Transforming growth factor beta 1 alters rat peritoneal macrophage mediator production and improves survival during endotoxic shock.

Transforming Growth Factor beta 1 (TGF-beta 1) is a potent anti-inflammatory cytokine involved in wound healing. Because of its anti-inflammatory actions, the effects of TGF-beta 1 were studied in vitro on rat macrophage inflammatory mediator production and in vivo on Salmonella enteritidis endotoxin (LPS) induced lethality. TGF-beta 1's effect on stimulated rat peritoneal macrophage (MO) production of prostacyclin and nitric oxide were assessed via measurement of their stable metabolites, 6-keto-PGF-1 alpha and nitrite, respectively. TGF-beta 1 (10 ng/ml) pretreatment (3 hours) resulted in significant reductions (p < 0.05) of LPS (50 micrograms/ml) stimulated, lipid A (1 and 10 micrograms/ml) stimulated, arachidonic acid (16 microM) stimulated, and Ca++ ionophore (10 microM) stimulated MO 6-keto-PGF 1 alpha production. These data suggest that TGF-beta 1 inhibits LPS stimulated MO 6-keto-PGF-1 alpha production by acting at, or distally to, phospholipase A2, possibly at the level of cyclooxygenase in the arachidonic acid cascade. In a similar study, TGF-beta 1 pretreatment led to a significant (p < 0.05) reduction of LPS stimulated MO nitrite production. In subsequent studies, the effects of TGF-beta 1 were studied in vivo on rats challenged with lethal doses of LPS. Rats pretreated with TGF-beta 1 (250 ng/rat i.v.) exhibited a significant increase (p < 0.01, n = 15 rats/group) in mean survival time compared to control rats. The increased survival time of TGF-beta 1 pretreated rats with LPS shock may be due, in part, to altered production of MO mediators.

Endotoxin tolerance is associated with decreased prostaglandin H synthases-1 and -2.

Lipopolysaccharide (LPS) induces macrophage protein and eicosanoid synthesis. Previous studies have shown that LPS induction of eicosanoid metabolism is transcription dependent and that prostaglandin (PG) H synthase is the committed step in the conversion of arachidonic acid (AA) to thromboxane (Tx) B2. LPS tolerance induced by sublethal in vivo injections of LPS renders rats resistant to LPS in vivo and macrophages refractory to LPS-stimulated eicosanoid synthesis in vitro. This study examined potential activity and content changes in constitutive and mitogen inducible PGH synthase in LPS-stimulated control and tolerant macrophages. TxB2 levels were measured to evaluate basal PGH synthase activity and stimulation by Salmonella enteritidis LPS (50 micrograms/ml), calcium ionophore A-23187, and AA. All tolerant macrophage groups demonstrated decreased TxB2 synthesis. TxB2 synthesis stimulated by AA in tolerant cells was decreased by 70% (P < 0.05) compared with control macrophages. In subsequent studies changes in PGH synthase content were examined in rat peritoneal macrophages from tolerant and control rats incubated with and without LPS. Immunoblot analysis of PGH synthase-1 (constitutive) demonstrated no increase in cells stimulated with LPS compared with basal but was diminished by 62 +/- 9% (n = 4, P < 0.05) in tolerant macrophages compared with control cells. Immunoblot analysis of PGH synthase-2 (mitogen inducible) demonstrated induction in response to LPS that was maximal between 12 and 24 h. PGH synthase-2 was also induced in tolerant macrophages in response to LPS but was less than in control cells. The results demonstrate that endotoxin tolerance is associated with reduced activity and content of PGH synthase-1 and a decreased LPS induction of PGH synthase-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of two magainin peptides on eicosanoid release from rat peritoneal macrophages.

