JOURNAL
OF SURGICAL
RRSRARCH
62,258-264
Platelet-Activating
(19%)
Factor Augments Tumor Necrosis Factor and Procoagulant Activity’ v2
RONALD V. MAIER, M.D., FACS,3
GREGB. HAHNEL, M.S., AND J. RAYMOND FLETCHER, M.D., FACS
Departments of Surgery, University of Washington, Seattle, Washington; and the University of South Alabama, Mobile, Alabama Submitted for publication October 22, 1990
Infusion of platelet activating factor (PAF) reproduces the host physiologic response to endotoxemia and sepsis. Tumor necrosis factor (TNF) and procoagulant activity (PCA) are two other potentially deleterious central inflammatory mediators produced in large quantities by tissue-fixed macrophages (Md). The relationship, if any, between PAF and TNF or PCA production is unknown. Rabbit alveolar M4 were treated in vitro with PAF alone and prior to endotoxin (LPS). PAF alone had no effect on M4 PCA or TNF. PAF ( 1O-s-1O-6 1M) cotreatment enhanced M& PCA and TNF levels in a dose response from two- to sixfold above that of LPS treatment alone. PAF (lo-* M) pretreatment of M# at T -4 to -6 hr produces an eight- to ninefold enhancement in both TNF and PCA levels. Thus, both coincubation and pretreatment or “priming” of the M4 with PAF prior to LPS stimulation greatly increase Mr$ production of PCA and TNF. The ability to augment the production of these two potent inflammatory mediators may explain in part the mechanism of action of PAF in UiUO.
0 1992 Academic Press, Inc.
INTRODUCTION
Infectious complications continue to be a major cause of morbidity and mortality in the critically ill surgical patient. Invasive infection, producing endotoxemia and sepsis, is a common etiology for multiple organ failure syndrome (MOFS) and, in particular, adult respiratory distress syndrome (ARDS) [ 11. The diffise organ injury seen during MOFS is thought to be caused by the host’s own inflammatory response to the invading organisms [2]. Many of the central mediators of this uncontrolled inflammatory response have been identified. Although these mediators are produced by many different cell
i Supported in part by NIH Grant GM35361. ’ Presented at the Tenth Annual Meeting of the Surgical Infection Society, Cincinnati, OH, June 14-16, 1990. 3 To whom request for reprints should be addressed at Department of Surgery, ZA-16, Harborview Medical Center, 325 Ninth Avenue, Seattle, WA 98104. 0022.4so4/92 51.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
258
types, the tissue-fixed macrophage (M$J), which is found in the various injured organs of MOFS, produces large quantities of these major inflammatory mediators [3]. Additionally, endotoxins or lipopolysaccharides (LPS), derived from the outer membrane of gram-negative bacteria, are extremely efficient in inducing the production of these inflammatory mediators by M@ and in reproducing the pathophysiologic changes seen clinically during sepsis and MOFS [4]. The Mb is capable of producing virtually all of the recognized mediators of the inflammatory response, including complement components, arachidonic acid metabolites, interleukin-1,6, and8 (IL-l, 6, and8), procoagulant activity (PCA), and tumor necrosis factor (TNF) [3,5-81. Selective inhibition of many of these inflammatory agents has been protective in experimental lethal endotoxemia [9,10]. TNF and PCA are thought to be central mediators and major contributors to the pathophysiology of sepsis [2,7,8,10-121. The tissue-fixed M$ is the primary source for both of these potent mediators, and LPS and other inflammatory stimuli induce the production of TNF and PCA by M$. PCA is produced by and expressed predominantly as a cell surface ectoenzyme. Activity can be demonstrated in association with the M4 in the microcirculation of animals within 15-30 min of LPS infusion and leads to potential diffuse organ ischemic injury during sepsis induced MOFS [2, 7, 111. Clinically, patients with MOFS and, in particular ARDS, have been shown to have diffuse microvascular thrombosis early during the organ injury phase [13]. The direct cellular effects of TNF during sepsis are unknown. However, infusion of TNF reproduces the derangements seen during endotoxemia and sepsis [ 121. In addition, passive immunization with antibody to TNF protects against lethal endotoxemia [lo]. TNF is found in the plasma of patients with overwhelming sepsis with gram-negative bacteria. And, unlike interleukin-1 and other inflammatory mediators, levels of TNF correlate with the survival outcome of sepsis [ 141. Both PCA and TNF are produced in enormous quantities by M& Together, they appear to contribute to the pathogenesis of endotoxin and sepsis. In addition to these mediators, platelet activating fac-
MAIER,
HAHNEL,
AND
FLETCHER:
tor (PAF) has been implicated recently as a central mediator of the inflammatory response [15-171. Infusion of PAF reproduces the host physiologic response to LPS and sepsis [18]. Inhibition of PAF using selective membrane receptor blockers ameliorates the pathophysiologic response to either PAF or LPS experimentally [ 16, 18-201. The mechanisms involved in PAF-induced alterations of normal physiology are not known. It is unclear whether PAF causes these changes by a direct cellular effect or primarily functions indirectly by stimulating Md and other cells to produce other critical mediators of the inflammatory response. PAF is known to interact with cells through a specific membrane receptor which is thought to function via a G-protein intracellular transduction process leading to hydrolysis of phosphytidylcholine release of Ca2+ from endoplasmic reticulum stores and activation of protein kinase C (PKC) [21-231. By measuring intracellular Ca2+ [Ca”]i, interaction of PAF with a membrane receptor that functions via phosphatidylcholine hydrolysis can be investigated. Receptor-mediated activation of the phosphytidylcholine pathway usually leads to direct cell activation. This is consistent with the known release of eicosanoids in response to PAF [20]. However, the interaction of PAF with M$J and the effect on production of other mediators is only partially elucidated. Investigation of the ability of PAF to enhance M$ production of TNF and/or PCA either directly or indirectly (by priming the M4 to subsequent stimuli) will help elucidate the mechanism of action of PAF in uiuo. Only by elucidation of these complex interactions can rational and safe therapeutic interventions be designed. In the present study, the effect of PAF on the production of TNF and PCA by a crucial M+ population involved in the pathogenesis of ARDS (the alveolar Md) is investigated. MATERIALS
AND METHODS
