Exuberant Ca2+ Signaling in Neutrophils: A Cause
for Concern
Mohammed M. Sayeed
Under inflammatory conditions after burn/trauma injuries, circulating neutrophils are frequently
hyperactive, contributing to excessive superoxide production and related tissue damage.
Although normal neutrophil activation is cooperatively controlled by Ca2+-independent and
Ca2+-linked signaling pathways, exuberant Ca2+-linked signaling appears to cause neutrophil
hyperactivation in the injury conditions.
eukocyte responses in mammalian organisms occur episodically not only after infectious challenges (bacterial, fungal,
viral, or parasitic), but also after seemingly noninfectious (nonseptic) injury conditions, such as trauma and burn. Although
early inflammatory phases after nonseptic challenges are probably independent of an invading pathogen, later phases are often
complicated by pathogen invasion of the host organisms. The
inflammatory response in the absence of a pathogen may be initiated by traumatized/burned tissue products serving as non-self
“antigens.” An advanced state of inflammation ensuing in mammalian hosts after nonseptic or septic injuries has been referred
to as the systemic inflammatory response syndrome (SIRS) (4). A
hallmark of SIRS is the release of inflammatory mediators primarily, but not exclusively, from the leukocytes. At present, there
is an exhaustive list of mediators identifiable in SIRS; inclusive in
this list are cytokines tumor necrosis factor-α (TNF-α), interleukin-1 (IL-l), IL-6, and IL-8; lipids platelet-activating factor
(PAF) and prostaglandin E2; and reactive molecular species
superoxide anion (O2–) and nitric oxide (8). If appropriately regulated in terms of their temporal release pattern and magnitude of
release, the mediators have the potential to play adaptive roles
in the host’s defense against the antigens/pathogens (9). Such
adaptive roles are played through the actions of mediators on
leukocytes, resulting in leukocyte responses that provide for neutralization and/or inactivation of the antigen/pathogen and protection of host cells/tissues.
However, the outcome of leukocyte responses resulting
from actions of mediators in SIRS is often damage and, ultimately, death of host cells/tissues. The host cell/tissue damage
could occur not only from the actions of mediators leading to
attenuated or disturbed leukocyte responses but also from
actions of mediators causing an excessive or exaggerated
response. Such maladaptive up- and downregulations of
leukocyte responses can arise from inappropriate mediator
release, mediator-receptor interactions, and/or receptormediated leukocyte signaling. An excessive neutrophil O2–generating response after trauma/burn conditions appears to
be inappropriate in the early phases of injury; it can potentially cause O2–-mediated host tissue damage (10). Such an
inappropriate neutrophil response could understandably be
M. M. Sayeed is in the Departments of Physiology and Surgery and the Burn
and Shock Trauma Institute, Loyola University Medical Center, Maywood, IL
60153.
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related to inappropriate activation of the signaling pathways
linked to O2– generation. Recent studies have supported a role
of altered Ca2+ signaling-related intracellular pathways in the
exaggerated neutrophil O2–-generating response (13). Figure 1
illustrates the role of burn/trauma-induced inflammatory
mediators in neutrophil activation and its consequences.
A broad understanding of the neutrophil signaling pathways
involving Ca2+ and related signaling components, and of
mechanisms by which these pathways control O2– production,
has the potential to elucidate the excessive O2–-generative
response. Such an understanding can also contribute to development of therapeutic strategies to abrogate O2–-related tissue
damage in injury conditions. This article focuses on the present state of knowledge of Ca2+ and related signaling pathways
and their role in O2– production in neutrophils and the impact
of alterations in the signaling mechanisms on the oxidant
response in trauma/burn injury conditions.
O2– production: role of inflammatory mediators
Although intracellular O2– release after phagocytic uptake of
pathogens by neutrophils leads to oxidant-mediated pathogen
killing and thus to an efficient host defense, excessive O2–
release in the environment in close proximity outside the neutrophil can cause oxidative host cell/tissue damage at sites
traversed by neutrophils during their migration from blood
to tissue regions of inflammation/infection (Fig. 1). Such neutrophil-caused oxidant injury is known to occur in pathological conditions of rheumatoid arthritis, myocardial infarction,
stroke, acute respiratory distress syndrome (ARDS), and tissue
ischemia (10). Inflammatory mediators, which originate from
cellular elements (e.g., tissue macrophages, dendritic cells) at
sites of inflammation and diffuse into blood, evidently stimulate neutrophils’ chemotactic response, which is responsible
for their migration to the inflamed tissue sites. The chemotactic mediators are also effective stimuli for neutrophil O2–
production. Although neutrophil chemotaxis results from
membrane skeletal and cytoskeletal changes contributing to
adhesion of neutrophils to other cells and the extracellular
matrix, the O2–-generative effector response is dependent on
the ultimate activation of the neutrophil plasma membrane
NADPH oxidase (O2 oxidoreductase).