Magainins are novel polycationic peptides with broad-spectrum antimicrobial activity, including activity against gram-negative bacteria. Gram-negative bacteremia can elicit endotoxic shock that is associated with the increased formation of eicosanoids. Inhibition of eicosanoid synthesis has been shown to improve the outcome of experimental endotoxic shock. The aim of the present study was to test the in vitro effects of two magainin peptides, MSI-97 (M1) and MSI-98 (M2), on eicosanoid synthesis by rat peritoneal macrophages (M phi) stimulated by Salmonella enteritidis lipopolysaccharide (LPS; 50 micrograms/ml) and Salmonella minnesota lipid A (5 micrograms/ml) and to compare their effects on LPS reactivity with a metachromatic dye. M1 (100 micrograms/ml) significantly (P < 0.05) reduced LPS-stimulated synthesis of thromboxane B2 (TXB2), without changing 6-keto-prostaglandin F1 alpha in M phi. Similarly, M2 (10 micrograms/ml) significantly attenuated M phi synthesis of TXB2 stimulated by either LPS or lipid A. However, at a higher concentration (100 micrograms/ml), M2 but not M1 significantly augmented LPS-induced increases in TXB2 and 6-keto-prostaglandin F1 alpha. Polymyxin B (40 micrograms/ml) inhibited LPS production and lipid A-stimulated TXB2 production. M1 (100 micrograms/ml) and polymyxin B (10 and 40 micrograms/ml) also inhibited calcium ionophore A23187 (10 microM)-induced synthesis of TXB2. The lipid A moiety of LPS reacts with dimethylmethylene blue dye, providing a metachromatic assay of LPS. This metachromatic reaction with lipid A was significantly reduced by polymyxin B and M2 at all concentrations. M1 was effective only at the highest M1:lipid A concentration ratio (2:1). Thus, M1 and M2 share some similarities with polymyxin B in inhibiting lipid A reactivity with the dye, which suggests that these magainins may also bind to lipid A. However, M1 was devoid of any inhibitory effects on dye reactivity with S. enteritidis LPS and M2 was inhibitory at only one concentration ratio (1:5). In conclusion, the varied effects of the magainin peptides on LPS, lipid A, and M phi eicosanoid synthesis appear to depend on the type of peptide used and on its concentration.

Endotoxin tolerance is associated with altered GTP-binding protein function.

Previous studies have suggested that guanine nucleotide regulatory (G) proteins modulate endotoxin-stimulated peritoneal macrophage arachidonic acid (AA) metabolism. Endotoxin-stimulated metabolism of AA by peritoneal macrophages is decreased in endotoxin tolerance (Rogers et al. Prostaglandins 31: 639-650, 1986). These observations led to a study of G protein function and AA metabolism by peritoneal macrophages in endotoxin tolerance. Endotoxin tolerance was induced by the administration of sublethal doses of endotoxin. AA metabolism was assessed by measurement of thromboxane B2 (TxB2), a cyclooxygenase metabolite. NaF (5 mM), an activator of G proteins, significantly stimulated TxB2 synthesis in control macrophages from 7.7 +/- 0.2 to 19.1 +/- 0.6 (SE) ng/ml (P less than 0.05) at 2 h and was partially inhibited by pertussis toxin, suggesting a G protein-dependent mechanism. Salmonella enteritidis endotoxin (50 micrograms/ml) stimulated a similar increase in TxB2 levels (23 +/- 0.4 ng/ml, P less than 0.05). In contrast to control macrophages, macrophages from endotoxin-tolerant rats stimulated with either NaF or S. enteritidis endotoxin had TxB2 levels that were only 30 and 2% of the respective stimulated control cells. Basal guanosine-triphosphatase (GTPase) activity (33 +/- 6 pmol.mg-1.min-1) in endotoxin-tolerant macrophage membranes was significantly lower (P less than 0.05) than control basal activity (158 +/- 5 pmol.mg-1.min-1). This suppression of macrophage GTPase activity was apparent 48 h after the first in vivo sublethal endotoxin injection (100 micrograms/kg ip). The reduced GTPase activity paralleled in vitro cellular hyporesponsiveness to endotoxin-stimulated TxB2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin-induced arachidonic acid metabolism requires de novo protein synthesis and protein kinase C activation.

The mechanisms whereby bacterial endotoxins stimulate arachidonic acid metabolism in macrophages are uncertain. Both protein kinase C activation and de novo protein synthesis occur in macrophages in response to endotoxin. In this study we evaluated the time course and role of protein kinase C and de novo protein synthesis in endotoxin stimulated arachidonic acid metabolism in resident rat peritoneal macrophages. Thromboxane (TX) B2 was measured as the representative arachidonic acid metabolite synthesized in response to Salmonella enteritidis endotoxin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate (PMA). The effect of inhibition of protein kinase C by 1-(5-isoquinolinsulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine on endotoxin- and A23187-induced TXB2 synthesis was examined. The potential roles of transcriptional and translational events in endotoxin- and A23187-stimulated TXB2 synthesis were determined by utilizing the transcriptional inhibitors camptothecin (10 microM) or actinomycin D (0.08 microM), and the translational inhibitor cycloheximide (0.1 microM). Whereas, A23187 stimulated maximal TXB2 synthesis within 15 min, endotoxin showed a more prolonged time course with a 12-fold increase in TXB2 synthesis above basal levels after 3 h (P less than 0.05). PMA induced an approx. 8-fold increase above basal TXB2 levels that was blocked by inhibition of transcription with actinomycin D. H-7 (10 microM to 50 microM) inhibited endotoxin- and A23187-stimulated eicosanoid synthesis. Staurosporine (0.2 microM) produced a selective 66% inhibition of endotoxin, but not A23187-stimulated TXB2 synthesis. Endotoxin-induced TXB2 production was significantly (P less than 0.05) inhibited by staurosporine, camptothecin, actinomycin D or cycloheximide at intervals from 30 min prior to, through 60 min after endotoxin stimulation. These studies suggest a role for protein kinase C activation and de novo protein synthesis in endotoxin signal transduction events leading to increased macrophage arachidonic acid metabolism. These intracellular events are essential in sustaining the prolonged inflammatory response to endotoxin.