Alveolar macrophages (M4) were obtained from male, 1.5-2.0 kg, New Zealand white rabbits (R,. and R. Rabbitry, Stanwood, WA), using a modification of bronchoalveolar lavage described by Brain and Frank [24]. Animals were housed in standard care facilities at the University of Washington, fed rabbit chow and water ad lib and used within 10 days of delivery. Animals were euthanized with an overdose of pentobarbital infusion, the trachea is isolated using sterile technique in situ, and bronchoalveolar lavage is performed using a 5-mm catheter placed just superior to the carina. The lungs are gently lavaged six times with 50-ml aliquots of normal saline at 4°C. The combined lavage fluid aliquots were centrifuged at 500g for 7 min at 4’C, the supernatant was discarded, and the M4 were resuspended in glutamine-supplemented RPMI-1640 media (GIBCO Corp., Grand Island, NY) with 100 pg/ml of gentamicin. M$ are plated immediately in 12-well tissue culture plates (Flow Laboratories, McLean, VA) at 5 X lo5 cells/ml/
PAF AUGMENTS
M$ FUNCTION
259
well. Preliminary experiments confirm that the cells are 98-99% M4 by Wright stain histology, presence of nonspecific esterase staining, and ability to phagocytize latex beads. The M$ were greater than 95% viable by trypan blue dye exclusion [2]. All animal studies are evaluated and approved by the Department of Comparative Medicine and the IACUC of the University of Washington. Experimental
Design
The M$ were allowed to adher for 30 min prior to initiation of experimental protocols. Cultures were treated at the various time points with either LPS (Escherichia coli Olll:B4) or PAF (L-cr-phosphatidylcholine, fi-acetyl-y-0-(octadec-9-cis-enyl)), both obtained from Sigma Chemical Co. (St. Louis, MO). A dose response of LPS from 1 rig/ml to 1 pg/ml was used, unless the LPS dose was held constant (10 rig/ml). The PAF was used in a dose range from 10-l’ to lo-’ M. For the pretreatment time course using PAF, the optimal dose of 10d6 M was utilized at all time points. The specific inhibitor of the PAF receptor utilized was BN52021 (IPSEN, Paris, France) over a dose response of 2 to 40 r.Lg/ml with PAF at lo-’ M. M$J cultures were treated in triplicate under the various experimental conditions. Reagents were added simultaneously at time zero except in the pretreatment assays where PAF was added at various time points prior to LPS. Cultures were harvested 24 hr after initiation of LPS treatment. Supernatants for TNF were gently aspirated and spun X60 set in a microfuge. The cell pellet was added to the remaining M4 monolayer, which was lysed for PCA assay by three cycles of freeze thawing and scraping. The lysates and supernatants were frozen at -70°C until analyzed. Previous studies have confirmed greater than 85% of PCA is membrane associated, expressed as an ectoenzyme on the exterior of the membrane and found intact in the M$ lysates [7]. Each of experimental conditions was tested for M$ cytotoxicity at 24 hr by 51Cr release and exclusion of trypan blue dye assays and found to cause injury to less than 10% of the M$ monolayer. PCA and TNF Assays Procoagulant activity (PCA) produced by the M4 was analyzed using a modified Quick one-step coagulation assay [7]. M$ lysates were suspended in Hanks BSS at 1 X lo6 cells/ml. A O.l-ml aliquot each of normal rabbit plasma (10 mMEDTA), CaCl, (20 mM), and M4 lysates from each experimental condition were combined and, with constant agitation in a 37°C water bath, time to visual clot formation was measured. Activity is derived from a standard curve generated from log dilutions of rabbit brain-derived thromboplastin. TNF was measured using the biologic cytotoxicity assay of Flick and Gifford [25]. Transformed mouse fibro-
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blasts (NCTC clone L929 American Type Culture Collection, Rockville, MD) were pretreated with actinomytin D (Sigma) at 1 pg/ml for 15 min in RPMI-1640 supplemented with 5% horse serum (Hyclone Laboratories, Logan, UT) and added to the serial dilutions of the conditioned media at 5 X lo4 cells/well. The plates were then placed in a 37”C, 5% CO, incubator. After 24 hr the cells were fixed and stained in 0.1% crystal violet in 20% methanol. Dried monolayers were solubilized in 0.1 M sodium citrate in 50% ethanol and the absorbance read at 550 nm in a microplate reader. One unit of TNF activity was defined as that activity that produced 50% cytolysis of the L929 monolayer. A linear regression was performed to determine the point between serial dilutions of the TNF samples where the 50% cytolysis endpoint occurred. The E. coli Olll:B4 LPS was tested against the L929 cell line and showed no direct cytotoxicity. Intracellular
Calcium Levels
To further elucidate the cellular mechanism of the PAF effect on M$ function, intracellular calcium concentrations, [Ca2’]i, were monitored using a fluorescent [Ca2+]i dependent probe, Fura- AM (Sigma) [26]. Rabbit alveolar M4 at 1 X lo6 cells/ml were loaded with 100 t&f Fura- AM. This was accomplished by a slow titration of the dye into a stirred suspension of the Md held in the dark at RT for 30 min. The slow titration of the dye reduced the level of entrapped Fura- AM in organelles. The cells were loaded at RT to reduce endocytosis of the dye. These steps are used to minimize the level of unhydrolysed dye in the M& After loading, the cells were allowed to hydrolyse the ester for an additional 15 min. The M4 were then centrifuged at 1OOOgfor 7 min through a 0.5% BSA pad in buffer (145 m&f NaCl, 5 n&f KCl, 10 mM Hepes, 0.5 mM Na,HPO,, 1 mM Ca2+, and 5 mM glucose). The cells were resuspended to 1 X lo6 cells/ml in the above buffer without the BSA and placed in polypropylene tubes on ice. A 300-~1 aliquot of the cell suspension (3 X lo5 cells) was added to 1.2 ml of RT buffer in a stirred cuvette (final concentration of 2 X lo5 cells/ml). LPS or PAF at 100X were added to yield final concentrations of 100 rig/ml and 1 X 10e6 M, respectively. A Shimadzu RF 5000U Spectrofluorophotometer with stirred cuvette and the wavelengths set to the following: excitation of 340 nm (slit = 5 nm) and emission at 510 nm (slit = 10 nm) was used to determine the [Ca2’]i. Internal controls for F (minimum) and F (maximum) were run on each sample. The maximum fluorescence value was obtained by permeabilizing the cells with digitonin, thus complexing essentially all Furawith calcium. The subsequent minimum fluorescence value was gained by adding EGTA in Tris Base, (final concentrations, 10 and 33 mM, respectively, at a final pH of 8.5), to chelate any free calcium. External FuraAM was quenched with 30 pM Mn and chelated with 60
VOL.
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52, NO. 3, MARCH
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1992
1
*
T
.
