
novo production of a range of potentially damaging,
but ephemeral, reactive oxygen intermediates. That
these functions are essential for host defence against
invading micro-organisms is illustrated by the propensity of patients with neutrophil deficiency syndromes such as neutropenia, chronic granulomatous
disease and leucocyte adhesion deficiency to develop
recurrent and often overwhelming infections. Paradoxically, it is now equally clear that inappropriate or
excessive activation of neutrophils may contribute to
inflammatory tissue injury in a variety of clinical
scenarios (e.g. the acute respiratory distress syndrome
(ARDS), rheumatoid arthritis and ischaemia-reperfusion injury). Thus modulation of the activation
status of the neutrophil is of key importance in
determining the balance between defence and injury.
The aim of this review is to outline the functional
consequences and in vivo significance of neutrophil
priming and thereafter discuss in detail the current
controversy that exists regarding the mechanistic basis
of priming.
1. Neutrophil priming by agents such as tumour necrosis
factor-a, granulocyte/macrophage colony-stimulating
factor and lipopolysaccharide causes a dramatic increase in the response of these cells to an activating
agent; this process has been shown to be critical for
neutrophil-mediated tissue injury both in vitro and in
vivo.
2. The principle consequence of priming, aside from a
direct effect on cell polarization, deformability and
integrinlselectin expression, is to permit secretagogueinduced superoxide anion generation, degranulation and
lipid mediator (e.g. leukotriene B4and arachidonic acid)
release. It is now recognized that most priming agents
also serve an additional function of delaying apoptosis
and hence increasing the functional longevity of these
cells at the inflamed site.
3. The potential mechanisms underlying priming are
discussed; current data suggest a dissociation between
priming and changes in receptor number and/or affinity,
Gprotein expression, phospholipase C and phospholipase A, activation and changes in intracellular Caz+
concentration. However, more recent studies support a
key role for protein tyrosine phosphorylation and
enhanced phospholipase D and phosphoinositide 3
kinase activity in neutrophil priming.
4. Recent work has also revealed the potential for
neutrophils to spontaneously and fully ‘de-prime’ after
an initial challenge with platelet-activating factor. This
ability of neutrophils to undergo a complete cycle of
priming-de-priming (and re-priming) reveals a previously unrecognized flexibility in the control of neutrophi1 behaviour at an inflamed site.
INTRODUCTION
Neutrophils comprise a fundamental component of
the non-specific immune response, being recruited
-
N E U T R O P H I L PRIMING
Priming refers to a process whereby the response of
neutrophils to an activating stimulus is potentiated,
sometimes greatly, by prior exposure to a priming
agent (Figure 1). Normal circulating neutrophils do
not express anywhere near their full microbicidal
capacity when challenged with biological activating
agents such as the bacterial formylated peptide Nformylmethionyl-leucyl-phenylalanine(fMLP) unless
they have first been primed. The other key concept
regarding priming is that these agents do not elicit the
effector function(s) on their own (although they may
Key words: neutrophil priming, phosphoinositidase C, phosphoinositide 3-kinase. phospholipase D. superoxide anions.
Abbreviations: AA, arachidonic acid; ARDS, acute respiratory distress syndrome; fMLP. N-formylmethionyl-leucyl-phenylalanine; GM-CSF.
granulocytemacrophagecolony-stimulating factor; Ins( I .4,5)P,. inositol I .4.S-trirphorphate; LF’S, lipopolysaccharide;LTB,. leukotriene 6,; PAF, plateletactivatingfactor; P13K. phosphoinositide3-hydroxykinase;PIC, phorphoinositidaseC; PKC, protein kinase C; PLA2, phospholipaseA,; PLC. phospholipase
C; PLD, phospholipase D;Ptdlns(4,5)P2, phosphatidylinositol4.5-birphosphate; Ptdlns(3,4,5)P3, phosphatidylinositol 3,4.5-trisphosphate; TNF-a, tumour
necrosis factor-a.
Correspondence: Dr E. R. Chilvers at present address: University of Cambridge School of Clinical Medicine, Department of Medicine, Box 157, Level 5.
Addenbrooke’r Hospital, Hills Road, Cambridge CB2 2QQ, U.K.
