Trauma/Critical Care Labs, Department of Surgery, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153
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ABSTRACT |
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The role of platelet-activating factor (PAF) in Ca2+ signaling and Ca2+-related enhancement of reactive oxygen intermediate (ROI) generation in neutrophils of burn-injured rats was ascertained by evaluating the effect of treatment of the rats with a PAF receptor antagonist. The treatment of rats with the antagonist also allowed us to evaluate the role of PAF in the priming of neutrophil ROI response with burn in vivo. A full skin thickness burn injury was produced in anesthetized rats by exposing 30% of total body surface area to 98°C water for 10 s. Sham and burn rats were killed 1 day later, and their blood was collected to obtain neutrophils. Fluorescence-activated cell sorter analysis was used to quantify ROI production by the neutrophils. Cytosolic-free Ca2+ concentration ([Ca2+]i) imaging technique was employed to measure neutrophil [Ca2+]i in individual cells and microfluorometry for the assessment of [Ca2+]i responses in suspensions of neutrophils. There was an overt enhancement of ROI generation by burn rat neutrophils. ROI release was accompanied by a marked elevation of [Ca2+]i signaling. The treatment of rats with PAF receptor antagonist before burn prevented the upregulation of both [Ca2+]i and ROI generation in neutrophils. These studies indicate that enhanced ROI production in neutrophils in the early stages after burn injury results from a PAF-mediated priming of the [Ca2+]i signaling pathways in vivo.
burn; rat; polymorphonuclear neutrophils; platelet-activating factor; reactive oxygen intermediates; calcium-protein kinase C signaling; platelet-activating factor blockade
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INTRODUCTION |
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NEUTROPHILS PLAY A CENTRAL
ROLE in generating toxic oxygen radicals during inflammatory
conditions. A membrane-bound multicomponent NADPH oxidase is
responsible for the production of oxygen radicals. In resting
neutrophils, this enzyme complex consists of unassembled plasma
membrane and cytosolic components (31, 44). After
neutrophil activation, the cytosolic components p40phox,
p47phox, p67phox, and Rac-2 translocate to the
membrane where they associate with flavocytochrome b558, a
heterodimer comprised of gp22phox and gp91phox,
and Rap1A to form the active oxidase complex (4, 40). The respiratory burst is enhanced by a number of proinflammatory mediators, such as tumor necrosis factor (TNF)- (53),
granulocyte/macrophage colony-stimulating factor (52), and
lipopolysaccharide (49), which generally do not stimulate
respiratory burst activity on their own. Some of the chemotactic
mediators that activate NADPH oxidase and stimulate respiratory burst
are activated serum complement C5a,
N-formylmethionyl-leucyl-phenylalanine (FMLP), and bioactive lipids PAF and leukotriene B4 (4). The
enhancement of respiratory burst activity is referred to as priming.
Previous in vitro studies have shown that stimulation of resting
neutrophils by a mediator results in either enhanced respiratory burst
or priming of the respiratory burst mechanism for a subsequent enhanced
production of O
into human volunteers (53).
It is known that PAF levels are increased in the course of injuries such as burn, sepsis, or ischemia and reperfusion (1, 10, 11, 19, 46). PAF is released from macrophages, neutrophils, and vascular endothelial cells (9, 26, 27, 35, 36). Studies of neutrophils, which were isolated from injured animals (13, 25) or injured patients (5, 58), have shown that PAF receptor modulations were involved in the respiratory burst responses during injury. Other studies have addressed the involvement of PAF in the neutrophil priming process. In the latter studies, neutrophils from healthy volunteers were exposed to sera from trauma patients in the presence or absence of the PAF receptor antagonist (PAFra) or neutrophils from trauma patients were themselves stimulated in vitro in the presence or absence of the PAFra (5). Thus the occurrence of neutrophil priming with burn or trauma injury was primarily ascertained through in vitro experiments. A major objective of this study was to evaluate the role of PAF priming of neutrophil in vivo with burn injury. PAF that caused modulation was examined in rats treated with a PAFra (PAF-16) before subjecting them to a 30% total body surface area (TBSA) full-thickness burn.
Despite the implications of certain inflammatory agents contributing to
neutrophil priming during the course of burn or trauma injury, little
is known about the intracellular signaling mechanisms causing such
priming. A role of protein kinase C (PKC) and subsequent protein
phosphorylations in the O isoform (29).