TNF
m PCA
a g 20 :: eCE m 0 10 us f 0
,001
.Ol
.I
1
LPS (uglml) FIG. 1. Production of TNF and PCA by alveolar M& after 24 hr in culture with various doses of E. coli Olll:B4 LPS. Data are given as fold increase over background or control levels. N = 8-10 (means k SEM). Asterisk indicates P < 0.05 compared to control by Dunnett’s test.
pM diethylenetriaminepenta acetic acid (DTPA) to measure the extracellular fluorescence and this value was used to correct for any dye leakage from the loaded cell. The formula used for calculating [Ca2’]i was: calcium (nM) = 224X (F-F min.)/(F-,-F)) where 224 is the binding constant for calcium [26]. Analysis
of Results
Data from these studies were analyzed by the Dunnett’s multiple-comparison test for comparison between various experimental groups and a control group. A P value of ~0.05 was considered significant. RESULTS
Rabbit alveolar M$ are obtained in a consistently homogeneous (>95%) and viable (>95%) condition. Approximately 2 X 10’ M4 are obtained per animal. If neutrophils comprise greater than 5% of the differential white cell count, the animal is considered to have been ill, in vivo stimulation of the Mb has occurred, and the preparation was discarded. Endotoxin (LPS) from E. coli Olll:B4 stimulates MI$ to produce both PCA and TNF. There is a significant increase in levels of both TNF and PCA in a similar bimodal response to LPS from 1 rig/ml to 1 pg/ml, Fig. 1. PAF alone had a minimal direct effect on the production of TNF or PCA by Mb. Throughout the dose range of 10-l’ to 10e5 M, PAF increased levels over background no greater than normal biologic variation, data not shown. In contrast, LPS alone increased levels 10 to 30+ fold above background, Fig. 1. Simultaneous treatment of M4 with LPS (10 rig/ml) and PAF over a dose curve of 10-l’ to lop5 M shows a significant increase in the production of TNF and PCA at lOA M; Fig. 2. The levels of TNF and PCA shown are the fold increase above the already lo- to ZO-fold increases produced in response to
MAIER,
-10
-9
-8
HAHNEL,
-7
PAF Concentration
AND
-6
FLETCHER:
PAF AUGMENTS
-5
Time (Hrs. of Pretreatment)
(M-log)
FIG. 2. Production of TNF and PCA by alveolar M&J after 24 hr LPS (10 rig/ml) and various doses of in culture with E. coli Olll:B4 PAF (both added at time zero). Data are given as the fold increase above that produced in response to LPS alone. N = 8-10 (means t SEM). Asterisk indicates P < 0.05 compared to control by Dunnett’s test.
LPS alone, Fig. 1. The reason for the large decrease in activity with PAF at 10e5 M is unknown but may represent toxicity and is similar to results from other investigators. Cotreatment of M4 with PAF ( lop6 M) and varying doses of LPS produced augmentation in the production of both TNF and PCA over the dose range of LPS, 1 rig/ml to 1 pg/ml, data not shown. However, the absolute magnitude of the augmentation was variable. There appears to be a maximal level of PCA and TNF production possible. In M$ treated with a submaximal LPS stimulus, PAF augmentation produced a greater effect, increasing production to highest levels possible. The greatest effect is seen in M+ treated with LPS doses that are minimally to nonstimulatory, where PAF augments production of the inflammatory mediators to near maximal. Pretreatment of M$ with PAF produced an even greater augmentation of LPS-induced PCA and TNF production than similar doses used simultaneously. Statistically significant priming effects of PAF on subsequent M4 inflammatory mediatory production persists for at least 4 to 6 hr following pretreatment, Fig. 3. Preliminary data show that the effect on M4 appears to be waning by 8 hr post-PAF exposure. The effect of PAF on M4 production of TNF and PCA was shown to be specific to a PAF-receptor dependent mechanism. The specific receptor antagonist, BN52021, was used to inhibit the augmentation effect seen with PAF treatment of M4, Fig. 4. Greater than 80% of the augmentation in PCA and greater than 75% of the augmentation in TNF production above that induced by LPS alone was inhibited by BN52021 (20 pg/ml). The effect of PAF on intracellular calcium, [Ca2’]i, in M4 was determined using a fluorescent [Ca2+]i-dependent probe, Fura- AM. PAF treatment of Md produced a rapid, short-lived spike in [Ca2+]i, Fig. 5. The duration and magnitude of the [Ca2+]i response is typical of re-
261
Mb FUNCTION
FIG. 3. Production of TNF and PCA by alveolar M$ after 24 hr LPS (10 rig/ml) and PAF (10e6 M) in culture with E. coli Olll:B4 added at various time points prior to the LPS. Data are given as fold increase in levels of PCA and TNF above that produced in response to LPS alone. N = 4-6 (means f SEM). Asterisk indicates P < 0.05 compared to control by Dunnett’s test.
stores. ceptor-mediated Ca2+ release from intracellular The peak of the [Ca2’]i spike was approximately 450 nM, which is consistent with other signal transduced events. The increase in [Ca2’]i has returned to near baseline by 60 sec. In contrast, LPS treatment of M4 does not cause a spike in [Ca”‘]i, Fig. 5. There is a small increase (approximately 30 n&f) in [Ca”+]i in response to LPS, which occurs over a prolonged period of time (greater than 5 min). Whether this response is responsible for physiologic or functional changes caused by LPS in the Mb are unknown. The combination of LPS plus PAF did not effect the [Ca”‘]i responses, regardless of the order in which the agents were given. No additive or priming effect was seen. DISCUSSION
PAF is an ether phospholipid released from multiple cells, including M& in response to physiologic stimulus
100
1
w PCA
80 3 6 zm .g g AZ E
40 20 0
0
2
20
40
BN 52021 @g/ml) FIG. 4. Effect of BN52021 inhibition on the augmentation by PAF of PCA and TNF production by alveolar Mb. PAF (10e6M), LPS (10 rig/ml) and various doses of BN52021 are added at time zero. Data are given as percentage of inhibition of the PAF augmentation of TNF and PCA production above that caused by LPS alone. N = 3 (means k SEM).
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160
300
150
300
Time (seconds)
FIG. 5. Effect of PAF (lo-’ M) and E. coli Olll:B4 LPS (10 ng/ ml) on levels of intracellular calcium, [CA”]& in alveolar Mb ueing Fura- AM. Changes in fluorescence are measured in a Shimadzu RF 5000U spectrofluorophotometer. Arrows mark time when PAF or LPS was added. Small spike response to LPS is artifact due to injection of fluid into the cuvette.
or cellular injury [27, 281. PAF appears to function as a potent vasoactive proinflammatory mediator that is released during endotoxemia and sepsis [16-191. Systemic effects of PAF demonstrated in animals include decreased peripheral vascular resistance, hypotension, increased pulmonary artery pressure, activation of platelets and leukocytes, and death [ 16,18,19]. These effects closely resemble the derangements seen during sepsis. In addition, specific PAF inhibitors produce an amelioration of the physiologic changes and an increased survival in lethal endotoxemia in animals [16,18,19]. Thus PAF, similar to TNF and other potent inflammatory mediators produced by tissue-fixed M& appears to be a central initiator of the pathophysiologic response to endotoxin and sepsis. PAF was first identified as a product released from basophils in response to antigen (as an IgE-dependent process) which led to release of histamine from platelets. Subsequently, PAF has been shown to be an ubiquitous membrane phospholipid-derived moiety found in all cells [27, 291. Lyso-PAF or 1-alkyl-2-lyso-glycero-3phosphorylcholine is both the immediate precursor and metabolite of PAF. Acetylation of C2 by acetyltransferase produces PAF and deacetylation by acetylhydrolase or phospholipase A2 (PLAB) produces lyso-PAF [29]. PAF production is dependent on both cell type and stimulus involved [27, 281. PAF is rapidly produced by inflammatory cells, such as neutrophils, in response to multiple stimuli [20,27]. The majority of PAF produced is released but a portion is retained intracellularly where it may function as an intracellular second messenger, further modulating cell activity. The PAF that is released interacts with other cells through a specific membrane receptor found on most types of cells [21,22]. The effect of PAF on multiple cell types is only beginning to be understood.
