Blockade of Prostaglandin Production Increases Cachectin Synthesis and Prevents Depression of Macrophage Functions After Hemorrhagic Shock
WOLFGANG ERTEL, M.D.,* MARY H. MORRISON, M.S.,* ALFRED AYALA, PH.D.,*t MICHELLE M. PERRIN,* and IRSHAD H. CHAUDRY, PH.D.*t
Although hemorrhage severely depresses macrophage functions, it is not known whether the increased TNF-a or PGE2 production is responsible for it. To study this C3H/HeN mice were bled to mean blood pressure of 35 mmHg for 60 minutes, resuscitated, and treated with either ibuprofen (1.0 mg/kg body weight) or vehicle (saline). Hemorrhage increased plasma prostaglandin E2 (PGE2) levels by 151.7% ± 40.0% (p < 0.05) and significantly decreased peritoneal macrophage (pMO) antigen presentation (AP) by 60.5% ± 7.3%, Ia expression by 523% ± 7.6%, and interleukin-1 (IL-1) synthesis by 60.5% ± 12.3% compared to shams. However ibuprofen treatment reduced PGE2 plasma levels by 61.3% ± 12.1% and significantly increased AP (+237.0% + 953%), Ia expression (+72.8% ± 27.5%), IL-1 synthesis (+235.7% ± 134.7%), and cachectin synthesis (+485.8% + 209.0%) compared to vehicle-treated animals. These results indicate that prostaglandins but not cachectin are involved in the suppression of pM4 functions following hemorrhage because blockade of prostaglandin synthesis improved depressed macrophage functions despite enhanced cachectin synthesis.
IMPLE HEMORRHAGE CAUSES a severe suppression of macrophage,' T-lymphocyte,2 and Blymphocyte3 functions. The suppression of macrophage antigen presentation,4 major histocompatibility complex (MHC) class II (Ia) expression,4 and interleukin1 (IL- 1)5 production after hemorrhage results in a depresS
sion of adequate T-lymphocyte induction, lymphokine production, and consequently may contribute to the enhanced susceptibility to sepsis.6 The mechanisms and potential mediators involved in the suppression of the immune responses after hemorrhage are not clearly understood. It has been suggested that after mechanical trauma7 or burn injury,8 prostaglandins of the E series, potent suppressor factors of cellular9 and humoral immunity,'0 may
Supported by National Institutes of Health grant RO1 GM 37127. Address reprints requests to Irshad H. Chaudry, Ph.D., Department of Surgery, B 424 Clinical Center, Michigan State University, East Lansing, MI 48824-1315. Accepted for publication May 23, 1990.
From the Departments of Surgery, * Microbiology and Public Health,t and Physiology,* Michigan State University, East Lansing, Michigan
play a detrimental role in inducing a defective immune response. In support of this suggestion are recent studies" that indicate that cyclooxygenase inhibitors can significantly improve the depressed T-cell proliferation and IL2 synthesis in vitro following major mechanical trauma. Furthermore studies by Faist et al.'2 demonstrated beneficial effects of in vivo indomethacin administration on different parameters of cellular immunity after major sur-
gery. Although the above studies indicate that prostaglandin E2 (PGE2) may be responsible for the depression of immunologic function after trauma, it is not known whether PGE2 is involved in the induction of the observed immunosuppression after simple hemorrhage. In addition it is not known whether the observed alterations in cachectin (TNF-a) synthesis,'3 a major mediator of the inflammatory response, are closely related to alterations of PGE2 production. In vitro studies of Kunkel et al.'4 demonstrated that PGE2 is a potent downregulator of tumor necrosis factor alpha (TNF-a) gene expression and synthesis. Whether alterations in TNF-a synthesis are beneficial or immunosuppressive is not clear. Studies investigating effects of TNF infusions on the host are controversial and the effects of TNF seem to be dose dependent. Sheppard et al. 5 demonstrated a decreased mortality rate after endotoxemia and sepsis following pretreatment with TNF, while studies by Tracey et al.'6 showed that TNF administration induced tissue injury and a shocklike state. The aim of this study, therefore, was to determine whether arachidonic acid metabolites and/or increased cachectin production are involved in the suppression of macrophage functions such as antigen presentation, Ia
ERTEL AND OTHERS
expression, and IL-I synthesis after hemorrhage, and whether the systemic administration ofthe cyclooxygenase inhibitor ibuprofen after hemorrhage has any beneficial effects on macrophage functions. Materials and Methods Animals