Some of the chemotactic mediators that activate the O2–generating NADPH oxidase system are activated serum
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L
complement fragment C5a, bacteria-derived N-formylated
methionyl peptides [e.g., formyl-methionyl-leucyl-phenylalanine (f-MLP)], bioactive lipids PAF and leukotriene B4 (LTB4),
and the chemokine IL-8 (1). Inflammatory cytokines TNF-α,
granulocyte colony-stimulating factor (GCSF), and granulocyte-macrophage colony-stimulating factor (GMCSF) also
contribute, but indirectly, to O2– production. The chemotactic
mediators f-MLP, C5a, PAF, LTB4, and IL-8 bind to neutrophil
plasma membrane receptors (the so-called serpentine receptors), which have seven intramembrane peptides in series,
with an NH2-terminal peptide as the extracellular and a
COOH-terminal peptide as the cytosolic domain. On the
other hand, TNF-α, GCSF, and GMCSF bind to a single transmembrane peptide receptor. The activation of NADPH oxidase is also effected by Fc and complement receptors (FcR
and CR, respectively), which are involved in the neutrophil’s
phagocytic and adhesive responses. Neutrophils express two
types of FcRs (which bind to the Fc component of immune
complexes), FcRII and FcRIII, and two types of CRs (which
bind to complement-pathogen complexes), CR1 and CR3.
CR3, known as CD11b/CD18 or β2-integrin, also binds to its
cognate ligand intracellular adhesion molecule 1 (ICAM-1),
leading to neutrophil adhesion to a target cell such as the
endothelial cell. Neutrophil adhesive molecule L-selectin,
which binds to the endothelial cell sialyl-Lewis ligand, also
participates in firm neutrophil adhesion to endothelium. Ligation of these phagocytosis- and adhesion-promoting molecules contributes to potentiation of neutrophil O2– production
(4). In addition, CD11b/CD18 and FcRIII may directly interact to enhance O2– production. Recent studies have shown
that there is a high degree of coordination between the
phagocytic/adhesive effector responses and NADPH oxidase
activation in neutrophils (2). The neutrophil’s receptor systems involved in O2– production are illustrated in Fig. 2.
The actions of various inflammatory mediators and the roles
of their receptors in neutrophil O2– production have mostly
been studied in vitro using neutrophils from healthy humans
and/or animals. Whereas the earlier studies obtained information pertaining to effects and roles of individual mediators,
more recent studies have focused on effects of multiple mediators sequentially impinging on neutrophils. The studies
focusing on the mediators’ cumulative effects have given us a
better understanding of both the potentially adaptive as well
as a maladaptive neutrophil respiratory burst responses that
may be elicited in pathophysiological inflammatory states,
including those associated with the trauma/burn injury conditions. The in vitro studies have clearly shown that stimulation
of resting neutrophils by a given mediator either results in O2–
generation or simply leads to “priming” of the O2– production
mechanism without actual O2– production (12, 15). O2– is
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FIGURE 1. Schematic illustration of septic and/or nonseptic injuries leading to release of inflammatory mediators that play roles in “priming” and subsequent “activation” of circulating neutrophils. Also shown is transendothelial migration (infiltration) of activated neutrophils into tissues and their interactions with extracellular matrix and host tissues that may become targets of “oxidant fire” (O2–) from neutrophils. Release of O2– intracellularly in a presumed “controlled” manner allows
for inactivation/neutralization of pathogens/antigens engulfed by neutrophils. Tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony stimulatory factor
(GMCSF), and granulocyte colony-stimulating factor (GCSF) mainly serve as priming agents; formyl-methionyl-leucyl-phenyl alanine (f-MLP), leukotriene B4
(LTB4), platelet-activating factor (PAF), complement fragment C5a, and interleukin (IL)-8 can act as both priming agents for resting neutrophils and activating agents
for preprimed neutrophils.