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The effects of PAF on M+ are only partially elucidated. It is known that both production of lyso-PAF and release of arachidonic acid (AA) are dependent in large part to early activation of PLAB. Activated PLA2 cleaves the acyl group on C2 leading to lyso-PAF [ 291. In addition, AA is commonly the acyl group found at C2. Since production of eicosanoids (AA metabolites) is controlled by accessibility of AA, generation of PAF is intimately linked to eicosanoid production [18, 20, 291. PAF has been shown to stimulate the release of eicosanoids from neutrophils in vitro and produce elevated levels of eicosanoids in vivo [18, 201. PAF inhibitors also prevent the increase in eicosanoids in response to LPS infusion experimentally [ 181, The inhibition of production of potent vasoactive eicosanoids, such as thromboxane, PGEB, and prostacyclin, is thought to be the mechanism of protection against the acute lethality of high dose LPS infusion. However, it is unclear whether PAF can directly activate the production of other inflammatory mediators by the M$ or is able to modulate, e.g., “prime” the M$J for subsequent response to inflammatory stimuli. The production of other potent mediators, such as TNF and PCA, may be more contributory to the diffuse organ injury and dysfunction seen in MOFS or ARDS than the acute hemodynamic effects caused by the vasoactive eicosanoids [2]. In the present series of experiments, the effect of PAF on M4 production of the two major inflammatory mediators, TNF and PCA, was investigated. Although small intracellular pools of functional TNF and PCA exist, increased levels of TNF and PCA require de nova protein synthesis. Treatment of M4 with PAF over a five log dose response had minimal effect on the production of both PCA and TNF. This dose curve includes concentrations expected in vivo from infusions of PAF that reproduce the pathophysiologic changes during endotoxemia. A potential small increase over background may be present but appears physiologically to be of no significance in comparison to the many fold increase in levels seen in response to LPS, Fig. 1. These data exclude a direct effect of PAF on M+ function as monitored by TNF and PCA production. The lack of response to PAF also supports Limulus assay data confirming the absence of LPS contamination in the PAF preparations. However, the large response to LPS in TNF and PCA production by Md is even further augmented by co- or pretreatment with PAF. Increases of lo- to 30-fold in response to LPS are pushed to 50- to loo-fold increases by the adjunct effect of PAF treatment. The most significant effects of PAF occur in Md stimulated with suboptima1 doses of LPS. There appears to be an overall maximal level of TNF and PCA production by M4 possible. PAF primes or is additive with the LPS effect to achieve essentially maximal stimulation, even when the M$ is suboptimally stimulated by LPS. In uiuo, this effect of PAF may function as an accessary mechanism to ensure a maximal M4 response in the production of inflamma-
MAIER,
HAHNEL,
AND
FLETCHER:
tory mediators. In addition, the data show that the PAF effect on the M4 lasts at least 6 hr. Thus PAF may function physiologically in those settings where an optimal response to a potentially insignificant stimulus is necessary, even hours after the initial stimulus that caused release of PAF has resolved. The ability of PAF released nondiscriminately, (e.g., during endotoxemia with widespread activation of neutrophils) to diffusely prime tissue-fixed M& such as the alveolar M4, may be partially responsible for the pathologic destructive response seen in inflammatory processes, such as ARDS and MOFS. PAF does not directly activate the M4 to produce TNF and PCA. However, M4 and other inflammatory cells are known to have a specific membrane receptor for PAF [Zl, 221. As an attempt to further define the mechanism of action of PAF on the M4, changes in M$ intracellular calcium, [Ca’+]i, were monitored [26]. The PAF receptor is thought to function via a G-protein intracellular transduction process leading to hydrolysis of phosphytidylcholine and release of inositol P3 (IP3) and diacylglycerol (DAG) [22]. IP3 causes release of Ca2+ from endoplasmin reticulum stores and a characteristic spike in [Ca2+]i. DAG causes activation of protein kinase C (PKC) [23]. Our results demonstrate that treatment of M4 with PAF leads to a transient spike in [Ca”‘]i consistent with the interaction of PAF with a specific receptor causing IP3 and DAG production [ 231. Interestingly, the spike in [Ca”‘]i in response to PAF does not lead to evidence of cell activation, at least as measured by TNF and PCA production. However, subsequent M+ function is modulated as documented by the enhanced production of TNF and PCA in response to LPS. In fact, although the [Ca2’]i spike is short lived, returning to baseline by 1 min, the priming effect on the M4 lasts for at least 6 hr. Receptor-mediated activation of the phosphytidylcholine pathway usually leads to direct cell activation. This M$ response to PAF is a unique observation that appears to demonstrate a dissociation between receptor mediated stimulation of IP3 release (and increase in [Ca2’]i) and the full activation of PKC, which are usually regarded as inherently linked events. Whether the increased [Ca”‘]i directly activates enzymes necessary for optimal TNF and PCA production or PKC (or some other kinase) enhances transduction of the subsequent LPS stimulus is unclear at present. Studies are planned to further elucidate this intracellular regulation pathway. Treatment of M4 with a specific PAF receptor antagonist, BN52021, confirmed the specificity of the PAF effect. BN52021 has been shown to be a highly selective and competitive inhibitor of the PAF receptor [la]. Treatment of the M4 with BN52021 blocked greater than 80% of the augmentation in production of PCA and greater than 75% of the TNF response to PAF. In addition, BN52021 blocked the increase in [Ca2’]i induced by PAF, data not shown. These data argue that the effects measured were due to a PAF-receptor interaction
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M# FUNCTION
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and not a nonspecific effect or contamination with other stimuli, such as LPS. The ability of PAF to prime or augment the M4 response to subsequent inflammatory stimuli, such as LPS in uitro, may explain, at least in part, the mechanism of action of PAF in uiuo. The ability to modulate the M$ response and alter the onset and outcome of MOFS requires elucidation of the cellular mechanisms involved. Only with this understanding can rational and safe therapeutic interventions be developed. REFERENCES 1.
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24.
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29.
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.P.c.e
1YYZ
Brain, J. D., and Frank, R. Alveolar macrophage adhesion: Wash electrolyte composition and free cell yield. J. Appl. Physiol. 34: 75, 1973. Flick, D. A., and Gifford, G. E. Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J. Immunol. Methods 68: 167,1984. Grynkiewiez, G., Poenie, M., and Tsien, R. Y. A new generation of Ca++ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440, 1985. Camussi, G., Bussoiino, F., Salvidio, G., and Baglioni, C. Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphoneclear neutrophils, and vascular endothelial cells to synthesize and release platelet-activating factor. J. Exp. Med. 166: 1390,1987. Prevost, M-C., Cariven, C., and Chap, H. Possible origins of PAF-acether and lyso-PAF-acether in rat lung alveoli secondary to hypoxia. Biochim. Biophy. Actu 962: 354, 1988. Braquet, P., Touqui, L., Shen, T. Y., and Vargaftig, B. B. Perspectives in platelet-activating factor research. Pharmacol. Reu. 39: 97,1987.