The Ca2+-dependent PKC isozyme has been shown to
phosphorylate the cytosolic proteins p47phox and
p67phox, which are then translocated to the membrane before
NADPH oxidase activation and O
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MATERIALS AND METHODS |
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Animals. Adult male Sprague-Dawley rats weighing 250-275 g were obtained from Harlan Sprague Dawley (Indianapolis, IN). The rats were acclimatized in the animal quarters for 3 days before use. The care of animals was in accordance with the guidelines set by Loyola University Chicago Medical Center Animal Care and Use Committee.
Thermal injury protocol. The animals were anesthetized with an intraperitoneal (ip) injection of pentobarbital sodium (45 mg/kg body wt). The hair on the animals' backs was clipped off. The animals were then placed in a supine position in a plastic template that exposed 30% of the TBSA. In the sham group, the exposed backs were immersed for 10 s in a water bath at room temperature. In the burn group rats, full- thickness skin scalds were inflicted by immersing the back of the animal to 95°C (203°F) water for 10 s. Rats were quickly dried after the exposure to hot water to avoid additional injury. The animals in each group were resuscitated with 10 ml ip of normal saline. The animals were housed in cages after thermal injury and killed at 24 h postinjury after being anesthetized (45 mg/kg) and exsanguinated through a cardiac puncture.
PAFra administration. PAF-16 antagonist (0.35 mg/kg body wt; Calbiochem, La Jolla, CA), dissolved in 1.0 ml of normal saline per animal, was injected intravenously 1 h before subjecting the animals to thermal injury. Preliminary experiments were performed to determine the dose of PAF-16 antagonist in our rat model of burn injury following IC50 values given in in vitro studies reported by Tokumura et al. (45).
Preparation of peripheral neutrophils.
At the time of death, rats were anesthetized with 40-50 mg/kg ip
pentobarbital, and the blood (10-12 ml) was collected into heparinized syringes by means of cardiac puncture. Neutrophils were
isolated from the heparinized blood using the standard Ficoll-Paque (Pharmacia) cell separation technique followed by dextran sedimentation and hypotonic red blood cell lysis. Neutrophils were then
washed and resuspended in HBSS (Hanks' balanced salt solution) buffer. Neutrophil preparations routinely contained 95% neutrophils, as
identified by the Giemsa stain, and were found to be
98% viable by
the trypan blue exclusion technique.
Flow cytometric assay for measurement of ROI.
Fluorescence-activated cell sorter (FACS) analysis determined the
ability of neutrophils to generate an oxidative burst by indirectly
measuring the increase in fluorescence generated by the oxidation by
O
Calcium measurements in cell suspensions.
The [Ca2+]i in suspension of neutrophils was
determined by microfluorometry. Neutrophils (3 × 106
cells) were loaded with 2 M fura 2-AM (Molecular Probes). After being
washed, the cells were resuspended in HBSS (GIBCO RBL), and the
fluorescence signals of a 0.5-ml stirred neutrophil suspension were
monitored in an F-2000 Hitachi spectrofluorometer using 340- and 380-nm
excitation wavelengths and a 510-nm emission wavelength. The
fluorescence ratio (R = F340/F380) was
calculated and converted to [Ca2+]i using the
equation described by Grynkiewicz et al. (21): [Ca2+]i = Kd[(R Rmin)/(Rmax
R)]b, where
Rmax = F340/F380 (with Ca2+), Rmin = F340/F380 (without Ca2+), and
b = F380 (without Ca2+)/F380
(with Ca2+). Kd = dissociation constant.
Fura 2 Ca2+ microscopic imaging. Neutrophils suspended in HBSS were loaded with 10 µM fura 2-AM (Molecular Probes) for 1 h at room temperature. A drop of neutrophil suspension (100 µl) was placed on a 1-µm-thick coverslip and examined with the 40× oil-immersion objective of an inverted Nikon microscope. Computerized fura 2 ratio imaging was then performed with the aid of MetaFluor software (series 4.5; Universal Imaging, West Chester, PA) and the associated hardware, including a SenSys charge-coupled device camera (Photometrics) and a Metaltek shutter. With this setup, real-time fluorescent images were generated by exposing cells to alternating 340- and 380-nm excitation wavelengths, automatically collecting associated fura 2 emissions through a 505-nm band-pass filter (30). The following steps were followed for each cell sample. First, cells were focused for optimal fluorescent fura 2 signal, and background correction was set up based on an adjacent blank part of the coverslip. Second, a field of cells was chosen, and the 340- to 380-nm ratio images were optimized. Third, a computer-controlled series of 30 subsequent images was acquired at 20-s intervals. Of the 30-point image series, the first five were kept as baseline images as cellular stimulation with 1 µM FMLP administered in vitro after the fifth point (100 s). Fourth, acquired data were then used to generate 340/380 ratio images and curves for analysis and presentation.