Effect of LTB4 receptor antagonists in endotoxic shock in the rat.

This study examined 1) the effects of infusion of LTB4 and 2) the potential role of LTB4 in the sequelae to endotoxic shock in the rat. Control rats were anesthetized with Ketamine/xylazine and given LTB4 (2 micrograms/kg) bolus i.v. followed by a 1 microgram/kg/min infusion for 10 min. LTB4 induced systemic hypotension and a three-fold increase in circulating band neutrophils which contributed to a 70% increase (P less than 0.05) in the total peripheral neutrophil count. LTB4 did not cause changes in circulating mature (segmented) neutrophils, lymphocytes, platelets, or hematocrits. Pretreatment (1 min) with LY233978, an LTB4 antagonist (10 mg/kg bolus i.v.), inhibited LTB4-induced systemic hypotension (-16.1 +/- 6.1 mmHg [n = 3] vs. -38.8 +/- 5.9 mmHg [n = 4], P less than 0.05). Salmonella enteritidis endotoxin (10 mg/kg bolus i.v.) induced systemic hypotension, hemoconcentration, leukopenia, and thrombocytopenia, which was greatest at 5 and 15 min postendotoxin. The leukopenia was characterized by lymphopenia, band neutropenia, and segmented neutropenia. LY233978 (10 mg/kg bolus i.v. immediately before endotoxin administration and followed by an infusion at 0.67 mg/kg/min for 90 min) attenuated endotoxin-induced hemoconcentration at 60 and 90 min postendotoxin (P less than 0.05), and systemic hypotension at 15 min postendotoxin (P less than 0.05). The LTB4-receptor antagonist LY255283 (10 mg/kg bolus i.v., 10 min before endotoxin followed by a 5 mg/kg bolus i.v. 30 min postendotoxin) completely inhibited endotoxin-induced systemic hypotension and partially attenuated endotoxin-induced hemoconcentration from 15 min to 90 min postendotoxin (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the LTD4 receptor antagonist LY203647 on endotoxic shock sequelae in the rat.

This study investigated the effect of a novel LTD4 receptor antagonist LY203647 on Salmonella enteritidis endotoxin-induced shock sequelae in anesthetized rats. LY203647 (30 mg/kg i.v.) or vehicle was given 10 min prior to endotoxin (10 mg/kg i.v.) or its vehicle, and the hematocrit, mean arterial pressure and circulating leukocyte counts were determined. LY203647 significantly inhibited endotoxin-induced hemoconcentration up to 90 min post-endotoxin (46.7 +/- 1.3 vs. 51.9 +/- 2.4% at 30 min post-endotoxin, 45.9 +/- 1.1 vs. 53.1 +/- 1.4% at 90 min post-endotoxin, N = 8-9, P less than 0.05). The endotoxin-induced decreases in mean arterial pressure were also attenuated by LY203647, -29 +/- 5 vs. -56 +/- 9 mm Hg at 60 min post-endotoxin and -42 +/- 4 vs. -60 +/- 9 mm Hg at 90 min post-endotoxin (N = 9-10, P less than 0.05). LY203647 also attenuated endotoxin-induced decreases in leukocyte count in arterial blood. A study of differential counts in circulating leukocytes (N = 3) showed that endotoxin induced complete disappearance in circulating neutrophils. The circulating lymphocyte count was decreased by 30 +/- 10 and 41 +/- 1% at 15 and 30 min post-endotoxin, respectively. LY203647 inhibited endotoxin-induced lymphopenia (P less than 0.05) but failed to alter endotoxin-induced neutropenia. These data suggest that LTD4 may play an important role in mediating hemoconcentration, hypotensive and lymphocytopenic sequelae of endotoxin shock.

Effects of a linseed oil enriched diet on endotoxin-induced sequelae: differential in vitro and in vivo effects.