A. M. Condliffe, E. Kitchen and E. R. C h i l v e n
462
Maaophage
4
GMCSF, TNFa
Endothelial Cell
hcteria
4
4
Importanceof priming in vivo
LPS
PAF, lNFa
Although priming was initially described as an in
vitro phenomenon, many priming agents have clear
biological relevance in vivo and are released in response
Figure I N e u t r o p h i l primingby inflammatory mediators
results i n enhanced superoxide production
Table I Variability of neutrophil priming agents
Priming agent
Time required to
induce maximal
priming
ATP
Substance P
lonomycin
lnositol hexakisphosphate
L-selectin cross4inking
PAF
CD18 cross-linking
TNF-a
Interleukin-8
Orthovanadate
Influenza A virus
LPS
GM-CSF
Interferon-y
IS s
I min
2 min
2 min
3 min
5 min
5 min
10 min
10 min
10 min
30 min
120 min
I20 min
120 min
Reference
[51
PI
m
PI
PI
[I01
[I 11
[I21
1131
1141
1151
[I1
[I61
[I71
do so when applied at very high concentrations), and
by definition, to be effective, they must be presented to
the cell for a variable period before the cell is exposed
to the activating stimulus. Hence, the neutrophil
respiratory burst that occurs in response to a secretagogue agonist and results in the release of superoxide
may be enhanced up to 20-fold by prior
anions (02-)
exposure of cells to a priming agent [l]. Likewise,
substantial priming of agonist-induced degranulation
[2] and the generation of lipid mediators {principally
arachidonic acid (AA), leukotriene B, (LTB,) and
platelet-activating factor (PAF) ; [3,4]} has also been
described. A wide variety of substances, both physiological and pharmacological, have now been shown to
act as priming agents (see Table l), and the differing
signal transduction routes utilized by these agents
reflect the varying preincubation times required to
initiate maximal priming, which range from a few
seconds (e.g. ATP) to over an hour [e.g. lipopolysaccharide (LPS), interferon-y] (Table 1).
to infection, trauma and haemorrhage. For example,
circulating endotoxin has been associated with the
development of ARDS [18], and persistent high levels
of plasma tumour necrosis factor-a (TNF-a) and
interleukin-6 have been linked to poor outcome in
septic shock [19]. Although such cytokines are detectable in the bloodstream only in extreme circumstances, locally generated mediators serve to upregulate the functional responses of extravasated
neutrophils; indeed, since cross-linking of neutrophil
adhesion receptors is itself a priming stimulus [9,11],
the process of extravasation per se may result in a
degree of priming.
Two approaches have been used to study priming in
vivo : firstly, investigators have infused priming agents
into laboratory animals or human volunteers and
studied granulocyte responses; and secondly, the
functional responses of neutrophils isolated from
patients with a variety of infectious or inflammatory
conditions have been assessed and compared with
control cells. Continuous endotoxin infusion has been
found to prime the respiratory burst of neutrophils
isolated from rat liver at 3 h and to a lesser extent at
30 h [20]. Intravenous injection of TNF-a into healthy
human volunteers resulted either in systemic neutrophi1 activation with elastase and lactoferrin release [211
or in neutrophil priming resulting in enhanced hypochlorous acid release [22]. Primed neutrophils have
also been identified in the peripheral blood of patients
after blunt trauma [23], ARDS [24], bacterial (Grampositive and Gram-negative) and fungal infections [25]
and in the joints of patients with active rheumatoid
arthritis [26].
Neutrophil priming has been shown to be critical for
the induction of endothelial injury, both in vitro and in
vivo. Smedley et al. [27] found that neutrophils
stimulated with activating agents alone (fMLP, the
complement component C5a, or very high concentrations of LPS) produced minimal damage to endothelial monolayers, but when the neutrophils were
first primed with lower concentrations of LPS and
subsequently stimulated with fMLP or C5a, extensive
endothelial cell injury ensued. Moreover, in a rabbit
model, intravascular administration of LPS in addition to fMLP greatly enhanced neutrophil vascular
sequestration within the lung, and the combination of
priming and activating agents led to lung damage, an
effect not seen when either substance was used alone;
this effect was completely abrogated by prior depletion
of neutrophils with nitrogen mustard [28]. Thus
priming appears to represent one of the key processes
regulating the functional responsiveness of neutrophils, both in vitro and in vivo, and may play a crucial
role in dictating the appropriateness of the neutrophil's
response at an inflamed site.