Inbred C3H/HeN male mice (Charles River Labs., Portage, MI), 6 to 8 weeks old, weighing 20 to 25 g were used in all experiments. Animals were fasted 12 hours before the experiments and were allowed to have food ad libidum thereafter. The care of all animals was in accordance with the guidelines set forth by the Animal Welfare Act and with the Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publications. Hemorrhage Model
Hemorrhage was induced according to the method of Stephan et al.2 Briefly, mice were slightly anesthetized with methoxyflurane and restrained in supine position. Both femoral arteries were catheterized with 10 polyethylene tubing under aseptic conditions using a minimal dissection technique. Mice were heparinized (150 U/kg/ body weight beef lung heparin [Upjohn Labs., Kalamazoo, MI]) and allowed to awaken. Blood pressure (BP) was constantly measured by attaching one of the catheters to a strain gauge pressure transducer coupled to a polygraph. The animals were bled ( -0.78 ± 0.12 mL/animal; 50% of estimated mouse blood volume) through the second catheter to a mean arterial BP of 35 ± 5 mmHg (mean BP before hemorrhage, 94.4 ± 8.3 mmHg). This BP was maintained for 60 minutes followed by infusion of the shed blood and Ringer's lactate solution (twice the shed blood volume) to provide adequate fluid resuscitation. In ibuprofen-treated animals, ibuprofen (Sigma Chemical Co., St. Louis, MO) was administered systemically as bolus (1 mg/kg/body weight) immediately after reinfusion of the shed blood. Vehicle-treated hemorrhaged animals received an equal volume of saline. After resuscitation the catheters were removed and the groin incisions closed. There were no deaths in either the vehicle-treated or ibuprofen-treated groups. Furthermore no significant changes in BP were detected after ibuprofen administration. Control (sham) animals underwent the same anesthetic and surgical procedures, including ligation of both femoral arteries, but hemorrhage was not induced. Preparation of Peritoneal Macrophages
Mice were killed 24 hours after hemorrhage and resuscitation. Peritoneal macrophages (pMO) were harvested by lavage using 10 mL of ice-cold Click's medium (Irvine Scientific, Santa Ana, CA). Cells were washed once (280g,
Ann. Surg. * March 1991
15 minutes, 4 C) and viability was determined by trypan blue exclusion. Macrophages were resuspended at a final concentration of X 106 pM0/mL in Click's medium.
Antigen Presentation Assay For the antigen presentation assay, pM4 were allowed to adhere onto 60-mm diameter plastic petri dishes at 37 C for 2 hours. Nonadherent cells were removed by repeated washing with Click's medium. The monolayer of pM/ then was incubated for 20 minutes (37 C, 5% C02, in the dark) with 30 ,Ag mitomycin C per milliliter (Sigma Chemical Co.). The plates were washed extensively with PBS, overlaid with 3 mL Click's medium plus 10% FBS (fetal bovine serum: Biologos Inc., Naperville, IL), and gently scraped off the surface using a rubber policeman. Cells were centrifuged, the viability determined, and pMq resuspended in Click's medium plus FBS. This protocol provided pM4 cultures containing more than 95% macrophages, which were positive by nonspecific esterase staining and which demonstrated typical macrophage morphology. No difference in cell yields was observed between control and hemorrhaged mice. The capacity of pMo to present the specific antigen conalbumin was assessed by coculturing pM4 with the mouse T-helper cell clone DlO.G4.1 (ratio pMo:DlO.G4.1 5000:20000 cells/ well),
previously described by Ayala et al.'7 The
DlO.G4.1 cell clone (provided by Dr. Charles Janeway, New Haven, CT) proliferates in the presence of conalbumin presented by MHC class II antigen identical macrophages. 18 The degree of DlO.G4. 1 proliferation correlates with the capacity of pM4 to present conalbumin. Determination of MHC Class II Antigen Expression
Major histocompatibility complex class II antigen (Ia) expression was determined as described by Ayala et al.'9 Macrophages were labeled with fluorescein-conjugated mouse lak alloantisera (Accurate Scientific, Westbury, NY). No fewer than 200 pMO were screened in each culture, recording both the number of fluorescent cells (Ia positive) and nonfluorescent cells. From these values, the percentage of Ia-positive cells were calculated. Assessment of IL-I, IL-6, and TNF Production The capacity of mouse pMq to produce monokines (i.e., IL- 1, IL-6, and TNF-a) was determined by incubating 1 X 106 pMo/mL/well in 24-well plates with 10 ,ug lipopolysaccharide (LPS, from Escherichia coli 055:B5, Difco Labs., Detroit, MI) per milliliter Click's medium with 10% FBS for 48 hours (37 C, 5% C02). The cell supernatants were collected at the end of the incubation period, filtered, aliquotted, and stored at -70 C until assayed.