Endotoxin stimulates macrophages to synthesize membrane-associated inflammatory mediators including eicosanoids and procoagulant activity (PCA). Alpha linolenic acid, a component of linseed oil, is metabolized to eicosapentaenoic acid, which may replace arachidonic acid in membrane phospholipids. Thus, ingestion of linseed oil may alter the generation of eicosanoids, PCA and other membrane-dependent responses. This study compared the in vitro endotoxin-induced synthesis of eicosanoids and expression of PCA by peritoneal macrophages obtained from rats fed a control diet and rats fed a diet enriched with linseed oil. The effect of endotoxin on in vivo plasma eicosanoid concentrations, leukogram, and microvascular permeability were determined. Endotoxin (5 ug/ml) stimulated synthesis of iTxB2, i6-keto PGF1 alpha and expression of PCA by macrophages in vitro. These in vitro responses of macrophages from linseed oil fed rats were significantly less than those of macrophages from control rats. In contrast, there were no significant differences in in vivo responses to endotoxin. The fatty acid composition of total lipids and phospholipids in liver and plasma from linseed oil fed rats and control rats were not different. These composite data suggest several possibilities: (1) linseed oil may have effects independent of alpha-linolenic acid on macrophage function, (2) linseed oil may alter the fatty acid composition of macrophage phospholipids prior to changing that of other tissues, and (3) the reduced in vitro responses of peritoneal macrophages may not reflect the systemic responses to endotoxin.

Endotoxin tolerance differentially alters hemodynamic responses to a thromboxane A2 mimetic and phenylephrine.

Repeated sublethal doses of endotoxin render rats tolerant to lethal doses of endotoxin and reduce thromboxane (Tx) A2 synthesis. Endotoxin-tolerant rats are also more resistant to lethal doses of U46619, a stable TxA2 mimetic. These observations raised the possibility that tolerance may alter hemodynamic responses to TxA2 via one or more mechanisms. Mean arterial pressure (MAP) responses to i.v. injections of the stable TxA2 mimetic U46619 at doses ranging from 0.17 to 8.4 micrograms/kg were determined. Despite an initial lower systemic vascular resistance in tolerant rats compared to control rats (2.4 +/- 0.3 vs 3.1 +/- 0.2 mm Hg/ml/min/100 g of body weight, respectively, p less than 0.05), the maximum pressor response to U46619 was significantly greater (p less than 0.05) at the higher doses of U46619 in tolerant rats compared to control rats. Tolerant and control rats also exhibited qualitatively different changes in MAP in response to U46619. Control rats manifested an initial hypotensive response (15 s) not observed in tolerant rats. In contrast, tolerant rats exhibited no depressor response, but a higher peak pressor response to U46619 than that seen in controls. Since prostaglandins may modulate vascular responses to U46619, subsequent studies were conducted in indomethacin-pretreated or essential fatty acid (EFA) deficient rats that were depleted of arachidonic acid substrate. Either indomethacin or EFA deficiency significantly prevented the initial hypotensive response in control rats, suggesting that prostaglandins may mediate this response to U46619. In additional studies, the MAP response in tolerant and control rats to the alpha 1-adrenergic agonist phenylephrine was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of leukotriene receptor antagonists on vascular permeability during endotoxic shock.