Mechanisms of neutrophil priming
FUNCTIONAL CONSEQUENCES O F
NEUTROPHIL PRIMING
Enhancement of the neutrophil respiratory burst
activity
Enhancement of the 0,- response to fMLP has been
viewed as the 'gold standard' priming response. Full
activation of ' respiratory burst' activity in the neutrophi1 results from translocation and assembly of the
cytosolic components of the NADPH oxidase enzyme
system ( ~ 4 7 p67phox,
~ ~ ' ~p2~1'"') with the membraneassociated flavocytochrome, cytochrome b,,,. This
translocation is dependent on the co-operative interaction of the various components, and involves
multiple ~47~~'"-directed
phosphorylation events [29]
and dissociation of the small GTP-binding protein
p21'"" from complexation with its chaperone inhibitor,
GDP-dissociation inhibitor. The activated NADPH
oxidase then catalyses the following reaction :
NADPH + H++ 20,
+ NADP'
+ 2H++ 20,-
Hence the NADPH produced by the cytosolic hexose
monophosphate shunt functions as an electron donor
to reduce two atoms of molecular oxygen [30]. The 0,formed in this reaction then rapidly dismutates to
form hydrogen peroxide, a reaction catalysed by
superoxide dismutase :
20,-
+ 2H++ 0,+ H,O,
Thereafter, in the presence of halide ions (preferentially chloride ions), myeloperoxidase released from
neutrophil azurophilic granules can catalyse the conversion of H,O, into hypohalous acids, such as
hypochlorous acid :
H,O,
+ HCl
+
HOCl + H,O
This set of sequential reactions triggered by the
NADPH oxidase is essential for efficient bacterial
killing. Deficiency of any of the oxidase components
results in chronic granulomatous disease ;neutrophils
from patients with this disease migrate and phagocytose normally but fail to generate a respiratory burst
with the resultant failure of intracellular killing leading
to recurrent bacterial (especially Staphylococcal) and
fungal infection, and early death. Since agoniststimulated 0,- generation is the most ' primable'
neutrophil function, with the potential to be upregulated at least 20-fold, the effects of priming agents
on the signalling pathways required for competent
assembly and activation of the NADPH oxidase
represent the obvious target to investigate the mechanisms underlying neutrophil priming.
Shape-change and deformability
When neutrophils are exposed to inflammatory
mediators (includingpriming agents) in a non-gradient
manner, they undergo a polarization or shape-change
463
response which is believed to represent frustrated
chemotaxis [31], and in many studies the extent of
shape-change has been shown to correlate closely with
priming of 0,- generation [31,32]. Shape-change
occurs as a result of modifications in cytoskeletal actin
and has the key effect of making neutrophils less
deformable; this 'stiffening' of the cell has been shown
to increase sequestration of neutrophils within the
pulmonary capillary bed, which in the lung represents
the main anatomical site of neutrophil efflux [33,34].
Even if these structural changes are not integral to
priming, prolonged retention of primed neutrophils
within the pulmonary capillary bed clearly increases
the risk of neutrophil-mediated damage to the vascular
endothelium.
Adhesion molecule expression and function
Intravascular sequestration of neutrophils at inflamed sites, and their subsequent trans-endothelial
migration, requires precise bipolar regulation of cell
adhesion molecule expression and function. These cell
adhesion molecules include members of the immunoglobulin superfamily, integrins and selectins. The
vascular selectins CD62-P (P-selectin) and CD62-E
(E-selectin) and the constitutively expressed leucocyte
antigen CD62-L (L-selectin) mediate the initial, lowaffinity ' rolling' interaction of neutrophils along the
vascular endothelium [35]. The firm adhesion that
follows to allow arrest of neutrophil movement is
principally a function of the leucocyte &integrins,
particularly CD 1 1b/CD 18 (Mac- l), and their endothelial cell ligand intercellular adhesion molecule- 1
(ICAM-1). Priming agents, in common with full
secretagogue agonists, have the capacity to enhance
neutrophil integrin expression and function and induce
CD62-L shedding; moreover, each priming agent
appears to produce a unique spectrum of effects, with
differing and preferential effects on integrin and
selectin expression and variable time-courses for the
manifestation of these effects [36]. Priming may therefore modulate the adhesive and migratory potential
of neutrophils with the effect of promoting recruitment
to an inflamed focus.
Degranulation and lipid mediator release
In contrast to the massive enhancement of secretagogue-induced 0,- release produced by priming, the
effects df this process on degranulation are far more
modest. For example, although a degree of priming of
elastase and myeloperoxidase release was demonstrated by Fittschen et al. [2] in response to LPS, for
other measured enzymes the effects of priming and
activating agents were simply additive. A more significant priming effect, however, has been shown for LTB,
and AA release [3,4], but the reasons for the differences
in 'primability' of these responses have not been
explored.