Statistical analysis. The data were analyzed using the Sigma Statistical program (SPSS, version 2.0; SigmaStat). P < 0.05 between two groups was considered statistically significant.
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RESULTS |
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ROI generated subsequent to activation of the NADPH oxidase system
were measured in neutrophils before and after stimulation with FMLP (1 µM) or PMA (100 ng/ml) via flow cytometric analyses. ROI production
was measured in units of mean channel fluorescence (MCF) of
dihydrorhodamine 123 labeling of FMLP- or PMA-stimulated neutrophils.
Figure 1 shows representative
FACS analyses after dihydrorhodamine 123 labeling of purified
neutrophils. MCF of an unlabeled cell (M1) was taken as the gating
control. Increases in MCF were evident in FITC-labeled sham animal
neutrophils stimulated with FMLP (Fig. 1A,
left) or PMA (Fig. 1A, right) over
burn animal neutrophils (Fig. 1B, left, or Fig.
1B, right). Nonstimulated FITC-labeled cells from
sham or burn animals did not show any detectable dihydrorhodamine 123 uptake, indicating little ROI production (data not shown).
PAFra treatment of burned animals significantly attenuated the
dihydrorhodamine 123 labeling of neutrophils stimulated with FMLP (Fig.
1C, left) or PMA (Fig. 1C,
right), compared with the labeling in the untreated burn
neutrophils.
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Figure 2 shows pooled MCF values from at
least 10 animals in the sham or burn group. The peak ROI production by
burn rat neutrophils stimulated with PMA (75 ± 4 MCF, means ± SE) or FMLP (70 ± 3 MCF) was significantly higher
(P < 0.05) than in the sham (PMA, 45 ± 3; FMLP,
35 ± 2) or the PAFra burn group (PMA, 50 ± 4; FMLP, 45 ± 4). There was no significant difference between the peak ROI
production in the sham- or PAFra-treated burn rats after PMA or FMLP
stimulation (Fig. 2, A and B). Thus PAFra
pretreatment effectively prevented the burn-caused enhancement of
neutrophil ROI production.
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The [Ca2+]i measurements in
suspensions of neutrophils, from sham and burn rats, are shown in Fig.
3. The burn injury caused elevations in
both basal [Ca2+]i and FMLP-mediated
[Ca2+]i responses. The basal
[Ca2+]i in neutrophils from the burn group
(130 ± 10 nM, means ± SE) was significantly higher
(P < 0.01) than in the sham group (100 ± 7 nM).
The FMLP-induced peak [Ca2+]i in neutrophils
from burn rats (480 ± 11 nM) was also significantly higher
(P < 0.05) than the [Ca2+]i
response to FMLP in the sham rats (320 ± 12 nM). In the sham rat
neutrophils, there was no effect of PAFra treatment on the FMLP-mediated peak [Ca2+]i (P > 0.05), but there seemed to be an increase in the basal [Ca2+]i after the treatment
(P < 0.05). The FMLP-mediated peak
[Ca2+]i in the PAFra-treated burn rats was
significantly less (P < 0.05) than in the untreated
burn group. There was no demonstrable effect of PAFra treatment
(P > 0.05) on the basal
[Ca2+]i in the burn rats. The difference
between the basal and the FMLP-induced peak
[Ca2+]i levels
([Ca2+]i) in the burn group (355 ± 11) was significantly higher (P < 0.05) compared with
[Ca2+]i values in the sham groups with
(230 ± 9) and without (235 ± 4) PAFra treatment (Fig. 3).
The pretreatment of burn rats with PAFra caused a significant
(P < 0.05) attenuation of
[Ca2+]i response (155 ± 8).
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The heightened [Ca2+]i response with FMLP in
neutrophil suspensions from the burn group relative to the sham group
was confirmed by [Ca2+]i measurements via
Ca2+ imaging of individual neutrophils (Fig.
4). Figure 4 shows both the
pseudocolor-digitized Ca2+ images and digitized fluorescent
ratios (F340/F380) obtained in neutrophils from
a representative sham and a representative burn rat. The frequency of
cells showing pseudoblue and pseudogreen color, representing quiescent
resting cells, seemed to be comparable in the sham-, burn-, and
PAFra-treated burn animals. Upon stimulation with FMLP, a significant
number of sham rat cells transformed into cells with pseudocolors in
the yellow and red range, corresponding to an image ratio of ~1.3.