Evidence has accumulated that sulfidopeptide leukotrienes are significant pathogenic mediators of certain hematologic and hemodynamic sequelae of endotoxic shock. In the present study, the effects of a selective LTD4/E4 receptor antagonist, LY171883 (LY), or a selective LTD4 receptor antagonist, SKF-104353 (SKF), were assessed on splanchnic and pulmonary localization of 99mTechnetium-labeled human serum albumin (99mTc-HSA) in acute endotoxic shock in the rat. Dynamic gamma camera imaging of heart (H), midabdominal (GI), and lung regions of interest generated time activity curves for baseline and at 5-35 min after Salmonella enteritidis endotoxin (10 mg/kg, i.v.). Slopes of GI/H and lung/H activity (permeability index, GI/H or lung/H X 10(-3)/min) provided indices of intestinal and lung localization. Rats received LY (30 mg/kg, i.v.), LY vehicle (LY Veh), SKF (10 mg/kg), or SKF vehicle (SK Veh) 10 min prior to endotoxin or endotoxin vehicle. In rats receiving the LY Veh and endotoxin (n = 8) or SKF Veh and endotoxin (n = 12), the splanchnic permeability indices to 99mTc-HSA were increased 11.2-fold and 5.1-fold, respectively (P less than 0.05) compared to vehicle control groups not given endotoxin (n = 5). Pulmonary permeability index for 99mTc-HSA was increased (P less than 0.05) to a lesser extent (3.2-fold) by endotoxin compared to vehicle controls. Pretreatment with SKF reduced the mesenteric permeability index to control levels (P less than 0.05) during the 5-35 min time interval post-endotoxin. LY reduced the mesenteric permeability index by 70%. Pulmonary relative permeability to 99mTc-HSA was not affected by LY pretreatment. Both splanchnic and lung relative permeability to the isotope was transient; at 135-225 min post-endotoxin, splanchnic localization of 99mTc-HSA (n = 4) was not significantly different from vehicle controls in these vascular beds. Relative localization of 99mTc-labeled red blood cells (RBC) in the splanchnic or lung region was not significantly altered by endotoxin (n = 7) or LY pretreatment (n = 6) compared to vehicle controls (n = 6). In additional studies, small intestinal luminal content of 99mTc-HSA and 111Indium (In)-labeled RBC were determined after i.v. administration of the isotopes, in a 4 cm segment of the upper small bowel. Radioactivity in the luminal lavage was normalized to activity in blood of the same animal. Endotoxin at 2 hr induced a 2.3-fold increase transluminal leakage of 99mTc-HSA (n = 5; P less than 0.03) compared to LY Veh (n = 5) or control (n = 5) rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered responses to modulators of guanine nucleotide binding protein activity in endotoxin tolerance.

The effects of cholera toxin or pertussis toxin and nonhydrolyzable GTP analogs on Salmonella enteritidis endotoxin stimulation of iTxB2 and i6-keto-PGF1 alpha synthesis in control and endotoxin tolerant rat peritoneal macrophages were determined. Pretreatment with pertussis toxin alone had no effect on basal macrophage iTxB2 or i6-keto-PGF1 alpha production, but pertussis toxin (0.1, 1.0 and 10 ng/ml) significantly inhibited endotoxin-stimulated iTxB2 and i6-keto-PGF1 alpha synthesis. Pretreatment with cholera toxin, which did not affect basal iTxB2 or i6-keto-PGF1 alpha synthesis, significantly enhanced endotoxin-induced synthesis of iTxB2 and i6-keto-PGF1 alpha. The effects of pertussis and cholera toxin with or without endotoxin were significantly (P less than 0.05) less in macrophages from endotoxin tolerant rats compared to control macrophages. GTP[gamma-S] (100 microM) significantly increased iTxB2 synthesis and significantly augmented endotoxin-stimulated iTxB2 synthesis in control macrophages (P less than 0.05). However, in macrophages from endotoxin tolerant rats the effect of GTP[gamma-S] on iTxB2 synthesis was significantly less (P less than 0.05) compared to control macrophages. Collectively, these data suggest that: (1) guanine nucleotide binding regulatory proteins mediate endotoxin-stimulated arachidonic acid metabolism in rat peritoneal macrophages; and (2) endotoxin tolerance induces alterations in guanine nucleotide binding protein activity.

Endotoxin-induced procoagulant activity, eicosanoid synthesis, and tumor necrosis factor production by rat peritoneal macrophages: effect of endotoxin tolerance and glucan.

Macrophages release pro-inflammatory substances that may augment intravascular coagulopathy associated with endotoxemia. In the present study, the effect of Salmonella enteritidis endotoxin on expression of procoagulant activity (PCA), eicosanoid metabolism, and production of tumor necrosis factor (TNF) by rat peritoneal macrophages was determined. Endotoxin induced significant dose-dependent increases in the concentrations of immunoreactive (i) thromboxane B2 (TxB2), 6-ketoprostaglandin F1 alpha (i6-keto-PGF1 alpha), and TNF in culture media. Calcium ionophore (A23187; 0.5 microM) induced an approximate two-fold greater increase (P less than 0.05) in iTxB2 and i6-keto-PGF1 alpha than that stimulated by the maximal endotoxin dose. Endotoxin (0.5, 5, and 50 micrograms/ml) induced similar increases in PCA in macrophage lysates which paralleled the production of iTxB2 and i6-keto-PGF1 alpha. In contrast to its marked effect on eicosanoid metabolism, A23187 elicited little increase in PCA. After the responses of peritoneal macrophages from normal animals were characterized, we hypothesized that procedures which alter the in vivo response of rats to endotoxin would similarly alter the in vitro responses of their peritoneal macrophages. In subsequent studies, the effect of altered endotoxin sensitivity on expression of PCA, eicosanoid synthesis, and TNF activity were assessed. Endotoxin tolerance, induced by repeated injection of sublethal doses of endotoxin in vivo, rendered rat peritoneal macrophages refractory to in vitro endotoxin-induced production of iTxB2, i6-keto-PGF1 alpha, PCA, and TNF activity. In contrast, pretreatment of rats with the macrophage stimulant glucan, which enhances endotoxin lethality, augmented the in vitro production of iTxB2, PCA, and TNF by endotoxin-stimulated peritoneal macrophages. These studies demonstrate that endotoxin-induced macrophage arachidonic acid metabolism is associated with expression of PCA and secretion of TNF. Additionally, macrophage synthesis of these pathogenic mediators is reduced under conditions associated with endotoxin resistance (endotoxin tolerance) and is augmented during endotoxin hypersensitivity (glucan stimulation).