OF SURGICAL
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Platelet-Activating
(19%)
Factor Augments Tumor Necrosis Factor and Procoagulant Activity’ v2
RONALD V. MAIER, M.D., FACS,3
GREGB. HAHNEL, M.S., AND J. RAYMOND FLETCHER, M.D., FACS
Departments of Surgery, University of Washington, Seattle, Washington; and the University of South Alabama, Mobile, Alabama Submitted for publication October 22, 1990
Infusion of platelet activating factor (PAF) reproduces the host physiologic response to endotoxemia and sepsis. Tumor necrosis factor (TNF) and procoagulant activity (PCA) are two other potentially deleterious central inflammatory mediators produced in large quantities by tissue-fixed macrophages (Md). The relationship, if any, between PAF and TNF or PCA production is unknown. Rabbit alveolar M4 were treated in vitro with PAF alone and prior to endotoxin (LPS). PAF alone had no effect on M4 PCA or TNF. PAF ( 1O-s-1O-6 1M) cotreatment enhanced M& PCA and TNF levels in a dose response from two- to sixfold above that of LPS treatment alone. PAF (lo-* M) pretreatment of M# at T -4 to -6 hr produces an eight- to ninefold enhancement in both TNF and PCA levels. Thus, both coincubation and pretreatment or “priming” of the M4 with PAF prior to LPS stimulation greatly increase Mr$ production of PCA and TNF. The ability to augment the production of these two potent inflammatory mediators may explain in part the mechanism of action of PAF in UiUO.
0 1992 Academic Press, Inc.
INTRODUCTION
Infectious complications continue to be a major cause of morbidity and mortality in the critically ill surgical patient. Invasive infection, producing endotoxemia and sepsis, is a common etiology for multiple organ failure syndrome (MOFS) and, in particular, adult respiratory distress syndrome (ARDS) [ 11. The diffise organ injury seen during MOFS is thought to be caused by the host’s own inflammatory response to the invading organisms [2]. Many of the central mediators of this uncontrolled inflammatory response have been identified. Although these mediators are produced by many different cell
i Supported in part by NIH Grant GM35361. ’ Presented at the Tenth Annual Meeting of the Surgical Infection Society, Cincinnati, OH, June 14-16, 1990. 3 To whom request for reprints should be addressed at Department of Surgery, ZA-16, Harborview Medical Center, 325 Ninth Avenue, Seattle, WA 98104. 0022.4so4/92 51.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
258
types, the tissue-fixed macrophage (M$J), which is found in the various injured organs of MOFS, produces large quantities of these major inflammatory mediators [3]. Additionally, endotoxins or lipopolysaccharides (LPS), derived from the outer membrane of gram-negative bacteria, are extremely efficient in inducing the production of these inflammatory mediators by M@ and in reproducing the pathophysiologic changes seen clinically during sepsis and MOFS [4]. The Mb is capable of producing virtually all of the recognized mediators of the inflammatory response, including complement components, arachidonic acid metabolites, interleukin-1,6, and8 (IL-l, 6, and8), procoagulant activity (PCA), and tumor necrosis factor (TNF) [3,5-81. Selective inhibition of many of these inflammatory agents has been protective in experimental lethal endotoxemia [9,10]. TNF and PCA are thought to be central mediators and major contributors to the pathophysiology of sepsis [2,7,8,10-121. The tissue-fixed M$ is the primary source for both of these potent mediators, and LPS and other inflammatory stimuli induce the production of TNF and PCA by M$. PCA is produced by and expressed predominantly as a cell surface ectoenzyme. Activity can be demonstrated in association with the M4 in the microcirculation of animals within 15-30 min of LPS infusion and leads to potential diffuse organ ischemic injury during sepsis induced MOFS [2, 7, 111. Clinically, patients with MOFS and, in particular ARDS, have been shown to have diffuse microvascular thrombosis early during the organ injury phase [13]. The direct cellular effects of TNF during sepsis are unknown. However, infusion of TNF reproduces the derangements seen during endotoxemia and sepsis [ 121. In addition, passive immunization with antibody to TNF protects against lethal endotoxemia [lo]. TNF is found in the plasma of patients with overwhelming sepsis with gram-negative bacteria. And, unlike interleukin-1 and other inflammatory mediators, levels of TNF correlate with the survival outcome of sepsis [ 141. Both PCA and TNF are produced in enormous quantities by M& Together, they appear to contribute to the pathogenesis of endotoxin and sepsis. In addition to these mediators, platelet activating fac-
MAIER,
HAHNEL,
AND
FLETCHER:
tor (PAF) has been implicated recently as a central mediator of the inflammatory response [15-171. Infusion of PAF reproduces the host physiologic response to LPS and sepsis [18]. Inhibition of PAF using selective membrane receptor blockers ameliorates the pathophysiologic response to either PAF or LPS experimentally [ 16, 18-201. The mechanisms involved in PAF-induced alterations of normal physiology are not known. It is unclear whether PAF causes these changes by a direct cellular effect or primarily functions indirectly by stimulating Md and other cells to produce other critical mediators of the inflammatory response. PAF is known to interact with cells through a specific membrane receptor which is thought to function via a G-protein intracellular transduction process leading to hydrolysis of phosphytidylcholine release of Ca2+ from endoplasmic reticulum stores and activation of protein kinase C (PKC) [21-231. By measuring intracellular Ca2+ [Ca”]i, interaction of PAF with a membrane receptor that functions via phosphatidylcholine hydrolysis can be investigated. Receptor-mediated activation of the phosphytidylcholine pathway usually leads to direct cell activation. This is consistent with the known release of eicosanoids in response to PAF [20]. However, the interaction of PAF with M$J and the effect on production of other mediators is only partially elucidated. Investigation of the ability of PAF to enhance M$ production of TNF and/or PCA either directly or indirectly (by priming the M4 to subsequent stimuli) will help elucidate the mechanism of action of PAF in uiuo. Only by elucidation of these complex interactions can rational and safe therapeutic interventions be designed. In the present study, the effect of PAF on the production of TNF and PCA by a crucial M+ population involved in the pathogenesis of ARDS (the alveolar Md) is investigated. MATERIALS
AND METHODS