produced by the primed neutrophils only after a subsequent
stimulation by either the same mediator, at a high concentration, or a different mediator; the response in the preprimed
cells is of a greater magnitude than that produced in
unprimed resting cells (Fig. 1). The chemotactic mediators
acting on the serpentine receptors are known to be capable
of eliciting O2– generation in both resting and primed neutrophils (7); the response, however, is much greater in primed
than in resting cells. On the other hand, the cytokines TNF-α,
GMCSF, and GCSF are only capable of priming (7). Neutrophils adhered to other cells (such as endothelial cells),
extracellular matrix substances, or to immune complexes
deposited into tissues in pathological conditions are also
known to exhibit enhanced O2– release (15). Such enhanced
oxidant responses in the adhered state are also ascribed to an
initial priming of neutrophils followed by the potentiated O2–
response. Both the serpentine and single transmembrane
peptide receptor agonists can cause the enhanced O2–
response in adhered-primed neutrophils (Figs. 1 and 4).
Although mediators that can cause priming and those that
can lead to potentiated O2– responses have been identified,
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the neutrophil intracellular mechanisms responsible for priming have not been adequately elucidated, particularly in
pathophysiological or pathological states in vivo (7).
Signaling to O2– production: role of Ca2+-dependent and
Ca2+-independent pathways
The activation of serpentine receptors in neutrophils leads
to their coupling within the membrane to the heterotrimeric
pertussis toxin-sensitive G protein, which complexes with
GTP in an amplified manner. The activated G proteins in turn
stimulate an array of intracellular phospholipases and kinases.
An early event following G protein activation is stimulation of
phosphatidyl inositol 4,5-bisphosphate (PIP2)-specific phospholipase C-β (PLC-β) which hydrolyzes PIP2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (5). The
resulting DAG serves as the second messenger to stimulate
protein kinase C (PKC), and IP3 induces a rapid release of Ca2+
from intracellular nonmitochondrial stores (calciosome or
endoplasmic reticulum), thereby elevating the concentration
of intracellular Ca2+ ([Ca2+]i). An initial transient increase in
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FIGURE 2. Schematic diagrams showing neutrophils’ Ca2+-dependent and Ca2+-independent signaling pathways triggered by ligands acting on cell surface receptors. A: 7-transmembrane-domain system specific for f-MLP, LTB4, C5a, PAF, IL-8. Ca2+-dependent pathways are activated via 7-transmembrane-domain receptor
system acting through G protein βγ-subunit. PLC-β, phospholipase C-β; PLD, phospholipase D; PA, phosphatidic acid; DAG, diacylglycerol; InsP3, inositol 1,4,5-tris
phosphate; Ptd Ins P2, phosphatidyl inositol bisphosphate; PKC-β, protein kinase C-β. Ca2+ influx is through a membrane ion channel. B: single transmembrane peptide-specific system for TNF-α, GMCSF, GCSF; Ca2+-independent pathways are triggered via 7-transmembrane-domain receptor system acting through α-subunit of
G protein and single transmembrane peptide receptor system. PTK, protein tyrosine kinases; Lyn, a PTK that is autophosphorylated and then interacts with target
protein Shc, leading to activation of protein Grb2, p21 Ras, and enzyme phosphatidyl inositol 3-kinase (Ptd Ins 3-K). C: alternative Ca2+-independent pathway via
7-transmembrane-domain system activating through G protein α-subunit. MAPK, mitogen activated protein kinase; ERK, extracellular signal related kinase; JNK,
c-Jun NH2-terminal kinases; MEK, MAPK-ERK kinase; MEK-K, MEK kinase. D: Fc system for immunoglobulin (Ig) bond immune complexes or heterodimer system
specific for β2-integrin (CD11b/ CD18) ligands. Ca2+-dependent and Ca2+-independent pathways are activated by Fc and heterodimer receptor system.
“The phosphorylation of the cytosolic protein
p47phox is…correlated with O 2– generation….”
