Agents Actions36 (1992)
0065-4299/92/020070-07 $1.50+ 0.20/0 9 1992 Birkhfiuser Verlag, Basel
Biphasic regulation by dexamethasone of IL-1- and LPS-stimulated endothelial prostacyclin production G. Prajgrod and A. Danon Dept. of Clinical Pharmacology,The Corob Center of Health Sciences, Ben-GurionUniversityand Soroka Medical Center, PO Box 653, Beer Sheva 84105,Israel
Abstract Body reaction to injury comprises two major pathways: the immune response, predominantly mediated by IL-1 and other cytokines, and neuroendocrine mechanisms, resulting in an increased glucocorticoid production. Each has distinct effects on prostaglandin (PG) production, which may in turn mediate both systemic and local inflammatory responses. The interactions, if any, between the two systems on PG synthesis have not been studied, Bovine aortic endothelial cell cultures were used and prostacyclin (PGI2) production was monitored. Cells were treated with dexamethasone (Dex) 10-6 M and IL-1 10-30 U/ml in one experiment, and lipopolysaccharide (LPS, 0.1-1.0 lag/ml) in another experiment, separately or in combination, for either 2 or 24 + 2 h. While Dex was without effect, IL-1 and LPS stimulated PGI 2 in a concentration- and time-dependent manner. Short exposure to Dex (2 h) enhanced the stimulation by IL-1 and LPS. On the contrary, more prolonged exposure (24+2 h) reversed the effects of IL-1 and LPS, resulting in PGI2 levels below the baseline. A biphasic regulation by Dex was also observed with increasing concentrations of LPS. Dex was actually ineffective by itself, but it enhanced PGI 2 production in combination with lower concentrations of LPS, while abolishing the influence of higher concentrations of this agonist. The data suggest that Dex may initially stimulate phospholipase Aa (PLA2) activity, while inhibiting it later. This biphasic behavior may be attributed to different concentrations of a PLA 2modulating protein, possibly lipocortin, that accumulate during exposure to Dex.
Introduction Vascular endothelium plays key roles both in inflammation and in hemostasis. It controls protein leakage and extravasation and allows leukocyte accumulation through a process initiated by leukocyte attachment to the endothelial cells. One of the many mediators of both the inflammatory and Supported by Grants (to AD) from the Basic Research Foundation, IsraelAcademyof Sciences,and the ChiefScientist, Ministry of Health.
hemostatic functions of these cells is prostacyclin (PGI2), which is produced principally in vascular endothelium. PGI z is an extremely potent antiaggregatory agent, has powerful vasodilator activity and plays an important role in cardiovascular physiology and, conversely, in circulatory disorders, such as ischemia and shock [1]. Many aspects of body response to injury are initiated and mediated by an increased production of interleukin-1 (IL-1) [2]. More recently, the contributions of interleukin-6 (IL-6) and tumor necrosis
71
Agents Actions 36 (1992)
factor (TNF) to the pathophysiology of inflammation are recognized [3]. These cytokines are induced in response to a variety of insults, such as antigen, infection, etc., and trigger the multiple manifestations of the so-called acute phase response. Both IL-1 and IL-6 are produced in endothelial cells when stimulated with lipopolysaccharide (LPS) [4, 5]. IL-1 is known to influence eicosanoid synthesis in various cells, notably of PGI2 synthesis in vascular tissue [6]. Moreover, IL-1 and related cytokines potentiate the effects of agonists on P G I 2 production [7]. The multitude of actions of cytokines on endothelial cells was recently reviewed [8]. Recent evidence indicates that IL-1 stimulates the hypothalamic-pituitary-adrenal axis with the production of ACTH and glucocorticoids (GC) [9-12]. The latter are believed to be anti-inflammatory and are widely used therapeutically for this indication. It is largely accepted that GC modulate the synthesis of prostaglandins (PGs). However, the precise influence of GC on vascular eicosanoid synthesis is not fully understood. Several authors reported suppression by GC of endothelial PG synthesis [ 13, 14]. In bovine pulmonary endothelial cell culture, GC inhibited stimulated PGI2 production, but were without effect in non-stimulated cells [15]. Other investigators reported inhibition of PGI2 production only after incubations with GC [16] or very high concentrations of hydrocortisone [173. In a previous paper we reported that adrenal GC altered the ex vivo response of the rat aorta to LPS. Thus, while LPS enhanced PGIz synthesis in adrenalectomized rats, it actually inhibited it in the intact rat [18]. In the present study we investigated the possible interactions between GC, on the one hand, and inflammatory inducers such as LPS and IL-1, on the other hand, on endothelial P G I 2 production. The data indicate that in these cells GC initially potentiate the PGIz-stimulating effect of LPS and IL-1, while, conversely, PGI2 synthesis declines following more prolonged exposure to GC.