A. M. Condliffe, E. Kitchen and E. R. Chilvers
464
Inhibition of neutrophil apoptosis
It has now become apparent that many priming
agents, particularly LPS and granulocyte-macrophage
colony-stimulating factor (GM-CSF), in addition to
upregulating neutrophil responsiveness, also delay the
onset of programmed cell death [37]. Prolongation of
the functional lifespan of the neutrophil is now thought
to represent a further important mechanism whereby
priming may enhance the neutrophil-mediated response to injury or infection. However, it should be
noted that priming is not universally associated with
delayed apoptosis since the potent priming agent TNFa has a bimodal effect on this process with accelerated
neutrophil apoptosis at early time points (< 8 h) and
delayed apoptosis at later times, while PAF has no
effect on the rate of constitutive cell death [38].
NEUTROPH IL PRIMIN G : POTENTIAL
MECHANISMS
The great diversity of agents capable of inducing
neutrophil priming (see Table 1) raises the possibility
that a single common priming mechanism may not
exist, indeed that there may be class- or even agentspecific variations in the signalling mechanisms whereby the primed state is achieved. The variable potency
displayed by different agents for priming (for example,
inositol hexakisphosphate induces a very weak priming signal while TNF-a can augment 0,- release 20fold) also suggests that this process is not an all-ornone phenomenon; in fact, priming agents may well
act primarily by recruiting additional cells into an
agonist-responsive pool [39]. Nevertheless, the involvement of the following signalling pathways in priming
has been the subject of intense study and although
considerable controversy remains, certain common
themes are now emerging.
Modulation of agonist receptors
Priming-induced upregulation of the number and/
or affinity of agonist receptors that trigger activation
represents an obvious potential mechanism to augment subsequent responses to a secretagogue agonist,
and indeed some priming agents have been shown
Table 2
Effects of priming agents on fMLP receptors
Priming agent
Incubation
time (min)
Effect on fMLP receptors
~~~~
TNF-a
TNF-a
10
15-60
LPS
60
60
LPS
LPS
GM-CSF
GM-CSF.
interleukin-0
Interferon
75
I20
30
I20
Non-significant increase
Increased (but dissociated
from prirning-ree text)
Non-significant decrease
2-3-fold increase
3-fold increase
Increased (decreased
affinity)
Increased
None
Reference
to have such effects on the G-protein-coupled fMLP
receptor (Table 2). However, such results need to
be interpreted with caution, not least because the
receptor-generated component of the signal may not
be the rate-limiting part of the response, and increasing
the receptor number may serve only to increase
‘receptor reserve’ and hence the sensitivity rather than
the magnitude of the agonist response. Furthermore,
O’Flaherty et al. [40] demonstrated that while TNF-a
increased fMLP binding to neutrophils, this effect
lagged behind the cytokine’s ability to prime fMLPinduced degranulation and hence could not be responsible for priming. Again, Roberts et al. [44]
showed that priming of the respiratory burst was
maximal with an interleukin-8 concentration of 10-50
ng/ml, whereas no increase in fMLP receptor number
was detectable at concentrations below 100 ng/ml.
Thus effects on fMLP receptor number can be clearly
dissociated from priming, and since neutrophils possess granule-associated fMLP receptors that are mobilized to the cell surface on degranulation, the presence
of increased surface fMLP-binding may simply reflect
stimulated exocytosis.
Few investigators have quantified the effects of
priming on other agonist receptors; however,
O’Flaherty et al. [40] showed that TNF-a priming of
the degranulation response to PAF and LTB, was
associated with a reduction in surface LTB, receptors
and only a transient increase in PAF receptors, again
arguing against a critical role for receptor modulation
in priming.
Heterotrimeric GTP-binding proteins (G-proteins)
In neutrophils the predominant G-protein a-subunit
is G,,, [45], although the closely related Gla3is also
present. The identity of the G,, subunits, and the
specific G-protein(s) coupling the fMLP receptor to its
various effectors are unknown. Both Gir2and GirSare
substrates for pertussis toxin, which inactivates them
by ADP-ribosylation. As pertussis toxin abolishes
fMLP-induced 0,- production it cannot be used to
dissect the potential involvement of G-proteins in
priming this response. However, functional responses
to phorbol myristate acetate or AA are not suppressed
by pertussis toxin, and Berkow and Dodson [46]
demonstrated that the slight priming effect of TNF-a
on phorbol myristate acetate-induced 0,- production
was not altered by pretreatment with this toxin.
Variable data have been obtained regarding GM-CSF
priming of AA-induced oxidase activity, which has
been reported to be either unaffected [47] or abolished
[48] by pertussis toxin: hence this does not appear to be
an ideal model system or tool for investigating the role
of G-proteins in priming.