The FMLP stimulation of burn rat neutrophils clearly caused their
transformation into the pseudocolors red and white range, corresponding
to the image ratio of >2. Imaging of individual neutrophils confirmed
that FMLP-induced [Ca2+]i elevations in the
burn group were markedly higher than cells in the sham group. Figure 4
also shows the elevation in the digitized fluorescent ratios,
representing [Ca2+]i as a function of time
after FMLP stimulation of neutrophils from sham and burn rats with and
without PAFra treatment. Although the Ca2+ image analyses
did not show a measurable effect of treatment with PAFra on the basal
[Ca2+]i or FMLP-mediated
[Ca2+]i elevation in the sham animals, these
analyses in burn rat neutrophils showed a pronounced effect of PAFra
treatment on FMLP-mediated [Ca2+]i responses
but not on basal [Ca2+]i. The image analyses
reinforce that PAFra treatment of burn rats led to a marked attenuation
of [Ca2+]i responses to FMLP.
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DISCUSSION |
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In the present investigation, we obtained evidence that PAF contributes to hyperactivation of circulating neutrophils in vivo in an early stage of inflammation following burn, namely, day 1 postburn. The hyperactivation of neutrophils is evident from enhanced ROI production in conjunction with upregulated Ca2+ signaling, which is essential for the respiratory burst through NADPH oxidase activation (4, 22, 42, 54). PAF is known for its ability to enhance adherence, chemotaxis, and respiratory burst of neutrophils (9, 28, 37, 38, 41, 48, 50). Clinical and laboratory studies have demonstrated that PAF metabolites are released in circulation during inflammatory conditions such as acute respiratory distress syndrome, burn, trauma, sepsis, and intestinal ischemia-reperfusion (1, 10, 11, 19, 20).
The stimulation of neutrophils with PMA would be expected to result in
a potentially maximal level of ROI production, due presumably to a
direct maximal stimulation of PKC and downstream signaling to NADPH
oxidase (15, 23, 32, 51, 56). The higher level of ROI
production with PMA than with FMLP in the sham rat neutrophils (Fig. 2)
supports the concept that FMLP contributes only partly to the
activation of PKC. The neutrophils in sham rats were likely not exposed
in vivo to neutrophil-activating chemotactic mediators such as would be
found in the burn-injured rats. Consequently, there is little
probability of their prior in vivo "priming" and/or activation by
such mediators. Thus the partial activation of sham rat neutrophils'
oxidant response by FMLP, relative to that by PMA, might represent
neutrophils' innate responses without their prior priming or
activation by the mediators. In the burn rat neutrophils, there was not
only an absence of a difference in the effects of PMA and FMLP on ROI
production but also a significantly higher level of ROI production with
either PMA or FMLP, compared with that in the sham rat neutrophils. The increase in ROI production with PMA in burn injury over the
PMA-mediated ROI production in the sham group suggests that a burn
inflammatory condition caused an upregulation of PKC signaling, leading
to high ROI production. The upregulation of PMA-mediated ROI in burn injury could possibly result from an in vivo overactivation of signaling events proximal to PKC. The fact that blockade of endogenous PAF in the burn animals effectively attenuated PMA caused ROI production to the level found in the sham animal neutrophils supports that PAF could modulate PKC signaling. A possible action of PAF released after burn could be priming of neutrophils in vivo such that a
secondary action of a neutrophil-activating agent, e.g., exogenous PMA
or FMLP, would produce a potentiated production of ROI compared with
that occurring in neutrophils without priming. The absence of the
difference in the effects of PMA and FMLP on ROI production by a burn
rat neutrophil could also be related to endogenous neutrophil priming
via actions of burn-related PAF. Priming per se may upregulate
signaling such that a subsequent activation of neutrophils via FMLP
leads to ROI production equal to that obtained with PMA. Previous in
vitro studies have shown that when neutrophils were primed with PAF and
subsequently stimulated with FMLP, the neutrophil O
In neutrophils, a key signaling step either parallel or upstream to PKC
activation is [Ca2+]i mobilization (23,
24). Because the Ca2+-dependent PKC isoform,
PKC-, seems to be abundant in neutrophils (29), it is
reasonable to assume that [Ca2+]i elevation
could play a role in PKC activation. However, there is also evidence
that PKC activation could occur via Ca2+-independent
upstream signaling events (17, 24). The greater magnitude
of the [Ca2+]i response to FMLP in the burn
rat neutrophils than that in the sham rat neutrophils (Fig. 3) may be
responsible for the higher potentiated PKC activation and consequent
ROI production. This consideration allows us to conclude that PAF
blockade in burn-injured rats may prevent the upregulation of the
Ca2+-linked PKC signaling pathway to prevent the
potentiated neutrophil ROI response. A number of previous studies have
implicated a role of PAF in the priming and/or activation of
circulating neutrophils during burn/trauma injury conditions (1,
10-13, 35, 36, 43, 50). However, these studies assessed the
PAF role primarily via measurements in isolated neutrophils from
healthy human volunteers and from injured patients or animals with such
injuries, incubated with PAF in the absence and/or presence of the
PAFra. In other studies, the investigators demonstrated neutrophil
priming with burn via measurements in healthy human volunteer blood
neutrophils incubated with plasma from burn patients along with PAFra
in vitro (5). The present study reinforces the role of PAF
in blood neutrophil priming via endogenous PAF blockade in vivo in
burn-injured animals.