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The IL- I activity in these cell cultures was determined by adding serial dilutions of the supernatant to the DI O.G4.1 cells (2 X 104 cells/well) in the presence ofConcanavalin A (2.5 ug/mL; Pharmacia Fine Chemicals, Piscataway, NJ). Proliferation of the DlO.G4.1 cells was measured with 3H thymidine incorporation.'7 Interleukin6 was determined by the amount of proliferation of the murine hybridoma cell line 7TDl (provided by Dr. Van Snick, Brussels, Belgium), which only grows in the presence of IL-6.20 The 7TD 1 cells (4 X 103/well) stimulated with serial dilutions ofthe pM4 supernatants were grown for 72 hours. At the end of the incubation period, MTTtetrazolium (Sigma Chemical Co.) produced during the last 4 hours was measured photometrically. Similarly TNF activity was measured by assessing pMo supernatants for WEHI- 164 clone 13 (provided by Dr. S. Kunkel, Ann Harbor, MI) cytotoxicity,2' as described by Ayala et al.'7 All samples were tested in triplicate. The relative unit(s) of monokine activity per milliliter were determined by comparison of the curves produced from the dilutions of the experimental supernatants to that generated by dilutions of a purified human IL- 1 standard (5 U/mL; Genzyme Corp., Boston, MA), a recombinant human IL-6 standard (200 U/mL; Amgene Corp., Thousand Oaks, CA), or a murine TNF standard (200 U/mL; Amgene Corp.) according to the methods of Mizel.22
Determination of PGE2 Plasma Levels
Prostaglandin E2 levels in plasma were measured with radioimmunoassay technique. Because contamination of the plasma by arachidonic acid was detected in preliminary measurements, PGE2 in plasma was purified using a two-column extraction procedure under vacuum. Briefly, 200 to 300 ,uL plasma, acidified to pH 3.5 with 10 to 15 ,uL 2N HCI, were applied to a 100-mg BondElut C18 column (Analytichem International, Harbor City, CA) that was prepared by washing with 1 mL methanol and 1 mL acidified water (pH 3.5). A 500-mg BondElut Silica extraction column (Analytichem International) was washed with 5 mL benzene:ethyl acetate (80:20). After washing the PGE2 bound to the C 18 column with 2 mL each of acidified water, 15% methanol and petroleum ether, the PGE2 was eluted with 1 mL ethyl acetate from the C 18 column and bound to the silica column. The bound PGE2 was washed with 1 mL each of benzene: ethyl acetate (80:20), benzene:ethyl acetate (60:40), benzene:ethyl acetate:methanol (60:40:2), and benzene:ethyl acetate:methanol (60:40:10). The PGE2 was eluted from the silica column with 3 mL benzene:ethyl acetate:methanol (60:40:30) and dried under nitrogen at room temperature. The PGE2 was reconstituted with assay buffer (0.9% NaCl, 0.01 mol/L ethylenediamine tetraacedic acid, 0.3% bovine gamma-globulin, 0.005% Triton X-100,
0,05% sodium azide, 25 mmol/L [millimolar] sodium phosphate buffer; pH 6.8). Plasma was assayed for PGE2 using an 1251 radioimmunoassay kit (NEN DuPont, Boston, MA) according to the manufacturer's directions. Samples were counted on a LKB 1282 Compugamma counter (LKB, Gaithersburg, MD). The concentrations of PGE2 were determined by calculating B/Bo (net CPM of standard/net counts per minute (cpm) of '0' standard) and interpolation of a PGE2 standard curve.