8% CO2/air atmosphere. Each 16 mm well contained 1 ml Dulbecco's modified Eagle's medium (DMEM, Biological Industries, Beth Haemek, Israel), supplemented with 10% fetal bovine serum, antibiotics and 2 ng/ml fibroblast growth factor (Sigma Chemical Co., St. Louis, MO.) The latter was added every 2 days until the cultures became confluent. Cultures were used 7 8 days after reaching confluency. Experiments were initiated by washing twice with serum-free DMEM. The cells were then incubated in 1 ml serum-free D M E M containing the appropriate concentrations of test agents. Dexamethasone sodium phosphate (Dex) (Teva Pharmaceuticals Ind.) was diluted in DMEM. LPS was from E. coli (serotype 0127:B8) (Sigma) and recombinant human I L - l e (rhIL-17) was obtained from Hoffmann-La Roche. Determination of 6-keto PGFI, (6-K ) The 6-K that accumulated in the media was measured in unextracted samples of DMEM, by single antibody radioimmunoassay (RIA) with dextrancoated charcoal precipitation. The RIA was performed in duplicate for each sample. Rabbit antiserum to 6-K was obtained from Bio-Yeda (Rehovot, Israel) and tritium-labeled 6-K (151 Ci/mmol) was supplied by the Radioehemical Center (Amersham, UK). The sensitivity of the assay was 1.5 pg/tube. The 6-K antiserum crossreacted with other PGs (at 50% displacement) as follows: PGE1, 22%; PGE2, 10%; PGFa~, 16%; PGFz~, 10%; other PGs < 1%. Data analysis Results are expressed as m e a n s + S E M for each experimental group. Comparisons were made by two-tailed Student's t-test and differences with P values lower than 0.05 were considered to indicate statistical significance. Results
Materials and methods
Cell cultures Cultures of bovine aortic endothelial cells (BAEC) were used [19] between passages 6 13. BAEC were plated in 24-well tissue culture plates (Costar, Cambridge, MA) and incubated at 37~ in humidified
The BAEC cultures as used synthesized considerable amounts of PGI2, measured as the stable dehydration product 6-keto PGFI~ (6-K). The cells also produced much smaller quantities of PGEe and thromboxane (results not shown), which were not measured in subsequent experiments. As shown in Fig. 1, Dex (10 - 6 M) was without effect at either
72
Agents Actions 36 (1992) A 6-K 3" ng/ml
,@+ *
+
2-
6-K ng/ml
-
Trgatment
I"I cu
*
I Dex I pM [ ] LPS 0.1 pg/ml [ ] LPS 1.0 pg/ml
@ p < 0,05 vs LPS +
p
0065-4299/92/020070-07 $1.50+ 0.20/0 9 1992 Birkhfiuser Verlag, Basel
Biphasic regulation by dexamethasone of IL-1- and LPS-stimulated endothelial prostacyclin production G. Prajgrod and A. Danon Dept. of Clinical Pharmacology,The Corob Center of Health Sciences, Ben-GurionUniversityand Soroka Medical Center, PO Box 653, Beer Sheva 84105,Israel
Abstract Body reaction to injury comprises two major pathways: the immune response, predominantly mediated by IL-1 and other cytokines, and neuroendocrine mechanisms, resulting in an increased glucocorticoid production. Each has distinct effects on prostaglandin (PG) production, which may in turn mediate both systemic and local inflammatory responses. The interactions, if any, between the two systems on PG synthesis have not been studied, Bovine aortic endothelial cell cultures were used and prostacyclin (PGI2) production was monitored. Cells were treated with dexamethasone (Dex) 10-6 M and IL-1 10-30 U/ml in one experiment, and lipopolysaccharide (LPS, 0.1-1.0 lag/ml) in another experiment, separately or in combination, for either 2 or 24 + 2 h. While Dex was without effect, IL-1 and LPS stimulated PGI 2 in a concentration- and time-dependent manner. Short exposure to Dex (2 h) enhanced the stimulation by IL-1 and LPS. On the contrary, more prolonged exposure (24+2 h) reversed the effects of IL-1 and LPS, resulting in PGI2 levels below the baseline. A biphasic regulation by Dex was also observed with increasing concentrations of LPS. Dex was actually ineffective by itself, but it enhanced PGI 2 production in combination with lower concentrations of LPS, while abolishing the influence of higher concentrations of this agonist. The data suggest that Dex may initially stimulate phospholipase Aa (PLA2) activity, while inhibiting it later. This biphasic behavior may be attributed to different concentrations of a PLA 2modulating protein, possibly lipocortin, that accumulate during exposure to Dex.