Resistance of essential fatty acid-deficient rats to endotoxin-induced increases in vascular permeability.

Resistance to endotoxin in essential fatty acid-deficient (EFAD) rats is associated with reduced synthesis of certain arachidonic acid metabolites. It was hypothesized that EFAD rats would manifest decreased vascular permeability changes during endotoxemia as a consequence of reduced arachidonic acid metabolism. To test this hypothesis, changes in hematocrit (HCT) and mesenteric localization rate of technetium-labeled human serum albumin (99mTc-HSA) and red blood cells (99mTc-RBC) were assessed in EFAD and normal rats using gamma-camera imaging. Thirty minutes after Salmonella enteritidis endotoxin, EFAD rats exhibited less hemoconcentration as determined by % HCT than normal rats (47 +/- 2% vs. 54 +/- 1% respectively, P less than 0.01). Endotoxin caused a less severe change in permeability index in the splanchnic region in EFAD rats than in normal rats (1.2 +/- 0.6 x 10(-3)min-1 vs. 4.9 +/- 1.7 x 10(-3)min-1 respectively, P less than 0.05). In contrast to 99mTc-HSA, mesenteric localization of 99mTc-RBC was not changed by endotoxin in control or EFAD rats. Supplementation with ethyl-arachidonic acid did not enhance susceptibility of EFAD rats to endotoxin-induced splanchnic permeability to 99mTc-HSA. Leukotrienes have been implicated as mediators of increased vascular permeability in endotoxin shock. Since LTC3 formation has been reported to be increased in EFA deficiency, we hypothesized that LTC3 may be less potent than LTC4. Thus the effect of LTC3 on mean arterial pressure and permeability was compared to LTC4 in normal rats. LTC3-induced increases in peak mean arterial pressure were less than LTC4 at 10 micrograms/kg (39 +/- 5 mm Hg vs. 58 +/- 4 mm Hg respectively, P less than 0.05) and at 20 micrograms/kg (56 +/- 4 mm Hg vs. 75 +/- 2 mm Hg respectively, P less than 0.05). LY171883 (30 mg/kg), an LTD4/E4 receptor antagonist, attenuated the pressor effect of LTC4, LTD4, and LTC3. Infusion of LTC4 (4 micrograms/kg/min) in normal rats induced a rise in HCT from 44 +/- 1% to 51 +/- 1% (P less than 0.01), which was greater (P less than 0.05) than the rise induced by LTC3 (47 +/- 1% to 49 +/- 1%). The results showing that EFAD rats are resistant to endotoxin-induced increases in HCT and vascular permeability raise the possibility that this may, in part, be a result of preferential LTC3 production that is less potent than LTC4.

Hemodynamic effects of leukotriene (LT) D4 and a LTD4 receptor antagonist in the pig.

Vascular bed reactivity to exogenous LTD4 and blockade of these responses by a selective LTD4 receptor antagonist, SK&F 104353, were studied. Yorkshire pigs (N = 13; 25.8 +/- 1.4 kg) were anesthetized with isoflurane, and monitored for cardiac output (CO), mean pulmonary artery pressure (MPAP), pulmonary capillary wedge pressure (PCWP), right ventricular stroke work (RVSW), mean arterial pressure (MAP), renal artery blood flow (RABF), pulmonary vasculature resistance (PVR), renal vascular resistance (RVR), and systemic vascular resistance (SVR). LTD4 (0.03, 0.1, 0.3, 1, 3 and 10.0 micrograms/kg) was given intravenously. The MPAP, MAP, and RABF were recorded at baseline and for 20 min after the injection of LTD4. SK&F 104353 was infused (3 mg/kg/h, i.v.) and the dose-response to LTD4 was repeated. LTD4 induced changes in the MPAP, PCWP, MAP, and RABF. SK&F 104353 attenuated LTD4 induced changes in the MPAP, PVR, RABF and RVSW. MPAP was increased 123 +/- 24% and 115 +/- 10%, respectively, in pigs given 3.0 and 10 micrograms/kg doses, and increased to only 36 +/- 9% at a 10 micrograms/kg dose of LTD4 in animals pretreated with SK&F 104353 (P less than 0.05). MPAP was not increased at the 3.0 micrograms/kg dose, and increased to only 36 +/- 9% at a 10 micrograms/kg dose of LTD4 in animals pretreated with SK&F 104353 (P less than 0.05). These changes were paralleled by a reduction in PVR. RABF was decreased 76 +/- 5% and 83 +/- 7%, respectively, at the 3.0 and 10 micrograms/kg dose of LTD4.(ABSTRACT TRUNCATED AT 250 WORDS)