[Ca2+]i elevation and PKC activation, involve activation of various protein kinases besides PKC (3, 5). The stimulation of
neutrophils through either the serpentine or single-transmembrane domain receptor leads to Ca2+-independent activation
of protein tyrosine kinases (PTKs). Diverse neutrophil effector
responses, including NADPH oxidase activation, are linked to
protein tyrosine phosphorylations. In case of stimulation of
neutrophils with the cytokine TNF-α, GMCSF, or GCSF, the
receptor’s cytoplasmic domain itself, which possesses the PTK
activity, catalyzes phosphorylation of tyrosine on some
selected target protein(s). The serpentine receptors activate the
src family nonreceptor tyrosine kinase, Lyn; the activation
process consists of Lyn autophosphorylation followed by
phosphorylation of target protein Shc (Fig. 2B). Lyn-Shc activation leads to stimulation of phosphatidyl inositol 3-kinase
(PI3K), which in turn could activate NADPH oxidase (2). A
somewhat specific blockade of PI3K by wortmannin or LY294002 is known to inhibit the neutrophil oxidative burst.
PI3K may be located upstream to Ca2+-signal-independent
PKC signaling. The target protein Shc can also be linked to
activation of the “small GTP-binding protein/GTPase,” Ras (2).
The events downstream to Ras activation in neutrophils are
not adequately elucidated. The f-MLP and C5a activation of
neutrophils leads to activation of p21 Ras, which in turn may
play a role in the activation of a group of serine/threonine
kinases, mitogen-activated protein kinases (MAPKs) (11).
Three subfamilies of MAPKs have been identified in mammalian cells: 1) the extracellular signal-related kinases
(ERKs/p42–44 MAPKs); 2) p38 MAPK; and 3) c-Jun NH2terminal kinases (JNKs) (11). All three MAPK subfamilies are
characterized by tripeptide sequences that can be phosphorylated both at threonine and tyrosine sites; such phosphorylations
are catalyzed by a family of dual-purpose tyrosine/threonine
kinases, known as MAPK-ERK kinases (MEKs). MEKs in turn
are activated via serine/threonine phosphorylation by MEK
kinases (MEKKs) (Fig. 2C). To date, studies have shown that
stimulation of neutrophils by C5a, IL-8, and f-MLP can activate ERKs and that f-MLP can stimulate both ERKs and p38
MAPK. The role of MAPKs in neutrophil effector response generation is not adequately understood. Whereas some studies
have dissociated ERK and p38 MAPK activation from neutrophil O2– production, other studies have supported p38
MAPK involvement with f-MLP stimulation of the respiratory
burst (11). A secondary ERK activation may also be required
for the respiratory burst oxidase activity.
The activation of neutrophil Fc receptors and β2-integrin/Lselectin adhesive molecules involves PTK activation as well
as [Ca2+]i elevation due primarily to Ca2+ release from the
intracellular stores (Fig. 2D) (5). In this case, src family PTK
may play a role in tyrosine phosphorylation of PLC-α isoform
which, like PLC-β, triggers Ca2+/PKC signaling. Thus adhesion
of neutrophils to immune complexes (via FcR), and to other
cells or matrix (via β2-integrin/L-selectin) can also lead to
NADPH oxidase activation through Ca2+-dependent as well
as Ca2+-independent pathways.
The O2–-generating NADPH oxidase is a multicomponent
enzyme comprised of a membrane-bound cytochrome b558
unit and several cytosolic factors that translocate to the membrane and form the active enzyme complex upon neutrophil
activation (10, 14). The phosphorylation of the cytosolic protein p47phox is one of the early and key events in NADPH
oxidase assemblage and is correlated with O2– generation
(10). Other cytosolic factors that appear to be important are
p67phox and Ras-related low-molecular weight GTP-binding
protein, Rac2. Both p67phox and Rac2 requirements for O2–
generation have been shown in human neutrophils. The
cytosolic protein p47phox can be phosphorylated at multiple
sites. All three cytosolic factors translocate to the membrane
and participate in the activation of NADPH oxidase. The
upstream signaling contributing to activation of the cytosolic
factors (p47phox, p67phox, and Rac2) is not definitively
known. PKC would appear to be involved in the activation of
p47phox. Both PKC activation and an inhibitory modulation
of protein phosphatases play a role in the p47phox phosphorylation and related O2– production. Rac activation appears to
require src family or other PTK. Thus the signaling components immediately proximal to NADPH oxidase activation
are phosphoprotein products of both Ca2+-dependent and
Ca2+-independent pathways.