An alternative approach to address the potential
role of G-proteins in mediating the primed response
has been to assess the effects of priming agents on Gprotein localization within the cell. Translocation of
Gizato the neutrophil membrane has been reported in
response to priming concentrations of LPS [49], GM-
465
Mechanisms of neutrophil priming
CSF [50] and TNF-a [51], and the time-course of GI,,
translocation after treatment with LPS, PAF and
TNF-a appears to correlate closely with that for
priming of the 0,response [52]. However, significant
amounts of GI,, is associated with the plasma membrane fraction under basal conditions where no 0,generation is observed in response to agonist stimulation, and the priming-associated augmentation of
membrane-associated G-protein in these studies was
at most 3-fold compared with a 10-20-fold increase in
0,-release. Finally, the most compelling evidence
against a central role for G-proteins as effectors of the
primed response is that certain key G-protein-linked
signalling events {notably inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3]generation, see below} are not enhanced
in primed cells, suggesting that increased G-protein
availability plays a minor, if any, role in priming.
Phospholipase C activation
The /?, isoform of phospholipase C (PLC) is the
major phosphoinositidase C (PIC) activity present in
neutrophils, and is activated predominantly by G,,
rather than G, subunits [53]to hydrolyse its membrane
phospholipid substrate phosphatidylinositol 4,5-bisphosphate ptdIns(4,5)P2]. This reaction generates
Ins( 1,4,5)P3, which induces Ca2+release from intracellular stores and diacylglycerol, which activates
protein kinase C. Diacylglycerolcan also be generated,
often in a more sustained fashion, by agonist stimulation of phospholipase D (PLD), which hydrolyses
phosphatidylcholine.
Ins(2,4,5)P3accumulation. The application of fMLP
to a neutrophil population results in a rapid but
transient elevation bf Ins( 1,4,5)P3,with peak levels at
1&15 s and thereafter a rapid return to basal levels.
Despite the clear demonstration that the resultant
calcium transient is necessary for activation of the
respiratory burst [54], remarkably few studies have
examined the potential for this second-messenger
pathway to be upregulated in the priming process. In
the three studies that have been published, GM-CSF
either had no effect on [48,55], or caused a small
increase in [56], resting and stimulated [3H]Ins(1,4,5)P3
levels. Unfortunately the [3H]inositollabelling protocols used in these studies that allow identification and
quantification of the separate inositol polyphosphates
do not label the phosphoinositides to isotopic equilibrium and may also have induced a degree of basal
priming. Using the Ins( 1,4,5)P3 mass assay described
by Challiss et al. [57] to avoid these problems, we
have demonstrated (A. M. Condliffe, E. R. Chilvers,
P. Hawkins and L. Stephens, unpublished work) that
the classic priming agents PAF and TNF-a have no
effect on baseline or fMLP-stimulated Ins( 1,4,5)P3
accumulation, which appears to be a fully competent
response even in unprimed cells which produce little or
on agonist challenge. Although in these studies
no 0,fMLP-stimulated Ins( 1,4,5)P3levels returned to baseline values by 30 s in both primed and unprimed cells,
an effect of priming on Ins(1,4,5)P3 flux cannot be
Table 3 Effect of priming agents on basal- and agoniststimulated neutrophil calcium transients
lcaz+l,
Priming agent/&+
indicator
LPs/fura2
LPs/fura2
LK/fluo-3
LPs/fUd
TNF-a/q uin2
T N F-a/fura2
PAF/fura2
PAFlfura2
GM-CSF/quinZ
GM-CSF/fud
GM-CSF/fura2
Substance P/fura2
ATP/quinZ
Vanadate/fura2
(Basal)
(Stimulated)
Reference
t
+
t
Not
+
+
t
t
+
t
t
+
+
+
excluded. This would require an analysis of total
[32P]/[3H]InsPxaccumulation measured in the presence of lithium to block the inositol monophosphatase
and hence prevent recycling of the inositol headgroup.
Elevation of [Ca"],. Binding of Ins(1,4,5)P3 to its
intracellular receptor induces a conformational change
in the receptor resulting in Ca2+ release from intracellular stores which in turn activates Ca2+influx
from the external medium (capacitative Ca2+entry).
While it is now well established that a rise in [Ca2+I1
is
an essential step in neutrophil activation and 0,generation in that neutrophils depleted of Ca2+do not
undergo a respiratory burst [54] and oxidase activation
only occurs if a threshold [Ca2+I1
of 250 nM is exceeded
[58], its role in priming is less clear.
Previous studies have demonstrated that calcium
ionophores such as ionomycin can act as priming
agents, and that elevation of [Ca2+I1
correlates closely
with the degree of priming [7]. However, as can be seen
in Table 3, there are conflicting reports on the effects of
individual priming agents on resting and stimulated
[Ca2+I1.For example, it is generally agreed that the
archetypal priming agent TNF-a does not mobilize
Caz+ or augment the Ca2+ transient generated in
response to other agonists, and similar results have
been reported for other agents such as substance P.