Essential fatty acid-deficient rats are resistant to oleic acid-induced pulmonary injury.

Because leukotrienes and prostaglandins are inflammatory mediators derived from arachidonic acid, their potential role in oleic acid-induced lung injury was evaluated in control and in essential fatty acid-deficient (EFAD) rats depleted of arachidonic acid substrate. In control rats, oleic acid (0.06 ml/kg iv) increased the pulmonary permeability index (measured by scintigraphy) from -10 +/- 13 x 10(-6) s-1 to 217 +/- 20 x 10(-6) s-1 and 118 +/- 13 x 10(-6) s-1 at 5 and 50 min (P less than 0.05), respectively. It also caused arterial hypoxemia at 30 min (P less than 0.05). Compared with saline controls, oleic acid increased bronchoalveolar lavage fluid levels of immunoreactive (i) LTC4/D4, iLTB4, (P less than 0.01), and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) (P less than 0.05). In EFAD rats, oleic acid failed to significantly increase the lung permeability index at 5 and 50 min. In contrast to control rats, oleic acid failed to cause hypoxemia in the EFAD rats. Bronchoalveolar lavage levels of iLTB4 and i6-keto-PGF1 alpha after oleic acid in EFAD rats were lower compared with oleic acid controls, whereas iLTC4/D4 in the oleic acid EFAD group was not decreased. Treatment with intraperitoneal ethyl arachidonate (400 mg over 2 wk) reversed the resistance of EFAD rats such that the pulmonary edema (P less than 0.05) was evident after oleic acid. This latter group also manifested a significant (P less than 0.05) rise in the bronchoalveolar lavage levels of iLTB4 and i6-keto-PGF1 alpha. These results suggest that arachidonic acid metabolites contribute to oleic acid-induced pulmonary permeability.

Alterations in Gc levels and complexing in septic shock.

Septic shock involves increased generation of eicosanoids from arachidonic acid. Gc (vitamin D-binding protein) has been recently found to bind the parent molecule arachidonic acid but can also complex actin released as a result of tissue damage which causes displacement of bound arachidonic acid. Possible changes in serum levels of Gc and extent of complexing were therefore investigated in patients with gram-negative sepsis. As compared to healthy controls, serum levels of Gc were significantly decreased in patients with septic shock (P less than 0.01). Moreover, the percentages of Gc circulating in complexed form were significantly increased (P less than 0.01) and correlated strongly with disease severity, with levels often greater than 90% in patients who died (normal mean 8% +/- 3). These results suggest a hitherto unsuspected role for Gc in septic shock syndrome.

Alterations in plasma levels and complexing of Gc (vitamin D-binding protein) in rats with endotoxic shock.

Septic shock is known to involve increased metabolism of arachidonic acid and generation of certain eicosanoids. Recently, a new extracellular pool of unsaturated fatty acids including arachidonate has been found in relation to group-specific component (Gc), a vitamin D-binding plasma protein that sequesters monomeric G-actin. Since complexing with G-actin displaces fatty acids, possible alterations in plasma levels of Gc and extent of complexing were sought in serial samples obtained from rats with shock induced by Salmonella enteritidis endotoxin (12.5-15 mg kg-1). Gc levels in animals receiving endotoxin exhibited bimodal alterations, with a significant reduction (P less than 0.001) at 1 hour, followed by a progressive elevation to 160% of starting concentrations at 6 days in animals that survived, whereas in sham-injected animals the change observed was a continuous rise to 147% at 6 days. A statistically significant increase in the percentage of Gc complexed was observed in all endotoxemic rats from 2 hours onward (P less than 0.01), in contrast to sham-injected animals, in which the percentage of Gc complexed remained at less than 5%. Levels in survivors peaked at 30 +/- 5.2% at 8 hours and then decreased to normal (2 +/- 0.9%) by 6 days (n = 7), whereas in nonsurvivors complexed Gc continued to rise until time of death (66-80%) at 6-12 hours (n = 4). Correlation of these results with glucose, transaminases, and immunoreactive TXB2 and 6-keto-PGF1 alpha indicated that decreased absolute levels of Gc represent a consistent early change in endotoxic shock and that the percentage of Gc complexed is an accurate prognostic indicator of severity.