Alveolar macrophages (M4) were obtained from male, 1.5-2.0 kg, New Zealand white rabbits (R,. and R. Rabbitry, Stanwood, WA), using a modification of bronchoalveolar lavage described by Brain and Frank [24]. Animals were housed in standard care facilities at the University of Washington, fed rabbit chow and water ad lib and used within 10 days of delivery. Animals were euthanized with an overdose of pentobarbital infusion, the trachea is isolated using sterile technique in situ, and bronchoalveolar lavage is performed using a 5-mm catheter placed just superior to the carina. The lungs are gently lavaged six times with 50-ml aliquots of normal saline at 4°C. The combined lavage fluid aliquots were centrifuged at 500g for 7 min at 4’C, the supernatant was discarded, and the M4 were resuspended in glutamine-supplemented RPMI-1640 media (GIBCO Corp., Grand Island, NY) with 100 pg/ml of gentamicin. M$ are plated immediately in 12-well tissue culture plates (Flow Laboratories, McLean, VA) at 5 X lo5 cells/ml/
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259
well. Preliminary experiments confirm that the cells are 98-99% M4 by Wright stain histology, presence of nonspecific esterase staining, and ability to phagocytize latex beads. The M$ were greater than 95% viable by trypan blue dye exclusion [2]. All animal studies are evaluated and approved by the Department of Comparative Medicine and the IACUC of the University of Washington. Experimental
Design
The M$ were allowed to adher for 30 min prior to initiation of experimental protocols. Cultures were treated at the various time points with either LPS (Escherichia coli Olll:B4) or PAF (L-cr-phosphatidylcholine, fi-acetyl-y-0-(octadec-9-cis-enyl)), both obtained from Sigma Chemical Co. (St. Louis, MO). A dose response of LPS from 1 rig/ml to 1 pg/ml was used, unless the LPS dose was held constant (10 rig/ml). The PAF was used in a dose range from 10-l’ to lo-’ M. For the pretreatment time course using PAF, the optimal dose of 10d6 M was utilized at all time points. The specific inhibitor of the PAF receptor utilized was BN52021 (IPSEN, Paris, France) over a dose response of 2 to 40 r.Lg/ml with PAF at lo-’ M. M$J cultures were treated in triplicate under the various experimental conditions. Reagents were added simultaneously at time zero except in the pretreatment assays where PAF was added at various time points prior to LPS. Cultures were harvested 24 hr after initiation of LPS treatment. Supernatants for TNF were gently aspirated and spun X60 set in a microfuge. The cell pellet was added to the remaining M4 monolayer, which was lysed for PCA assay by three cycles of freeze thawing and scraping. The lysates and supernatants were frozen at -70°C until analyzed. Previous studies have confirmed greater than 85% of PCA is membrane associated, expressed as an ectoenzyme on the exterior of the membrane and found intact in the M$ lysates [7]. Each of experimental conditions was tested for M$ cytotoxicity at 24 hr by 51Cr release and exclusion of trypan blue dye assays and found to cause injury to less than 10% of the M$ monolayer. PCA and TNF Assays Procoagulant activity (PCA) produced by the M4 was analyzed using a modified Quick one-step coagulation assay [7]. M$ lysates were suspended in Hanks BSS at 1 X lo6 cells/ml. A O.l-ml aliquot each of normal rabbit plasma (10 mMEDTA), CaCl, (20 mM), and M4 lysates from each experimental condition were combined and, with constant agitation in a 37°C water bath, time to visual clot formation was measured. Activity is derived from a standard curve generated from log dilutions of rabbit brain-derived thromboplastin. TNF was measured using the biologic cytotoxicity assay of Flick and Gifford [25]. Transformed mouse fibro-
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blasts (NCTC clone L929 American Type Culture Collection, Rockville, MD) were pretreated with actinomytin D (Sigma) at 1 pg/ml for 15 min in RPMI-1640 supplemented with 5% horse serum (Hyclone Laboratories, Logan, UT) and added to the serial dilutions of the conditioned media at 5 X lo4 cells/well. The plates were then placed in a 37”C, 5% CO, incubator. After 24 hr the cells were fixed and stained in 0.1% crystal violet in 20% methanol. Dried monolayers were solubilized in 0.1 M sodium citrate in 50% ethanol and the absorbance read at 550 nm in a microplate reader. One unit of TNF activity was defined as that activity that produced 50% cytolysis of the L929 monolayer. A linear regression was performed to determine the point between serial dilutions of the TNF samples where the 50% cytolysis endpoint occurred. The E. coli Olll:B4 LPS was tested against the L929 cell line and showed no direct cytotoxicity. Intracellular
Calcium Levels
To further elucidate the cellular mechanism of the PAF effect on M$ function, intracellular calcium concentrations, [Ca2’]i, were monitored using a fluorescent [Ca2+]i dependent probe, Fura- AM (Sigma) [26]. Rabbit alveolar M4 at 1 X lo6 cells/ml were loaded with 100 t&f Fura- AM. This was accomplished by a slow titration of the dye into a stirred suspension of the Md held in the dark at RT for 30 min. The slow titration of the dye reduced the level of entrapped Fura- AM in organelles. The cells were loaded at RT to reduce endocytosis of the dye. These steps are used to minimize the level of unhydrolysed dye in the M& After loading, the cells were allowed to hydrolyse the ester for an additional 15 min. The M4 were then centrifuged at 1OOOgfor 7 min through a 0.5% BSA pad in buffer (145 m&f NaCl, 5 n&f KCl, 10 mM Hepes, 0.5 mM Na,HPO,, 1 mM Ca2+, and 5 mM glucose). The cells were resuspended to 1 X lo6 cells/ml in the above buffer without the BSA and placed in polypropylene tubes on ice. A 300-~1 aliquot of the cell suspension (3 X lo5 cells) was added to 1.2 ml of RT buffer in a stirred cuvette (final concentration of 2 X lo5 cells/ml). LPS or PAF at 100X were added to yield final concentrations of 100 rig/ml and 1 X 10e6 M, respectively. A Shimadzu RF 5000U Spectrofluorophotometer with stirred cuvette and the wavelengths set to the following: excitation of 340 nm (slit = 5 nm) and emission at 510 nm (slit = 10 nm) was used to determine the [Ca2’]i. Internal controls for F (minimum) and F (maximum) were run on each sample. The maximum fluorescence value was obtained by permeabilizing the cells with digitonin, thus complexing essentially all Furawith calcium. The subsequent minimum fluorescence value was gained by adding EGTA in Tris Base, (final concentrations, 10 and 33 mM, respectively, at a final pH of 8.5), to chelate any free calcium. External FuraAM was quenched with 30 pM Mn and chelated with 60
VOL.
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1
*
T
.