Introduction Vascular endothelium plays key roles both in inflammation and in hemostasis. It controls protein leakage and extravasation and allows leukocyte accumulation through a process initiated by leukocyte attachment to the endothelial cells. One of the many mediators of both the inflammatory and Supported by Grants (to AD) from the Basic Research Foundation, IsraelAcademyof Sciences,and the ChiefScientist, Ministry of Health.
hemostatic functions of these cells is prostacyclin (PGI2), which is produced principally in vascular endothelium. PGI z is an extremely potent antiaggregatory agent, has powerful vasodilator activity and plays an important role in cardiovascular physiology and, conversely, in circulatory disorders, such as ischemia and shock [1]. Many aspects of body response to injury are initiated and mediated by an increased production of interleukin-1 (IL-1) [2]. More recently, the contributions of interleukin-6 (IL-6) and tumor necrosis
71
Agents Actions 36 (1992)
factor (TNF) to the pathophysiology of inflammation are recognized [3]. These cytokines are induced in response to a variety of insults, such as antigen, infection, etc., and trigger the multiple manifestations of the so-called acute phase response. Both IL-1 and IL-6 are produced in endothelial cells when stimulated with lipopolysaccharide (LPS) [4, 5]. IL-1 is known to influence eicosanoid synthesis in various cells, notably of PGI2 synthesis in vascular tissue [6]. Moreover, IL-1 and related cytokines potentiate the effects of agonists on P G I 2 production [7]. The multitude of actions of cytokines on endothelial cells was recently reviewed [8]. Recent evidence indicates that IL-1 stimulates the hypothalamic-pituitary-adrenal axis with the production of ACTH and glucocorticoids (GC) [9-12]. The latter are believed to be anti-inflammatory and are widely used therapeutically for this indication. It is largely accepted that GC modulate the synthesis of prostaglandins (PGs). However, the precise influence of GC on vascular eicosanoid synthesis is not fully understood. Several authors reported suppression by GC of endothelial PG synthesis [ 13, 14]. In bovine pulmonary endothelial cell culture, GC inhibited stimulated PGI2 production, but were without effect in non-stimulated cells [15]. Other investigators reported inhibition of PGI2 production only after incubations with GC [16] or very high concentrations of hydrocortisone [173. In a previous paper we reported that adrenal GC altered the ex vivo response of the rat aorta to LPS. Thus, while LPS enhanced PGIz synthesis in adrenalectomized rats, it actually inhibited it in the intact rat [18]. In the present study we investigated the possible interactions between GC, on the one hand, and inflammatory inducers such as LPS and IL-1, on the other hand, on endothelial P G I 2 production. The data indicate that in these cells GC initially potentiate the PGIz-stimulating effect of LPS and IL-1, while, conversely, PGI2 synthesis declines following more prolonged exposure to GC.
8% CO2/air atmosphere. Each 16 mm well contained 1 ml Dulbecco's modified Eagle's medium (DMEM, Biological Industries, Beth Haemek, Israel), supplemented with 10% fetal bovine serum, antibiotics and 2 ng/ml fibroblast growth factor (Sigma Chemical Co., St. Louis, MO.) The latter was added every 2 days until the cultures became confluent. Cultures were used 7 8 days after reaching confluency. Experiments were initiated by washing twice with serum-free DMEM. The cells were then incubated in 1 ml serum-free D M E M containing the appropriate concentrations of test agents. Dexamethasone sodium phosphate (Dex) (Teva Pharmaceuticals Ind.) was diluted in DMEM. LPS was from E. coli (serotype 0127:B8) (Sigma) and recombinant human I L - l e (rhIL-17) was obtained from Hoffmann-La Roche. Determination of 6-keto PGFI, (6-K ) The 6-K that accumulated in the media was measured in unextracted samples of DMEM, by single antibody radioimmunoassay (RIA) with dextrancoated charcoal precipitation. The RIA was performed in duplicate for each sample. Rabbit antiserum to 6-K was obtained from Bio-Yeda (Rehovot, Israel) and tritium-labeled 6-K (151 Ci/mmol) was supplied by the Radioehemical Center (Amersham, UK). The sensitivity of the assay was 1.5 pg/tube. The 6-K antiserum crossreacted with other PGs (at 50% displacement) as follows: PGE1, 22%; PGE2, 10%; PGFa~, 16%; PGFz~, 10%; other PGs < 1%. Data analysis Results are expressed as m e a n s + S E M for each experimental group. Comparisons were made by two-tailed Student's t-test and differences with P values lower than 0.05 were considered to indicate statistical significance. Results
Materials and methods
Cell cultures Cultures of bovine aortic endothelial cells (BAEC) were used [19] between passages 6 13. BAEC were plated in 24-well tissue culture plates (Costar, Cambridge, MA) and incubated at 37~ in humidified
The BAEC cultures as used synthesized considerable amounts of PGI2, measured as the stable dehydration product 6-keto PGFI~ (6-K). The cells also produced much smaller quantities of PGEe and thromboxane (results not shown), which were not measured in subsequent experiments. As shown in Fig. 1, Dex (10 - 6 M) was without effect at either
72
Agents Actions 36 (1992) A 6-K 3" ng/ml
,@+ *
+
2-
6-K ng/ml
-
Trgatment
I"I cu
*
I Dex I pM [ ] LPS 0.1 pg/ml [ ] LPS 1.0 pg/ml
@ p < 0,05 vs LPS +
p
