Theresa M. Stevens

Effects of recombinant interleukin-1 beta on phospholipase A2 activity, phospholipase A2 mRNA levels, and eicosanoid formation in rabbit chondrocytes

We have investigated the effects of recombinant interleukin-1 beta (rIL-1 beta) on phospholipase A2 activity (PLA2), PLA2 messenger RNA levels, and eicosanoid production in rabbit chondrocytes. Phospholipase A2 activity increased 5 fold with exposure to 1.6 x 10(-11) M rIL-1 beta for 20 hours. An mRNA specific for an extracellular PLA2 was detected by RNA blot hybridization after treatment of the cells with rIL-1 beta. Hydroxylated derivatives of arachidonic acid, including prostaglandins, leukotriene B4, 5-hydroxyeicosa 6E, 8Z, 11Z, 14Z-tetraenoic acid, 12-hydroxyeicosa 5Z, 8Z, 10E, 14Z-tetraenoic acid and 15-hydroxyeicosa 5Z, 8Z, 11Z, 13E-tetraenoic acid increased in cells after rIL-1 beta treatment.

Induction of antioxidant enzyme activities by a phenylurea derivative, EDU

Oxygen free radicals have the potential to mediate cell injury. Defenses against such radicals include the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). The purposes of this study were (1) to develop an in vitro model using human cells in which to investigate a potential pharmacologic agent as an inducer of these antioxidant enzymes; (2) to investigate the phenylurea derivative N-[2-(2-oxo-1-imidazolindinyl)ethyl]-N-phenylurea (EDU) in this model with paraquat (PQ) serving as the positive control; and (3) to determine if induction of the antioxidant enzymes by EDU occurs in vivo. Human gingival fibroblasts (Gin-1) were used as the target cell in vitro; PQ and EDU, an inducer of SOD and CAT activities in plants, were evaluated as antioxidant enzyme inducers. Total SOD activity in Gin-1 cells increased 2-fold (p less than 0.05) in the presence of 1.0 mM PQ for 18-48 hr compared with untreated controls. Gin-1 cells incubated with 0.25-2.0 mM PQ for 24 hr had significantly increased total SOD (1.5 to 2.0-fold; p less than 0.05). CAT activity increased with 1.0 and 2.0 mM PQ (p less than 0.05). In the presence of PQ, GSH-PX activity decreased (p less than 0.05) in a concentration-dependent manner, indicating inactivation of this enzyme. No toxicity, indicated by lactate dehydrogenase released into the incubation medium, was noted at PQ concentrations below 5.0 mM. In the presence of 0.125-2.0 mM EDU, total SOD activity in Gin-1 cells significantly increased (1.5 to 2.0-fold; p less than 0.05). CAT activity significantly increased in a dose-dependent manner (p less than 0.05), while GSH-PX activity remained constant following exposure to 0.125-2.0 mM EDU. Intraperitoneal administration of EDU to rats twice a day for 2 days at 100 mg/kg induced SOD activity in heart, liver, and lung compared to controls (p less than 0.05). CAT activity increased in the liver 56% and in the lung 36% (p less than 0.05). GSH-PX activity remained constant. Our findings indicate that Gin-1 cells are a useful model in which to study inducers of antioxidant enzymes in vitro and that the phenylurea compound EDU induces SOD and CAT activities both in vitro and in vivo.

Phospholipase A2 (PLA2) activity in rabbit chondrocytes.

The effects of recombinant interleukin-1β (rIL-1β), recombinant tumor necrosis factor (rTNFα) and two growth factors, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on PLA2 activity and prostaglandin E2 (PGE2) release were investigated using rabbit chondrocytes. Cellular PLA2 activity increased 2–10×above controls in the presence of 8×10−12M (5 U) rIL-1β or 5×10−9M rTNFα after 20 hr incubation. PLA2 activity remained constant with 1–50 ng/ml of either growth factor. PGE2 release significantly increased (p<0.05) when the chondrocytes were incubated with rIL-1β, bFGF and EGF alone, but not with rTNFα above. These data suggest PLA2 activity and PGE2 release are not coordinately regulated in rabbit chondrocytes.