Likewise, despite consistent reports that PAF mobilizes Ca2+, and may augment subsequent agonistinduced Ca2+ transients, Koenderman et al. [68]
reported that PAF-induced priming was only partially
inhibited under [Ca2+],-bufferingconditions. Furthermore, in experiments by Wymann et al. [69], application of fMLP 2 min after a low 'priming' concentration of phorbol myristate acetate resulted in a
rapid (2 s) enhancement of chemiluminescence that
preceded the rise in fura-2 fluorescence, again suggesting that priming of the oxidative burst does not
rely on enhanced calcium fluxes. Taken together, these
results imply that while an elevation in [CaZ+lican
induce priming and indeed is essential for full receptormediated NADPH oxidase assembly and activation, it
is not an obligate step for priming and hence is unlikely
466
A.
M.Condliffe, E. Kitchen and E. R. Chilvers
to represent a 'final common pathway' mediating
priming.
Diacylglycerol and protein kinase C . Diacylglycerol
produced by the action of PLC and/or PLD activates
members of the protein kinase C (PKC) family [70],
inducing phosphorylation of several substrates (including ~ 4 7 and
~ activation
~ ~ ~ of) the NADPH oxidase. Neutrophils have been shown to possess at least
two PKC isoforms, the conventional P,-PKC (the
most abundant isoform) and the novel r]-PKC [71].
While it is important to emphasize that phorbol esters
(pharmacological activators of classical PKCs) used
alone can fully activate the NADPH oxidase, in subactivating concentrations these agents prime neutrophils, indicating a possible role for PKC in the priming
process.
The effects of priming agents on the PKC-activator
diacylglycerol are somewhat controversial. It has been
shown that GM-CSF alone produces only a small
increase in neutrophil diacylglycerol mass but can
markedly increase its accumulation in response to
fMLP [55,72]; this effect presumably reflects enhanced
fMLP-induced PLD activation as PIC-mediated
PtdIns(4,5)P2 hydrolysis is not increased. In contrast,
Bauldry et al. [73] found that treatment of polymorphonuclear cells with TNF-a alone could not
enhance the fMLP-induced diacylglycerol response in
the absence of cytochalasin B.
Whereas some of these studies suggest that priming
may enhance agonist-stimulated diacylglycerol generation, subsequent studies have failed to show translocation or activation of PKC in primed cells: LPS
[59], TNF-a [46] and GM-CSF [65] all fail to induce
PKC translocation under priming conditions. Thus
the weight of evidence remains against a significant
role for augmented PKC activation in priming.
PLD activation
PLD catalyses the cleavage of the terminal phosphodiester bond of phosphatidylcholine to yield phosphatidic acid and an inactive choline molecule, and
fMLP stimulation of a phosphatidylcholine-specific
PLD activity is a well-documented response in neutrophils [74]. Phosphatidic acid is converted to diacylglycerol by phosphatidate phosphohydrolase [75],
and this results in a second, often more sustained
phase of diacylglycerol generation than that resulting
from the activation of PIC [75,76]. Inhibition of PLDmediated phosphatidic acid production prevents
neutrophil fMLP-induced 0,- release [77].
Bourgoin et al. [55] and Bauldry et al. [73] both
studied the effects of priming (with GM-CSF and
TNF-a respectively) on PLD activity and demonstrated that while treatment of neutrophils with the
priming agent alone did not result in accumulation of
phosphatidic acid, the subsequent fMLP-induced accumulation was augmented and prolonged in primed
cells. In the latter study, phosphatidic acid production
was found to correlate closely with 0,- generation.
While other priming agents have not been examined in
this model, these two studies provide good evidence
that regulation of agonist-induced PLD activity may
represent an important target for TNF-a and GMCSF priming activity. Moreover, Waite et al. [78] have
recently identified a novel phosphatidic acid-stimulated protein kinase activity in human neutrophils
which is capable of phosphorylating (among other
substrates) ~ 4 7 ~ ~ " " .
Phospholipase A, (PLA,) activation
PLA, releases free AA from cellular phospholipids
[79] in what is believed to be the rate-limiting step
in the synthesis of biologically active eicosanoids.