Signaling modulations in burn inflammation: relationship
to neutrophil priming
Although there is little feasibility of directly measuring O2–
production by tissue neutrophils under pathological conditions in vivo, there is evidence that circulating neutrophils
under such conditions have an enhanced ability to produce
O2– and to inflict oxidative damage to host tissues (10).
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[Ca2+]i is attributable to emptying of intracellular Ca2+ stores;
this transient increase is followed by a somewhat sustained
influx of Ca2+ from the extracellular space. The influx of Ca2+
appears to be regulated by the state of filling/depletion of
intracellular Ca2+ stores. Although the downstream molecular
targets of elevated [Ca2+]i in neutrophils are not well understood, Ca2+ signal is known to synergize the stimulation of
PKC by DAG. In neutrophils, the most abundant PKC isoform
is PKC-β, whose activation through translocation to the
plasma membrane is dependent on Ca2+. A direct activation
of PKC, bypassing signaling through PLC, via neutrophil stimulation with phorbol 12-myristate 13-acetate (PMA) is known
to stimulate NADPH oxidase. PKC activation can also occur
in neutrophils independently of the upstream Ca2+ signal,
since chemotactic mediators are known to stimulate phosphatidylcholine-specific phospholipase D (PLD) to lead to
generation of the PKC activator DAG (Fig. 2A). Whereas earlier studies had shown involvement of PLC/Ca2+/PKC signaling
in neutrophil O2– generation (6), later studies provided evidence
that signaling to O2– production could also occur independently of the activation of this pathway. Ca2+/PKC-independent pathways, demonstrated under conditions that abrogate
Whereas a number of studies of blood neutrophils harvested
from human trauma and burn patients have ascertained the
enhanced O2– response and have evaluated the roles of various inflammatory mediators in its generation (1), fewer studies have investigated intracellular signaling mechanisms
responsible for the enhanced response. Recent studies from
the author’s laboratory assessed the signaling and O2–
responses in blood neutrophils from rats with 25–30% total
body surface area burns (13). The rat model of thermal injury
allows for evaluation of effects of burn/trauma inflammation
during an early (1–3 days postburn) and a late (7–10 days
postburn) injury phase. In neutrophils isolated in physiological media, O2– generation was measured after stimulating
neutrophils with the chemotactic mediator f-MLP. The f-MLPstimulated O2– production 1–3 days postburn was substantially
greater than the marginal response observed in control, sham,
or burn rats 7–10 days postinjury. Thus neutrophil respiratory
burst occurred during the early but not late burn inflammatory
phase. This is in keeping with the concept that hyperactivation
of neutrophils in the early periods after severe burns is followed by decreased neutrophil responsiveness (1).
[Ca2+]i in neutrophils was measured before and after f-MLP
stimulation using fura 2 fluorescence spectroscopy and
imaging techniques (13). A substantially greater elevation in
[Ca2+]i occurred on neutrophil stimulation with exogenous
f-MLP in the 1–3 day postburn rat groups than in control,
sham, or 7–10 day postburn groups. These results clearly
indicated enhanced responsiveness of Ca2+ signaling in
neutrophils during early burn inflammatory phase. Assessments of PKC activation (translocation of the protein kinase
activity from cytosol to membrane) in control, sham, and
burn rat neutrophils, with and without f-MLP stimulation,
yielded results indicating a close relationship between PKC
and Ca2+ signaling changes. The enhanced Ca2+ and PKC
signaling in the early phase after burn was correlated with
the increased O2– production response (Fig. 3). The observed
enhanced signaling and effector response can be rationally
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attributed to priming of neutrophils, in vivo, during early
periods of burn inflammation.
Previous studies have shown that neutrophils that were first
primed by TNF-α or GMCSF and then stimulated with Fc
ligand (immune complexes) exhibited a greater elevation in
[Ca2+]i compared with that observed in unprimed neutrophils.