TNF
m PCA
a g 20 :: eCE m 0 10 us f 0
,001
.Ol
.I
1
LPS (uglml) FIG. 1. Production of TNF and PCA by alveolar M& after 24 hr in culture with various doses of E. coli Olll:B4 LPS. Data are given as fold increase over background or control levels. N = 8-10 (means k SEM). Asterisk indicates P < 0.05 compared to control by Dunnett’s test.
pM diethylenetriaminepenta acetic acid (DTPA) to measure the extracellular fluorescence and this value was used to correct for any dye leakage from the loaded cell. The formula used for calculating [Ca2’]i was: calcium (nM) = 224X (F-F min.)/(F-,-F)) where 224 is the binding constant for calcium [26]. Analysis
of Results
Data from these studies were analyzed by the Dunnett’s multiple-comparison test for comparison between various experimental groups and a control group. A P value of ~0.05 was considered significant. RESULTS
Rabbit alveolar M$ are obtained in a consistently homogeneous (>95%) and viable (>95%) condition. Approximately 2 X 10’ M4 are obtained per animal. If neutrophils comprise greater than 5% of the differential white cell count, the animal is considered to have been ill, in vivo stimulation of the Mb has occurred, and the preparation was discarded. Endotoxin (LPS) from E. coli Olll:B4 stimulates MI$ to produce both PCA and TNF. There is a significant increase in levels of both TNF and PCA in a similar bimodal response to LPS from 1 rig/ml to 1 pg/ml, Fig. 1. PAF alone had a minimal direct effect on the production of TNF or PCA by Mb. Throughout the dose range of 10-l’ to 10e5 M, PAF increased levels over background no greater than normal biologic variation, data not shown. In contrast, LPS alone increased levels 10 to 30+ fold above background, Fig. 1. Simultaneous treatment of M4 with LPS (10 rig/ml) and PAF over a dose curve of 10-l’ to lop5 M shows a significant increase in the production of TNF and PCA at lOA M; Fig. 2. The levels of TNF and PCA shown are the fold increase above the already lo- to ZO-fold increases produced in response to
MAIER,
-10
-9
-8
HAHNEL,
-7
PAF Concentration
AND
-6
FLETCHER:
PAF AUGMENTS
-5
Time (Hrs. of Pretreatment)
(M-log)
FIG. 2. Production of TNF and PCA by alveolar M&J after 24 hr LPS (10 rig/ml) and various doses of in culture with E. coli Olll:B4 PAF (both added at time zero). Data are given as the fold increase above that produced in response to LPS alone. N = 8-10 (means t SEM). Asterisk indicates P < 0.05 compared to control by Dunnett’s test.
LPS alone, Fig. 1. The reason for the large decrease in activity with PAF at 10e5 M is unknown but may represent toxicity and is similar to results from other investigators. Cotreatment of M4 with PAF ( lop6 M) and varying doses of LPS produced augmentation in the production of both TNF and PCA over the dose range of LPS, 1 rig/ml to 1 pg/ml, data not shown. However, the absolute magnitude of the augmentation was variable. There appears to be a maximal level of PCA and TNF production possible. In M$ treated with a submaximal LPS stimulus, PAF augmentation produced a greater effect, increasing production to highest levels possible. The greatest effect is seen in M+ treated with LPS doses that are minimally to nonstimulatory, where PAF augments production of the inflammatory mediators to near maximal. Pretreatment of M$ with PAF produced an even greater augmentation of LPS-induced PCA and TNF production than similar doses used simultaneously. Statistically significant priming effects of PAF on subsequent M4 inflammatory mediatory production persists for at least 4 to 6 hr following pretreatment, Fig. 3. Preliminary data show that the effect on M4 appears to be waning by 8 hr post-PAF exposure. The effect of PAF on M4 production of TNF and PCA was shown to be specific to a PAF-receptor dependent mechanism. The specific receptor antagonist, BN52021, was used to inhibit the augmentation effect seen with PAF treatment of M4, Fig. 4. Greater than 80% of the augmentation in PCA and greater than 75% of the augmentation in TNF production above that induced by LPS alone was inhibited by BN52021 (20 pg/ml). The effect of PAF on intracellular calcium, [Ca2’]i, in M4 was determined using a fluorescent [Ca2+]i-dependent probe, Fura- AM. PAF treatment of Md produced a rapid, short-lived spike in [Ca2+]i, Fig. 5. The duration and magnitude of the [Ca2+]i response is typical of re-
261
Mb FUNCTION
FIG. 3. Production of TNF and PCA by alveolar M$ after 24 hr LPS (10 rig/ml) and PAF (10e6 M) in culture with E. coli Olll:B4 added at various time points prior to the LPS. Data are given as fold increase in levels of PCA and TNF above that produced in response to LPS alone. N = 4-6 (means f SEM). Asterisk indicates P < 0.05 compared to control by Dunnett’s test.
stores. ceptor-mediated Ca2+ release from intracellular The peak of the [Ca2’]i spike was approximately 450 nM, which is consistent with other signal transduced events. The increase in [Ca2’]i has returned to near baseline by 60 sec. In contrast, LPS treatment of M4 does not cause a spike in [Ca”‘]i, Fig. 5. There is a small increase (approximately 30 n&f) in [Ca”+]i in response to LPS, which occurs over a prolonged period of time (greater than 5 min). Whether this response is responsible for physiologic or functional changes caused by LPS in the Mb are unknown. The combination of LPS plus PAF did not effect the [Ca”‘]i responses, regardless of the order in which the agents were given. No additive or priming effect was seen. DISCUSSION
PAF is an ether phospholipid released from multiple cells, including M& in response to physiologic stimulus
100
1
w PCA
80 3 6 zm .g g AZ E
40 20 0
0
2
20
40
BN 52021 @g/ml) FIG. 4. Effect of BN52021 inhibition on the augmentation by PAF of PCA and TNF production by alveolar Mb. PAF (10e6M), LPS (10 rig/ml) and various doses of BN52021 are added at time zero. Data are given as percentage of inhibition of the PAF augmentation of TNF and PCA production above that caused by LPS alone. N = 3 (means k SEM).
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300
150
300
Time (seconds)
FIG. 5. Effect of PAF (lo-’ M) and E. coli Olll:B4 LPS (10 ng/ ml) on levels of intracellular calcium, [CA”]& in alveolar Mb ueing Fura- AM. Changes in fluorescence are measured in a Shimadzu RF 5000U spectrofluorophotometer. Arrows mark time when PAF or LPS was added. Small spike response to LPS is artifact due to injection of fluid into the cuvette.
or cellular injury [27, 281. PAF appears to function as a potent vasoactive proinflammatory mediator that is released during endotoxemia and sepsis [16-191. Systemic effects of PAF demonstrated in animals include decreased peripheral vascular resistance, hypotension, increased pulmonary artery pressure, activation of platelets and leukocytes, and death [ 16,18,19]. These effects closely resemble the derangements seen during sepsis. In addition, specific PAF inhibitors produce an amelioration of the physiologic changes and an increased survival in lethal endotoxemia in animals [16,18,19]. Thus PAF, similar to TNF and other potent inflammatory mediators produced by tissue-fixed M& appears to be a central initiator of the pathophysiologic response to endotoxin and sepsis. PAF was first identified as a product released from basophils in response to antigen (as an IgE-dependent process) which led to release of histamine from platelets. Subsequently, PAF has been shown to be an ubiquitous membrane phospholipid-derived moiety found in all cells [27, 291. Lyso-PAF or 1-alkyl-2-lyso-glycero-3phosphorylcholine is both the immediate precursor and metabolite of PAF. Acetylation of C2 by acetyltransferase produces PAF and deacetylation by acetylhydrolase or phospholipase A2 (PLAB) produces lyso-PAF [29]. PAF production is dependent on both cell type and stimulus involved [27, 281. PAF is rapidly produced by inflammatory cells, such as neutrophils, in response to multiple stimuli [20,27]. The majority of PAF produced is released but a portion is retained intracellularly where it may function as an intracellular second messenger, further modulating cell activity. The PAF that is released interacts with other cells through a specific membrane receptor found on most types of cells [21,22]. The effect of PAF on multiple cell types is only beginning to be understood.