Statistics For statistical calculations unpaired Student's t test with Bonferroni correction was used. Values were considered significant if p < 0.05. Values are reported as mean ± standard error of the mean. Results
Antigen Presentation Antigen presentation by pMo obtained from hemorrhaged animals without ibuprofen treatment (12,337 ± 2061 cpm) revealed a significant (p < 0.01) suppression compared to sham-operated animals (30,190 ± 2252 cpm) (Fig. 1). However treatment with ibuprofen completely restored (p < 0.05) antigen presentation by pMo from hemorrhaged animals (35,938 ± 7305 cpm) to control values. Ia Expression In parallel with antigen presentation, the capacity of pMo to express the MHC class II (Ia) antigen was markedly (p < 0.01) decreased in hemorrhaged animals (20.7% CPM 50000.
% la positive rl 00
Antigen presentation =J la expression
FIG. 1. Capacity of pM4 to present the specific antigen conalbumin to the murine T-helper cell clone DlO.G4.1 [CPM] and to express MHC class II (Ia) antigen [%Ia positive pMO] 24 hours after hemorrhage. Hemorrhaged saline-treated mice (hem; n = 6) were compared to sham-operated animals (sham; n = 6) and hemorrhaged animals with ibuprofen treatment (1 mg/kg BW; hem + IBU; n = 6). The data are presented as the mean ± SEM. **p < 0.01 hem versus sham; #p . 0.05/##p c 0.01 hem versus hem + IBU.
ERTEL AND OTHERS
± 2.7% Ia positive cells) by 60% compared to sham animals (51.5% ± 3.0%) (Fig. 1). Administration of ibuprofen significantly (p < 0.01) improved pMo capability to present Ia by 58% (32.7% ± 1.1%) when compared to the vehicle-treated group (20.7% ± 2.7%). Nonetheless a complete restoration of Ia expression was not observed after ibuprofen treatment.
Interleukin-J Synthesis Interleukin- 1 synthesis by pM4 from the vehicle-treated hemorrhaged group (4.15 ± 1. 10 U/mL) was significantly (p < 0.05) decreased compared to sham-operated animals (11.6 ± 1.97 U/mL) (Fig. 2). Macrophages from ibuprofen-treated hemorrhaged animals demonstrated a high, but not significant, increase in IL- 1 production of 108% (8.64 ± 1.73 U/mL) compared to the vehicle-treated group.
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1.50. 1.00. 0.50. sham
FIG. 3. Production of tumor necrosis factor (cachectin) [U/mL] from pMo 24 hours after hemorrhage. Hemorrhaged saline-treated animals (n = 6) were compared to sham-operated animals (n = 6) and hemorrhaged animals with ibuprofen treatment (1 mg/kg BW; n = 6). The WEHI 164 clone 13 cell line was used for TNF measurements in pMo supernatants. The data are presented as the mean ± SEM. *p < 0.05 sham versus hem + IBU. #p < 0.05 hem versus hem + IBU. a
Interleukin-6 Synthesis Interleukin-6 production by pMo from hemorrhaged mice (310 ± 6 U/mL) was reduced by 47% when compared to sham animals (589 ± 102 U/mL) (Fig. 2). Administration of ibuprofen immediately after hemorrhage resulted in a further decrease of IL-6 production by pMo (184 ± 26 U/mL), which was statistically different (p < 0.01) from sham- and vehicle-treated animals.
erated animals (0.60 ± 0.12 U/mL) was observed (Fig. 3). However ibuprofen treatment significantly (p < 0.05) increased TNF production by 239% (1.49 ± 0.27 U/mL) compared to the vehicle-treated group and by 148% compared to sham-operated animals.