Furthermore, these studies showed that, whereas [Ca2+]i elevation in unprimed neutrophils stimulated with the Fc ligand
was due primarily to Ca2+ release from the intracellular
stores, an influx of Ca2+ from the extracellular fluid contributed to the observed Ca2+ signal in the primed neutrophils
(15). The upregulation of neutrophil Ca2+/PKC signaling, in
our study, also could have resulted from Ca2+ influx, because
it was abolished when the rats were treated with the Ca2+
entry blocker diltiazem (Fig. 3). The diltiazem treatment of
rats also abrogated the enhanced O2– response in the burn rat
neutrophils (Fig. 3). Because Ca2+ blocker prevented the
upregulation of both Ca2+/PKC signaling and O2– production in neutrophils that would be presumably primed in vivo
after burn injury, Ca2+ influx blockage appeared to have intercepted the priming process. A role of Ca2+ signaling upregulation in enhanced O2– production has also been shown in
neutrophils primed with growth hormone and subsequently
stimulated with f-MLP (12). Ca2+ signaling upregulation in the
primed neutrophils can apparently contribute to enhanced
O2– response via potentiating the PKC pathway to NADPH
oxidase activation. In addition, in the case of stimulation of
previously primed neutrophils by a serpentine receptor agonist, NADPH oxidase may understandably be activated to a
maximum possible level due to potential parallel activation
of both Ca2+-dependent and Ca2+-independent pathways. A
somewhat similar maximum potentiation of the O2– response
may also occur due to parallel activation of the Ca2+-dependent and -independent pathways after the stimulation of the
primed neutrophils with the Fc receptor ligands.
Figure 4 shows a diagrammatic representation of the
above-described potential stimulations of neutrophils in a
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FIGURE 3. Intracellular Ca2+ concentration ([Ca2+]i), PKC, and O2– responses in blood neutrophils (stimulated with 1 µM f-MLP) from sham and burn rats. Burn
rats were subjected to 25–30% total body surface area scald burns (98ºC for 5 s), and their blood neutrophils were obtained on day 3 postburn. Sham rats were
treated similarly to burn group except 25–30% of total body surface area was exposed to 37ºC. O2– was measured using ferricytochrome reduction assay, [Ca2+]i
was determined using fura 2 microfluorometry, and PKC activation was determined measuring translocation of protein kinase activity into cell membrane fraction. Also shown is effect of treatment of rats with Ca2+ blocker diltiazem (DZ) on [Ca2+]i, PKC, and O2– responses. DZ was administered to rats at 2 and 24 hours
after sham or burn procedures. Neutrophils were obtained from animals 3 days after sham or burn procedures. Figure was drawn using data published in Ref. 13.
milieu of mediators such as those found in the inflammatory
phase of burn/trauma injury. The resting neutrophil might be
initially stimulated by ligands binding to the single- and/or
seven-transmembrane-domain receptors leading to neutrophil priming. During priming, the signaling pathways
involved would primarily be the PTK-dependent and Ca2+independent pathways if single peptide receptors were activated. On the other hand, if both the single and sevenpeptide receptors were to be triggered, there is the possibility that both Ca2+-independent and Ca2+-dependent pathways could be activated in a parallel manner. Yet in both scenarios, O2– production would be no more than a marginal
response as observed in neutrophils harvested from burn rats
before they were stimulated with f-MLP in vitro. The primed
neutrophils might be activated, in vivo, via actions of the
seven-transmembrane-domain ligands or Fc/CR/β2-integrin
ligands to lead to an overt and potentially intense O2– generation due to cumulative activations of the Ca2+-dependent
and Ca2+-independent pathways (as shown in Fig. 4). The
finding of the dependency of the O2– response in burn rat neutrophils on Ca2+ influx allows for a speculation that not only
a cumulative but also an interactive/synergic activation of the
Ca2+-dependent and Ca2+-independent pathways leads to the
overt O2– response. Whereas the maximum potentiation of
primed neutrophils after stimulation with the serpentine
receptor agonist could plausibly occur in circulation where
both priming and “nonpriming” mediators might be present,
the potentiation with Fc receptor stimulation is likely with the
formation of immune complexes in inflammatory conditions.
The efficacy of diltiazem treatment of postburn rats in modulating Ca2+ signaling upregulation and respiratory burst has provided clues to potential therapeutic regimens for abrogating
neutrophil priming in injury conditions (13). Diltiazem’s effect
was likely due to its ability to block Ca2+ influx in neutrophils.