Arachidonic acid turnover in peritoneal macrophages is altered in endotoxin-tolerant rats.

Peritoneal macrophages from endotoxin-tolerant rats have been found to exhibit depressed metabolism of arachidonic acid (AA) to prostaglandins and thromboxane in response to endotoxin. The effect of endotoxin tolerance on AA turnover in peritoneal macrophages was investigated by measuring [14C]AA incorporation and release from membrane phospholipids. Endotoxin tolerance did not affect the amount of [14C]AA incorporated into macrophages (30 min-24 h). However, the temporal incorporation of [14C]AA into individual phospholipid pools (15 min-24 h) was altered. In endotoxin-tolerant macrophages, [14C]AA incorporation into phosphatidylcholine (PC) (2, 4, 24 h) and phosphatidylethanolamine (PE) (8 h) was increased, while the incorporation into phosphatidylserine (PS) (2-24 h) was reduced (P less than 0.005) compared to control macrophages. There was no change in [14C]AA incorporation into phosphatidylinositol (PI). Following 2 or 24 h of incorporation of [14C]AA, macrophages were incubated (3 h) with endotoxin (50 micrograms/ml) or A23187 (1 microM), and [14C]AA release was measured. Endotoxin-tolerant macrophages released decreased (P less than 0.05) amounts of [14C]AA in response to both endotoxin and the calcium ionophore A23187 compared to controls. Control macrophages in response to endotoxin released [14C]AA from PC, PI and PE. In contrast, tolerant cells released [14C]AA only from PC (P less than 0.05). A23187 released [14C]AA from all four pools in the control cells, but only from PC and PE in the tolerant cells. These data demonstrate that endotoxin tolerance alters the uptake and release of AA from specific macrophage phospholipid pools. These results suggest that changes in AA turnover and/or storage are associated with endotoxin tolerance.

The role of eicosanoids in mediating blood flow alterations in endotoxin shock.

The vasoactive metabolites of arachidonic acid TxA2 and sulfidopeptide-leukotrienes appear to play a role in mediating hemodynamic alterations in endotoxic shock. The ultimate goal of these and other studies in shock is, however, to understand the mechanisms underlying the pathogenic changes in human shock states in order to develop effective treatment. Recent studies (see reviews by Ball, 1988) have implicated eicosanoids in pathogenesis of shock in man confirming the directions provided by the bolus endotoxin studies and suggest additional investigations of hemodynamics following interventions designed to alter eicosanoid synthesis in shock.

The rat in sepsis and endotoxic shock.

Compared to the experimental studies, interpretation of eicosanoid levels in human sepsis is complicated by a large number of uncontrolled variables. Further clinical studies are needed to establish the relationship between the septic rat model and the human septic condition. The rat model is useful for uncovering fundamental pathophysiologic processes and should be useful in developing therapeutic interventions in human circulatory shock. Studies on the rat model offered the first indication that eicosanoids were involved in the shock syndrome (Cook et al., 1980). This led to clinical trials to determine the role of these mediators in human sepsis (Reines et al. 1982). The rat affords a model system which allows for pharmacologic manipulation and testing of a large number of hypotheses in a practical and cost effective manner. The recent evidence that leukotrienes are released during circulatory shock in the rat and that attenuation of this release alters the pathophysiologic outcome (Ball et al., 1986), lays the groundwork for future clinical studies. Finding that eicosanoids and leukotrienes are elevated in rat models of adult respiratory distress syndrome (Hammarstrom, 1983) has opened further new areas of clinical inquiry. Although rat models involving bolus endotoxin administration have not been predictive of the human septic syndrome, they have proven useful in the determination of the cellular sources and mechanisms of eicosanoid and leukotriene action. From the evidence presented, the septic shock rat model would seem to be the best model in predicting the outcome of therapies in human sepsis. Limited clinical trials have assessed the therapeutic efficacy of pharmacologic agents which alter eicosanoid and leukotriene metabolism in sepsis. In view of the continued high mortality of patients with septic shock and the failure of conventional therapies (Sprung et al., 1984; DuToit et al. 1985), further clinical and experimental studies are desirable.