Priming agents can enhance the synthesis of bioactive
lipids (AA, PAF, LTB,) in response to activating
stimuli [3,47,80]. In eosinophils, in marked contrast to
the situation in neutrophils, PLA, plays a key role in
activating the NADPH oxidase, a response that is
both Ca2+- and phosphoinositide 3-hydroxykinase
(PI3K)hdependent. Hence, activation and/or translocation of PLA, has been suggested as a potential
mechanism of priming. However, agonist-stimulated
PLA, activation and AA release, unlike 0,- generation, are events that are entirely dependent on Ca2+
influx and are unaffected by the PI3K inhibitor wortmannin (see later); hence PLA, activity, although
'primable', appears to be mediated via a discrete set of
signalling pathways not involved in NADPH oxidase
assembly [81].
Investigations into the mechanism(s) of PLA, priming have demonstrated that a PLA, activity is translocated from the cytosolic to the plasma-membrane
fraction in LPS-primed neutrophils [82], and Doerfler
et al. [4] in confirming this finding also identified that
LPS induced the phosphorylation of PLA,, an event
previously correlated with PLA, activation [83]. However, LPS priming of LTB, release appeared to be
agonist-selective and was not observed using fMLP as
the secretagogue agonist [3], and again none of these
studies was able to link PLA, activation with priming
of the respiratory burst.
Worthen et al. [64]correlated LPS-induced synthesis
of intracellular PAF with priming of the respiratory
burst, and suggested that PAF could be responsible.
However, Stewart et al. [84] demonstrated that neither
blockade of PAF receptors (with WEB 2086) nor
inhibition of LTB, synthesis (with CGS 8515) influenced the priming of fMLP-mediated 0,- generation
by TNF-a or GM-CSF.
Finally, Ely et al. [85] have now published data
demonstrating that the release of physiological
amounts of AA in the neutrophil does not induce 0,generation or degranulation, or influence fMLP or
primed fMLP responses. It therefore seems unlikely
that mobilization of AA from cellular phospholipids
plays a major role in neutrophil priming activation.
Protein phosphorylation and neutrophil priming
The cytosolic oxidase component ~ 4 7 becomes
~ ~ " ~
extensively phosphorylated on cellular activation with
Mechanisms of neutrophil priming
the phosphorylation targets consisting of a group of
serines in the carboxy-terminus of the peptide [29]. The
enzyme(s) responsible have not been definitively identified. Activation of neutrophils by fMLP and other
stimuli is associated with increased tyrosine phosphorylation of multiple protein substrates [86]. Tyrosine
phosphorylation has also been described in response
to several priming agents, including GM-CSF [67],
TNF-a [46,63], substance P [63] and PAF [87]. One of
the targets appears to be mitogen-activated protein
kinases [88]. In several of these studies, the time-course
of tyrosine phosphorylation was consistent with a role
in priming, and manipulation of tyrosine phosphorylation levels using tyrosine phosphatase or tyrosine
kinase inhibitors resulted in priming and inhibition of
priming respectively [14]. It therefore seems likely that
tyrosine phosphorylation plays an important, if as yet
uncharacterized, role in priming.
P13K
Many neutrophil secretagogue agonists stimulate
PI3K with resultant formation of phosphatidylinositol
3,4,5-trisphosphate [PtdIns(3,4,5)P3] from PtdIns(4,5)P, [89]. Indeed, agonist-stimulated PtdIns(3,4,5)P3 accumulation was first identified in the
neutrophil and much of the cardinal biochemistry
regarding PI3K activation and PtdIns(3,4,5)P3 signalling has been undertaken in this cell. The ability of
wortmannin, a relatively specific and irreversible inhibitor of neutrophil PI3K activity, to abolish both
fMLP- and phagocytosis-induced respiratory burst
activity without any effect on agonist-induced [Ca2+Ii
flux, PKC-mediated NADPH-oxidase activation or
granule exocytosis [54,90] has provided strong evidence to support a second-messenger role for PtdInsgeneration. In addition to the well(3,4,5)P, in 0,characterized and seemingly ubiquitous pllO/p85
PI3K, neutrophils also possess a novel Gh-regulated
p120/p101 PI3K activity [91]. Moreover, we have
recently demonstrated that priming of human neutrophils by TNF-a results in a substantial enhancement
of fMLP-stimulated PtdIns(3,4,5)P3 accumulation
(A. M. Condliffe, E. R. Chilvers, P. Hawkins and
L. Stephens, unpublished work), further implicating
PtdIns(3,4,5)P3as a messenger critical to signalling the
primed state. This finding fits well with the recent data
indicating that PtdIns(3,4,5)P3 can activate the small
GTP-binding protein p21'" [92], which is an essential
component of the NADPH oxidase, and can activate a
p21-activated kinase which phosphorylates p47ph0x
NEUTROPHIL DEPRlMlNG
Studies of human peripheral blood neutrophils
primed with LPS or G-CSF [94] suggest that the
capacity for enhanced respiratory burst activity is
maintained for at least 24 h; in vivo infusion of
endotoxin also engenders a primed state in sheep
467
peripheral blood and bone marrow-derived neutrophils that lasts for at least 24 h [95]. However, the
assumption that neutrophil priming is an irreversible
process was recently challenged by the demonstration
that both hypotonic shock and cell-swelling could
induce a hyperresponsive state of the human neutrophi1 NADPH oxidase that was only temporary [96,97].