Our present finding of an increase in neutrophil basal
[Ca2+]i with burn injury is in agreement with
previous studies (39). This increase was interpreted to
indicate an upregulation of Ca2+ signaling with burn injury
in vivo. In this study, although PAFra effectively abrogated
FMLP-mediated [Ca2+]i response, it did not
have an effect on the burn-induced increase in basal
[Ca2+]i. This implies that while endogenous
PAFra could modulate Ca2+ signaling triggered by exogenous
FMLP in the presumably burn-preprimed neutrophils, it could not prevent
an endogenous activation of neutrophil Ca2+ signaling
reflective in the measured level of basal
[Ca2+]i. It is plausible that such endogenous
Ca2+ signaling activation in burn rat neutrophils may not
be related to endogenous PAF action. The lack of effect of PAFra on the
basal [Ca2+]i increase in burn rat
neutrophils may also be related to the observed decrease in the
Ca2+ response (FMLP-mediated
[Ca2+]i
basal
[Ca2+]i) below the level of
[Ca2+]i response in the sham rat
neutrophils. This is accounted by the observed decrease in
Ca2+ (sham vs. PAFra-treated burn) in the absence of a
difference between the FMLP-mediated peak
[Ca2+]i responses in the sham and
PAFra-treated burn rat neutrophils.
Several studies have suggested that early neutrophil hyperactivation following burn, trauma, and ischemia-reperfusion episodes contributes to the development of multiple organ failure (2, 3, 14, 32). PAF release is associated with hemodynamic instability and organ or cellular dysfunction in a wide range of human (1, 10, 19, 20, 33, 58) and animal (11, 18) conditions of systemic inflammation. Previous studies have also indicated that PAFra, administered early in gram-negative bacteremia or endotoxemia in rodents, reduces pulmonary hypertension and capillary leak (13). These findings implicate PAF as a significant early mediator of cellular dysfunction, microvascular leak, and pulmonary vasoconstriction in the acute stages of sepsis.
PAF-mediated priming of neutrophils following postischemic gut injury in patients has been shown to be related to decreased circulating levels of PAF-acetylhydrolase (PAF-AH), which hydrolyzes PAF to lyso-PAF (35). Furthermore, reduced levels of PAF-AH have been associated with the development of multiple organ failure (MOF) (35). Other findings suggest that endogenous PAF and interleukin (IL)-8 sequentially prime neutrophils in patients with trauma at the risk of MOF (5). Plasma of such patients, when treated with PAF antagonist, inhibited priming of neutrophils and blocked their capacity to release superoxide (5). Studies have also associated PAF production in an acute phase of injury with an increase in neutrophil cytosolic Ca2+ (7). In these studies, the investigators found IL-6 and PAF via activation of phospholipase A2 primed neutrophils in the gut (7). However, IL-6 alone could not directly prime neutrophils for superoxide release; it synergized with PAF in vitro to prime neutrophils (8). It has been hypothesized that ischemia of gut (6) or gut ischemia-reperfusion injury (8) serves as a priming bed for circulating neutrophils which then initiates MOF.
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ACKNOWLEDGEMENTS |
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We thank Drs. Xiapong Ren, Shafeeq Khan, Shahla Namak, and Farah Haque for technical assistance.
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FOOTNOTES |
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This research was supported by National Institute of General Medical Sciences Grants RO1-GM-56865 and RO1-GM-53235.
Address for reprint requests and other correspondence: M. M. Sayeed, Dept. of Surgery, Loyola Univ. Chicago Medical Center, 2160 S. First Ave., Maywood, IL 60153 (E-mail: msayeed{at}luc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 February 2001; accepted in final form 16 May 2001.
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