Tumor Necrosis Factor-a Synthesis
Prostaglandin E2 Plasma Levels
Twenty-four hours after hemorrhage
Plasma levels of PGE2
significantly increased by
significant difference in TNF production bly pMo from hemorrhaged animals (0.44 ± 0.18 U/mL) arnd sham-op-
156% in hemorrhaged animals (63.7 ± 8.2 pg/mL) compared to the shams (24.9 ± 5.8 pg/mL) (Fig. 4). Plasma
levels of PGE2 from ibuprofen-treated animals (23.1 ± 12.8 pg/mL) demonstrated a massive reduction by 64% and the values were comparable to the shams (Fig. 4).
12.0. 8.0 4.0
FIG. 2. Production of interleukin- [U/mL] and interleukin-6 [U/mL] from pMO 24 hours after hemorrhage. Hemorrhaged saline-treated mice (n = 6) were compared to sham-operated animals (n 6) and hemorrhaged animals with ibuprofen treatment (1 mg/kg BW; n 6). Cytokine levels in supernatants were determined using specific bioassays (Dl O.G4. 1 for IL- 1, 7TDI for IL-6). The data are presented as the mean ± SEM. *p < 0.05/**p c 0.01 hem versus sham; ##p < 0.01 hem versus hem + IBU. =
Discussion Our data demonstrate that increased PGE2 plasma levels following hemorrhage correlated with the depressed macrophage antigen presentation, Ia expression, and IL1 synthesis. Ibuprofen, a cyclooxygenase inhibitor, prevented the hemorrhage-induced suppression of peritoneal macrophage functions, despite a significant increase in cachectin synthesis. Clinical studies showed a 1 0-fold increase of PGE2 production by blood macrophages up to 21 days after major trauma.23 In addition elevated serum levels of PGE2 after bum injury in humans have been reported.24 Furthermore experimental studies demonstrated that blockade of prostaglandin synthesis after burn injury in mice improved the depressed IL-2 synthesis25 in vitro. These results from animal and clinical experiments, therefore, support the notion that PGE2 in vitro is capable of suppressing dif-
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FIG. 4. Plasma levels of PGE2 [pg/mL] 24 hours after hemorrhage. Hemorrhaged saline-treated animals (n = 3) were compared to sham-operated animals (n = 3) and hemorrhaged animals with ibuprofen treatment (1 mg/kg BW; n = 3). PGE2 levels were determined with radioimmunoassay technique. The data are presented as the mean ± SEM. *p < 0.05 hem versus sham.
ferent immune responses such as IL- 12627 and TNF14 production, MHC class II antigen expression,28 antigen presentation,21 lymphokine synthesis by T lymphocytes30 or B-lymphocyte proliferation,3' and immunoglobulin production. '0 Although we demonstrated in previous studies that simple hemorrhage caused a marked depression of the immune responses and macrophage functions,"4 it remained unknown whether increased PGE2 synthesis by macrophages played any major role in inducing the observed immunosuppression and enhanced susceptibility to sepsis. This was investigated in this study by measurement of PGE2 plasma levels as well as via administration of the cyclooxygenase inhibitor ibuprofen immediately after hemorrhage and resuscitation. Such studies were performed 24 hours after hemorrhage because recent findings revealed a significant suppression of various immune functions at this time point.4'6"7 The dose of 1 mg ibuprofen per kilogram body weight used in this study is similar to that used in previous studies.'2'32 Our data demonstrate that even simple hemorrhage without significant tissue trauma caused a significant elevation of plasma PGE2 levels. Furthermore the observed suppression of antigen presentation capacity, Ta expression, and IL-1 synthesis correlated well with increased PGE2 plasma levels. Thus it appears that there is a close relationship between elevated PGE2 levels and decreased macrophage functions. This suggestion is supported by our studies that demonstrated that in vivo blockade of PGE2 synthesis with ibuprofen significantly improved the depressed macrophage functions. This finding further confirms the immunosuppressive role of PGE2 after hem-