The efficacy of Ca2+ entry blockers may be limited to neutrophil
priming phenomena in which respiratory burst is sensitized by
Ca2+ influx into the primed neutrophils. An alternative way of
abrogating neutrophil priming would be to target the initial
priming process itself resulting from the actions of primer agonists on resting neutrophils. Inasmuch as initial priming may be
triggered in vivo via activation of a variety of neutrophil receptors,
namely Fc, β2-integrin, serpentine, and single-transmembranedomain receptors, therapeutic abrogation of initial priming
would seem at the outset to require multiple regimens. However,
as discussed in the preceding section, priming through all of
these receptor systems seems to be more or less universally
dependent on activation of PTKs. Thus PTK blockers and Ca2+
blockers both remain as prospective therapeutic agents against
neutrophil priming in pathophysiological conditions prevailing
in early inflammatory phase of trauma/burn injuries.
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FIGURE 4. Schema showing priming of resting neutrophils and activation of primed neutrophils. Resting neutrophil receptors identified are 7- and single-transmembrane-domain, Fc, and β2-integrin (also known as CR3 or CD11b/CD18) types. Neutrophil priming can occur via actions of TNF-α, GMCSF, and/or GCSF
receptors (A and C) to prime neutrophils, or via f-MLP, LTB4, C5a, IL-8, PAF (B); whereas former ligands act primarily via activation of PTK, latter presumably activate both PTK and PLC. PLC activation leads to Ca2+-dependent signaling, whereas PTK activation can lead to Ca2+-independent priming of NADPH oxidase without actual O2– production. Activation of primed cells can occur via 7-transmembrane-domain (A and B) or FcR/β2-integrin receptors (C), leading to NADPH oxidase activation, resulting in overt O2– production. PLC on open background indicates PLC-β, and PLC on shaded background indicates PLC-γ.
Concluding remarks
I gratefully acknowledge the invaluable editorial assistance of my wife
Delphine Sayeed and the skillful help of Megan Flanagin in the preparation
of the figures.
The research in my laboratory was supported by National Institutes of
Health grants GM-53235 and GM-568501.
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Key Roles of Renal Aquaporins in Water Balance
and Water-Balance Disorders
Søren Nielsen, Tae-Hwan Kwon, Jørgen Frøkiær, and Mark A. Knepper
The discovery of aquaporins by Agre and co-workers provided an answer to the long-standing
biophysical question of how water can pass cell membranes. The identification and characterization
of several aquaporins expressed in the kidney has allowed detailed insight, at the molecular
level, into the fundamental physiology and pathophysiology of renal water metabolism.
R
enal regulation of body water balance involves reabsorption
of water in the proximal nephron and vasopressin-regulated
S. Nielsen, T.-H. Kwon, and J. Frøkiær are in the Department of Cell Biology,
Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark, and
M. A. Knepper is in the Laboratory of Kidney and Electrolyte Metabolism,
National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, MD 20892.
136
News Physiol. Sci. • Volume 15 • June 2000
water reabsorption in the collecting duct. At least six aquaporins (AQP1, -2, -3, -4, -6, and -7) are presently known to be
expressed in the kidney (Table 1). AQP1 is highly abundant in
the proximal tubule and descending thin limb, and several studies have now underscored its important role in constitutive
water reabsorption in these segments. AQP1 is absent in other
tubule segments. At least three aquaporins are known to be
expressed in the kidney collecting duct, and they participate in
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Neutrophil intracellular signaling triggered by neutrophil
activating agents have been extensively studied in vitro. The
signaling pathways leading to neutrophil O2– generation
include both the classical PLC-mediated Ca2+/PKC sequence
as well as a variety of novel protein kinases not dependent on
the Ca2+ signal, namely PTK, MAPK, and PI3K. The recent burst
of knowledge about the roles of the novel kinases in modulating target proteins implicated in the O2– response generation
and the extensive presence of the Ca2+-independent pathways
has appropriately taken the limelight away from Ca2+ signaling.
The Ca2+-independent pathways do play critical roles not only
in actual generation of O2– by neutrophils but also in priming
neutrophils and enhancing their potential to produce O2– without an actual production. Nevertheless, the activation of
preprimed neutrophils could potentially occur via either the
Ca2+-independent or the Ca2+-dependent sequences as redundant pathways. Recent studies in neutrophils harvested from
inflammatory conditions prevailing after burn/trauma injuries
indicate that the activation of the primed neutrophils, in vivo,
may result from potentiating interactions between the Ca2+linked and the Ca2+-independent pathways.