Extracellular phospholipase A2 activity in cell free peritoneal lavage fluid from mice with zymosan peritonitis

The kinetics and appearance of extracellular phospholipase A2 (PLA2) activity and its relationship to the appearance of arachidonic acid (AA) metabolites in the peritoneal cavity of mice injected with zymosan is described. AA metabolites levels including leukotriene C4 (LTC4) were maximum 15–30 min after zymosan. Peak PLA2 activity was also found 15–30 min after zymosan and was significantly increased above levels found in saline control exudates (3.83±0.89 and 0.35±0.11, respectively,p≤0.05). Results show that an extracellular PLA2 is present in zymosan peritonitis.

Pretreatment with EDU decreases rat lung cellular responses to ozone

Abstract The phenylurea compound EDU (N-[2-(2-oxo-1-imidazolindinyl)ethyl]-N′-phenylurea) has been shown to protect plants from the damaging effects of ozone exposure. Models of rat lung injury, based on acute exposure to 2 ppm ozone for 3 hr and on exposure to 0.85 ppm ozone for 2 days, were used to determine whether EDU pretreatment of rats protected lungs from oxidant injury. Rats were pretreated with 100 mg/kg body wt EDU by ip administration for 2 days prior to and on the days of ozone exposure. No adverse toxicological effects of EDU pretreatment were observed. Lung superoxide dismutase (SOD) and catalase (CAT) activities were significantly enhanced from 636 to 882 U/lung and from 599 to 856 U/lung, respectively. One day following acute exposure (2 ppm for 3 hr), an ozone-induced increase of polymorphonuclear leukocytes (PMNs) from 0.01 to 1.18 million cells/lung was decreased to 0.68 million by EDU pretreatment. No alteration occurred in the degree of lung permeability indicated by increased lavage fluid albumin. EDU pretreatment also significantly decreased ozone-induced increases in PMN recovery after 2 days exposure to 0.85 ppm ozone from 5.54 to 2.12 million cells/lung. However, in this second case, EDU pretreatment reduced the observed ozone damage, indicated by a decrease in lavage fluid albumin and by a decrease in the macrophage and lymphocyte infiltration associated with this length of ozone exposure. The observation that EDU-treated cultured pulmonary arterial endothelial cells increased SOD and CAT activities identified a potential lung site of EDU interaction. These data demonstrated that although EDU pretreatment appears not to prevent initial ozone damage, it does reduce the infiltration of PMNs and might therefore prevent amplification of the injury associated with this cell type.

Phospholipase A2 (PLA2) activity in undifferentiated U937 cells

PLA2 activity has been described in U937 cells. The present study characterized PLA2 activity in these undifferentiated cells. Cells were grown in suspension culture, harvested by centrifugation, and washed and homogenized in a neutral buffer containing standard proteinase inhibitors. A low speed supernatant was fractionated either by acid extraction or by sucrose density gradient centrifugation. PLA2 activity was measured using either L-α-1-palmitoyl-2-arachidonoyl [1-14C]-phosphatidylcholine or heat-inactivated [3H]oleic acid-labeledE. coli as substrates. Substrate-specific PLA2 activity was found in the acid-extracted and in the 25% sucrose fractions. Standard inhibitors were investigated with these PLA2 activities. Our results suggest undifferentiated U937 cells contain three distinct PLA2 activities. This is the first indication that more than one PLA2 activity is present in undifferentiated U937 cells.