A similar phenomenon was subsequently identified
after exposure of neutrophils to the highly charged
Ca2+-chelator inositol hexakisphosphate [98]. Recently, it has been demonstrated that receptor (PAF)mediated priming of neutrophils is also fully reversible,
with cells able to return to a non-polarized morphology with low CD1 lb/CD18 activity and minimal
0,release to fMLP. Furthermore, the cells which had
'deprimed ' could subsequently be fully ' reprimed ' by
either TNF-a or PAF [32]. This ability of neutrophils
to undergo a complete cycle of priming/depriming/
repriming permits a previously unrecognized flexibility
in the control of neutrophil behaviour at an inflammatory site, and provides a potential mechanism for
damage limitation and 'cell rescue' at an inflamed site.
A MODEL OF NEUTROPHIL PRIMING
The variety of priming agents, and the complexity of
the data reviewed above, suggest that priming is not a
simple event, and that several complementary signal
transduction processes are involved in the overall
response. The final common pathway of priming of the
neutrophil respiratory burst is the activation of the
NADPH oxidase, and many signals may impinge on
this process (Figure 2). For example, the phosphorylation of p47phox
appears to require the co-ordinated
activity of a number of kinases including PKC,
p42/p44"*Pk, p21-activated kinase and a phosphatidic
acid-activatedprotein kinase, and this event may allow
conformational changes promoting membrane translocation and oxidase activation. Rac must also dissociate from its GDP-dissociation inhibitor to access
the other oxidase components, and this dissociation
may be promoted by signalling molecules derived from
PIC and PI3K and perhaps PLA, activation [99].
Likewise, the increased membrane expression of agonist receptors and G-proteins may be involved in
enhancing the sensitivity of the cell to stimulating
agonists and in combating receptor desensitization by
supplying new signalling units to the plasma membrane. It is clear therefore that the redundancy and
cross-talk that exist within this system allow widely
differing agents to modulate and arrive at a final
common effector pathway, although the disparate
routes taken may explain the variable and selective
priming properties of these agents. It is also likely that
priming of responses other than 0,-generation may
rely on different mechanisms; for example, phosphorylation and Ca2+-dependent translocation of
PLA, may be more important in priming the release of
lipid mediators, and Ca2+ transients may dominate
degranulation responses.
A.
468
M.Condliffe. E. K i t c h e n and E. R. Chilvers
MAPK
f
Tyrosinekin-
\
'
PLD'
YDAG
-D
I~s(I,~,s)P~+
Inueasea receptor
a n d G-Drotein
expres;ion
Y
phosphorylation of
[Ca2+Ii-
\
\ k PI3K - - - - - - Unknown
m e d i ators
I
- - - - - -+ M e m b r a n e
translocation of
Oxidase
oxidase
+activation
components
I
\
\\
\\r '
Figure 2
PKC
- .
'
rac/GDI
f
/
dissociation
/
/
--A
-
O t h e r mechanisms
/
Possible signalling routes leading to neutrophil priming
Abbreviations: DAG. diacylglycerol; GDI. GDP-dissociation inhibitor: MAPK. mitogen-activated protein kinase; PA, phosphatidic acid; PAK.
p2I -activated kinase.
CONCLUSION
This review has outlined the importance of priming
as a key regulator of neutrophil function and highlighted the crucial role priming plays in dictating
neutrophil-mediated tissue damage it? vivo. The recent
finding that priming is reversible further underlines the
potential for therapeutic intervention which, if successful, could offer the possibility of modulation rather
than ablation of neutrophil activity, and facilitate the
safe and timely removal of these cells from the inflamed
site via apoptosis. Finally, although the signalling
pathways orchestrating NADPH oxidase priming
remain elusive, upregulated PLD and PI3K activity
appear to be attractive candidate targets.
ACKNOWLEDGMENTS
E.R.C. was supported by a Wellcome Trust Senior
Research Fellowship in Clinical Science. A.M.C. was
supported by an MRC Training Fellowship, and E.K.
by a Wellcome Trust Prize Studentship.
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