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The effects of PAF on M+ are only partially elucidated. It is known that both production of lyso-PAF and release of arachidonic acid (AA) are dependent in large part to early activation of PLAB. Activated PLA2 cleaves the acyl group on C2 leading to lyso-PAF [ 291. In addition, AA is commonly the acyl group found at C2. Since production of eicosanoids (AA metabolites) is controlled by accessibility of AA, generation of PAF is intimately linked to eicosanoid production [18, 20, 291. PAF has been shown to stimulate the release of eicosanoids from neutrophils in vitro and produce elevated levels of eicosanoids in vivo [18, 201. PAF inhibitors also prevent the increase in eicosanoids in response to LPS infusion experimentally [ 181, The inhibition of production of potent vasoactive eicosanoids, such as thromboxane, PGEB, and prostacyclin, is thought to be the mechanism of protection against the acute lethality of high dose LPS infusion. However, it is unclear whether PAF can directly activate the production of other inflammatory mediators by the M$ or is able to modulate, e.g., “prime” the M$J for subsequent response to inflammatory stimuli. The production of other potent mediators, such as TNF and PCA, may be more contributory to the diffuse organ injury and dysfunction seen in MOFS or ARDS than the acute hemodynamic effects caused by the vasoactive eicosanoids [2]. In the present series of experiments, the effect of PAF on M4 production of the two major inflammatory mediators, TNF and PCA, was investigated. Although small intracellular pools of functional TNF and PCA exist, increased levels of TNF and PCA require de nova protein synthesis. Treatment of M4 with PAF over a five log dose response had minimal effect on the production of both PCA and TNF. This dose curve includes concentrations expected in vivo from infusions of PAF that reproduce the pathophysiologic changes during endotoxemia. A potential small increase over background may be present but appears physiologically to be of no significance in comparison to the many fold increase in levels seen in response to LPS, Fig. 1. These data exclude a direct effect of PAF on M+ function as monitored by TNF and PCA production. The lack of response to PAF also supports Limulus assay data confirming the absence of LPS contamination in the PAF preparations. However, the large response to LPS in TNF and PCA production by Md is even further augmented by co- or pretreatment with PAF. Increases of lo- to 30-fold in response to LPS are pushed to 50- to loo-fold increases by the adjunct effect of PAF treatment. The most significant effects of PAF occur in Md stimulated with suboptima1 doses of LPS. There appears to be an overall maximal level of TNF and PCA production by M4 possible. PAF primes or is additive with the LPS effect to achieve essentially maximal stimulation, even when the M$ is suboptimally stimulated by LPS. In uiuo, this effect of PAF may function as an accessary mechanism to ensure a maximal M4 response in the production of inflamma-
MAIER,
HAHNEL,
AND
FLETCHER:
tory mediators. In addition, the data show that the PAF effect on the M4 lasts at least 6 hr. Thus PAF may function physiologically in those settings where an optimal response to a potentially insignificant stimulus is necessary, even hours after the initial stimulus that caused release of PAF has resolved. The ability of PAF released nondiscriminately, (e.g., during endotoxemia with widespread activation of neutrophils) to diffusely prime tissue-fixed M& such as the alveolar M4, may be partially responsible for the pathologic destructive response seen in inflammatory processes, such as ARDS and MOFS. PAF does not directly activate the M4 to produce TNF and PCA. However, M4 and other inflammatory cells are known to have a specific membrane receptor for PAF [Zl, 221. As an attempt to further define the mechanism of action of PAF on the M4, changes in M$ intracellular calcium, [Ca’+]i, were monitored [26]. The PAF receptor is thought to function via a G-protein intracellular transduction process leading to hydrolysis of phosphytidylcholine and release of inositol P3 (IP3) and diacylglycerol (DAG) [22]. IP3 causes release of Ca2+ from endoplasmin reticulum stores and a characteristic spike in [Ca2+]i. DAG causes activation of protein kinase C (PKC) [23]. Our results demonstrate that treatment of M4 with PAF leads to a transient spike in [Ca”‘]i consistent with the interaction of PAF with a specific receptor causing IP3 and DAG production [ 231. Interestingly, the spike in [Ca”‘]i in response to PAF does not lead to evidence of cell activation, at least as measured by TNF and PCA production. However, subsequent M+ function is modulated as documented by the enhanced production of TNF and PCA in response to LPS. In fact, although the [Ca2’]i spike is short lived, returning to baseline by 1 min, the priming effect on the M4 lasts for at least 6 hr. Receptor-mediated activation of the phosphytidylcholine pathway usually leads to direct cell activation. This M$ response to PAF is a unique observation that appears to demonstrate a dissociation between receptor mediated stimulation of IP3 release (and increase in [Ca2’]i) and the full activation of PKC, which are usually regarded as inherently linked events. Whether the increased [Ca”‘]i directly activates enzymes necessary for optimal TNF and PCA production or PKC (or some other kinase) enhances transduction of the subsequent LPS stimulus is unclear at present. Studies are planned to further elucidate this intracellular regulation pathway. Treatment of M4 with a specific PAF receptor antagonist, BN52021, confirmed the specificity of the PAF effect. BN52021 has been shown to be a highly selective and competitive inhibitor of the PAF receptor [la]. Treatment of the M4 with BN52021 blocked greater than 80% of the augmentation in production of PCA and greater than 75% of the TNF response to PAF. In addition, BN52021 blocked the increase in [Ca2’]i induced by PAF, data not shown. These data argue that the effects measured were due to a PAF-receptor interaction
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and not a nonspecific effect or contamination with other stimuli, such as LPS. The ability of PAF to prime or augment the M4 response to subsequent inflammatory stimuli, such as LPS in uitro, may explain, at least in part, the mechanism of action of PAF in uiuo. The ability to modulate the M$ response and alter the onset and outcome of MOFS requires elucidation of the cellular mechanisms involved. Only with this understanding can rational and safe therapeutic interventions be developed. REFERENCES 1.
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M. G., Varese, L., and Bosia, A. Intravascular release of platelet activating factor in children with sepsis. Thromb. Res. 48: 619, 1987. 18. Fletcher, J. R., Disimone, A. G., and Earnest, M. A. Platelet
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