Acute lung inflammation in rats induced by phorbol myristate acetate (PMA)

Intratracheal administration of PMA produces acute lung injury in part due to the generation of O2-derived free radicals. This study evaluated the role of the antioxidant enzyme superoxide dismutase (SOD) in PMA-induced lung injury in the rat. PMA was instilled into rats intratracheally (20–60 μg/kg), and the lungs were lavaged 4 hr later. Total number of cells recovered from lavage after PMA treatment was not different from the total number recovered from controls; lavagable PMNs increased in a dose-dependent manner. Albumin in lavage fluid (an index of lung vascular permeability) was significantly increased at 60 μg/kg PMA. SOD (10,000 U)+PMA (60 μg/kg) reduced the albumin level but significantly increased both total number of cells and number of PMNs recovered from lavage fluid. To investigate the possibility that SOD decreases the ability of PMNs to adhere, PMN aggregation was measuredin vitro. The results indicated that 10,000 U SOD can inhibit PMA-induced aggregation by 50%. In contrast, aggregation to other stimuli (e.g., fMet-Leu-Phe, A23187) was unaffected by SOD. We conclude SOD prevents PMA-induced lung permeability and diminishes PMN adherence.

Phospholipase A2 (PLA2) activity in bovine pulmonary artery endothelial cells

We have investigated the role of recombinant human interleukin-1β (rIL-1β) and recombinant human tumor necrosis factorα (rTNF-α) on PLA2 activity, protein synthesis and eicosanoid production in bovine pulmonary artery endothelial cells. Cellular PLA2 activity increased 4-fold and production of PGE2 increased 3-fold at 1–2 hrs in the presence of 10 units/ml rIL-1β. PLA2 activity increased 3-fold at 30 min and PGE2 production increased 2-fold with 5×10−9M rTNF-α. The data show that endothelial cells respond more rapidly to rIL-1β (2–6 hr) and rTNF-α (30 min) than do chondrocytes and synovial cells (6–16 hrs), suggesting endothelial cells may play a primary role in initiating the inflammatory response.

Biochemical and Pharmacologic Characterization of a Phosphatidylinositol-Specific Phospholipase C in Rat Neutrophils

The phosphatidylinositol (PI)‐specific phospholipase C (PLC) activity contained in sonicates of casein‐elicited (4‐6 hr) rat neutrophils has been identified and characterized. With phosphatidylinositol (PI) as the substrate, PLC activity is found both in the supernate and pellet of a 100,000g spin of the neutrophil sonicate. Further fractionation of the crude sonicate by centrifugation on discontinuous sucrose gradients indicates that the PLC activity is predominantly cytosolic with lesser amounts of activity found in the plasma membrane and granule enriched fractions. Hydrolysis of PI by the sonicate PLC is linear for 15‐20 min at 37° and also with respect to the amount of sonicate protein added. The enzyme shows selectivity for PI with little, if any, hydrolytic activity towards other phospholipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS), or phosphatidylcholine (PC). The PLC activity has a pH optimum of 5.5‐6.0, is enhanced 1.5‐3‐fold by the addition of deoxycholate, and is Ca+ + dependent. Kinetic analysis of the PLC hydrolysis of PI yields an apparent Km of 240 ± 85 μM and a Vmax of 34.3 ± 17.0 nmol/min/mg protein (n = 3). Similarly, when phosphatidylinositol 4,5 bisphosphate (PIP2) is used as substrate, an apparent Km of 109 ± 66 μM and a Vmax of 14.3 ± 10.4 nmol/min/mg (n = 3) protein is obtained. These data suggest that PIP2 may be a slightly better substrate for the PMN PLC relative to PI. Finally, a variety of drugs previously reported to inhibit platelet PLC activity in vitro were tested for their ability to inhibit rat PMN PLC. Of the compounds tested, none were potent (i.e., IC50 values 100 μM) inhibitors of the PMN PLC.

Characterization of extracellular phospholipase A2 (PLA2) activity in fluid and peritoneal cells from casein-treated rats

Extracellular phospholipase A2 activity (PLA2) found in the fluid and cells of the peritoneal cavity of rats injected with casein is described. PLA2 activities from both the fluid and cells require Ca2+ and have pH optima of 7. Acid-extraction increased PLA2 activity in the polymorphonuclear leukocyte (PMN) homogenates 20-fold but not the PLA2 activity in the extracellular fluid. Acid extraction also increased the sensitivity of the PLA2 activities to standard inhibitors. Since the PLA2 activities described in this model have characteristics similar to other inflammatory PLA2s, including human synovial fluid PLA2, casein stimulation should prove useful for testing potential inhibitors.