orrhage. The observed immunoprotective effects of ibuprofen are in line with previous findings concerning the beneficial effect of cyclooxygenase inhibitors in the therapy of depressed cellular immune functions following burn injury in mice32 and major surgery in humans.'2 However none of these studies investigated the effect of cyclooxygenase inhibitors on macrophage functions such as antigen presentation and monokine production. The enhancement of depressed macrophage functions such as antigen presentation and MHC class II antigen expression are important because macrophages play a key role in eliciting an adequate cellular and humoral immune response to microorganisms. Whether enhanced cachectin production by Kupffer cells33 correlating with elevated TNF plasma levels is responsible for the observed immunosuppression is so far not understood. Nonetheless it is known that very high levels of cachectin, as found during endotoxemia and sepsis, cause hypotension, cachexia, and increased mortality.34'35 In addition blockade of TNF activity by monoclonal antibodies decreased rates of mortality after endotoxemia.36 Our data show that blockade of PGE2 synthesis with ibuprofen resulted in significantly increased TNF production. Therefore PGE2 may be a major downregulating agent for TNF synthesis not only in vitro, as shown by Kunkel et al.,'4 but also in vivo. The improvement of macrophage functions despite an elevated TNF production by macrophages following ibuprofen treatment further indicates that TNF, at least in amounts present during and after hemorrhage, does not have any immunosuppressive effect. In fact, TNF-a may even have a stimulatory effect on cellular immune responses as it is shown in several in vitro studies, where TNF-enhanced Fc receptor expression,37 B-cell proliferation and differentiation,38 T-lymphocyte growth,39 as well as macrophage Ia expression.40 Therefore it can be hypothesized from our data that a certain amount of TNF is necessary to activate immune and nonimmune cells, initiate, support, and maintain inflammatory responses following tissue injury induced by hemorrhage. In contrast overproduction of this monokine, as observed during sepsis,4' or TNF-a infusion in high amounts'6 35 are detrimental for the host. Therefore TNF-a and cachectin might be the two different sides of the same biologic coin. Although many studies concerning TNF-a and its effects have been carried out, little is known about the role and regulation of IL-6 production following hemorrhage and resuscitation. Studies from Guo et al.42 and Waage et al.43 demonstrated a significant correlation between endotoxemia, mortality after sepsis, and the degree of increased IL-6 levels in plasma. Whether increased IL-6 production only represents an important parameter in severe inflammation and infection or whether IL-6 is involved directly in the induction of immunosuppression
ERTEL AND OTHERS
is not known. In previous studies we demonstrated a peak of IL-6 plasma levels 2 hours after hemorrhage'3 and a significant increase of IL-6 synthesis by peritoneal macrophages (unpublished data). However 24 hours after hemorrhage the production of IL-6 from peritoneal macrophages was decreased compared to sham animals. Treatment with ibuprofen further decreased IL-6 synthesis. Whether this represents a direct effect of the anti-inflammatory agent ibuprofen on IL-6 synthesis or an indirect effect by blocking the production of other mediators that induce IL-6 synthesis remains unknown. The decreased IL-6 synthesis in ibuprofen-treated hemorrhaged animals correlated well with the increased production of IL- 1 and TNF-a. These data confirm the observations of Schindler et al.,44 who described a suppressive effect of IL-6 on IL-1 and TNF-a synthesis by peripheral blood cells and indicate a potential down-regulating function of IL-6 after hemorrhage. Nonetheless the role of altered IL6 production after hemorrhage must be investigated further. In summary our data demonstrate that arachidonic acid metabolites such as PGE2 but not cachectin are directly involved in the suppression of different macrophage functions after hemorrhage. In vivo blockade of PGE2 production by ibuprofen following hemorrhage significantly improved macrophage functions and did not affect the hemodynamic response to resuscitation. Therefore the cyclooxygenase inhibitor ibuprofen represents a safe and potent agent in the therapy of hemorrhage-induced im-
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