Cell-specific Regulation of Expression of Plasma-type Platelet-activating Factor Acetylhydrolase in the Liver
1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês
10.1074/jbc.272.44.27543
ISSN1083-351X
AutoresKatherine M. Howard, Joseph E. Miller, Masao Miwa, Merle S. Olson,
Tópico(s)Adrenal Hormones and Disorders
ResumoPlatelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that causes hypotension, increases vascular permeability, and has been implicated in anaphylaxis, septic shock and several other inflammatory responses. PAF is hydrolyzed and inactivated by the enzyme PAF-acetylhydrolase. In the intact rat, a mesenteric vein infusion of lipopolysaccharide (LPS) served as an acute, liver-focused model of endotoxemia. Plasma PAF-acetylhydrolase activity increased 2-fold by 24 h following LPS administration. Ribonuclease protection experiments demonstrated very low levels of plasma-type PAF-acetylhydrolase mRNA transcripts in the livers of saline-infused rats; however, 24 h following LPS exposure, a 20-fold induction of PAF-acetylhydrolase mRNA was detected. In cells isolated from endotoxin-exposed rat livers, Northern blot analyses demonstrated that Kupffer cells but not hepatocytes or endothelial cells were responsible for the increased PAF-acetylhydrolase mRNA levels. In Kupffer cells, plasma-type PAF-acetylhydrolase mRNA was induced by 12 h, peaked at 24 h, and remained substantially elevated at 48 h. Induction of neutropenia prior to LPS administration had no effect on the increase in PAF-acetylhydrolase mRNA seen at 24 h. Although freshly isolated Kupffer cells contain barely detectable levels of plasma-type PAF-acetylhydrolase mRNA, when Kupffer cells were established in culture, PAF-acetylhydrolase expression became constitutively activated concomitant with cell adherence to the culture plates. Alterations in plasma-type PAF-acetylhydrolase expression may constitute an important mechanism for elevating plasma PAF-acetylhydrolase levels and an important component in minimizing PAF-mediated pathophysiology in livers exposed to endotoxemia. Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that causes hypotension, increases vascular permeability, and has been implicated in anaphylaxis, septic shock and several other inflammatory responses. PAF is hydrolyzed and inactivated by the enzyme PAF-acetylhydrolase. In the intact rat, a mesenteric vein infusion of lipopolysaccharide (LPS) served as an acute, liver-focused model of endotoxemia. Plasma PAF-acetylhydrolase activity increased 2-fold by 24 h following LPS administration. Ribonuclease protection experiments demonstrated very low levels of plasma-type PAF-acetylhydrolase mRNA transcripts in the livers of saline-infused rats; however, 24 h following LPS exposure, a 20-fold induction of PAF-acetylhydrolase mRNA was detected. In cells isolated from endotoxin-exposed rat livers, Northern blot analyses demonstrated that Kupffer cells but not hepatocytes or endothelial cells were responsible for the increased PAF-acetylhydrolase mRNA levels. In Kupffer cells, plasma-type PAF-acetylhydrolase mRNA was induced by 12 h, peaked at 24 h, and remained substantially elevated at 48 h. Induction of neutropenia prior to LPS administration had no effect on the increase in PAF-acetylhydrolase mRNA seen at 24 h. Although freshly isolated Kupffer cells contain barely detectable levels of plasma-type PAF-acetylhydrolase mRNA, when Kupffer cells were established in culture, PAF-acetylhydrolase expression became constitutively activated concomitant with cell adherence to the culture plates. Alterations in plasma-type PAF-acetylhydrolase expression may constitute an important mechanism for elevating plasma PAF-acetylhydrolase levels and an important component in minimizing PAF-mediated pathophysiology in livers exposed to endotoxemia. Platelet-activating factor (PAF) 1The abbreviations used are: PAF, platelet-activating factor; LPS, lipopolysaccharide; BSA, bovine serum albumin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MOPS, 4-morpholinepropanesulfonic acid; bp, base pair(s); kb, kilobase pair(s). is a potent proinflammatory phospholipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) involved prominently in diverse pathophysiological episodes. In fact, PAF has been implicated as a mediator of inflammation, allergic reactions, and shock (for review, see Chao and Olson (1Chao W. Olson M.S. Biochem. J. 1993; 292: 617-629Crossref PubMed Scopus (425) Google Scholar)). Tissue PAF levels are modulated by regulation of key steps in both the biosynthetic and degradative pathways. The degradation of PAF occurs through the hydrolysis of the acetyl group at the sn-2 position of PAF and produces biologically inactive lyso-PAF. PAF-acetylhydrolase catalyzes the hydrolytic reaction, and this enzyme is present in mammalian blood (2Farr R.S. Cox C.P. Wardlow M.L. Jorgensen R. Clin. Immunol. Immunopathol. 1980; 15: 318-330Crossref PubMed Scopus (153) Google Scholar, 3Stafforini D.M. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1987; 262: 4223-4230Abstract Full Text PDF PubMed Google Scholar, 4Stafforini D.M. McIntyre T.M. Carter M.E. Prescott S.M. J. Biol. Chem. 1987; 262: 4215-4222Abstract Full Text PDF PubMed Google Scholar), blood cells (5Stafforini D.M. Prescott S.M. Zimmerman G.A. McIntyre T.M. Lipids. 1991; 26: 979-985Crossref PubMed Scopus (90) Google Scholar, 6Stafforini D.M. Rollins E.N. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1993; 268: 3857-3865Abstract Full Text PDF PubMed Google Scholar, 7Korth R. Bidault J. Palmantier R. Benveniste J. Ninio E. Lipids. 1993; 28: 193-199Crossref PubMed Scopus (38) Google Scholar), and various tissues (5Stafforini D.M. Prescott S.M. Zimmerman G.A. McIntyre T.M. Lipids. 1991; 26: 979-985Crossref PubMed Scopus (90) Google Scholar, 8Lee T.C. Malone B. Wasserman S.I. Fitzgerald V. Snyder F. Biochem. Biophys. Res. Commun. 1982; 105: 1303-1308Crossref PubMed Scopus (125) Google Scholar, 9Nijssen J.G. Roosenboom C.F. van den Bosch H. Biochim. Biophys. Acta. 1986; 876: 611-618Crossref PubMed Scopus (57) Google Scholar). Also, PAF-acetylhydrolase has been isolated from the peritoneal cavity of guinea pigs after endotoxin shock (10Karasawa K. Yato M. Setaka M. Nojima S. J. Biochem. 1994; 116: 374-379Crossref PubMed Scopus (22) Google Scholar). Both intracellular and extracellular PAF-acetylhydrolase isoforms have been described. The molecular cloning and characterization of the human plasma PAF-acetylhydrolase was recently reported (11Tjoelker L.W. Wilder C. Eberhardt C. Stafforini D.M. Dietsch G. Schimpf B. Hooper S. Le Trong H. Cousens L.S. Zimmerman G.A. et al.Nature. 1995; 374: 549-553Crossref PubMed Scopus (485) Google Scholar). This 44-kDa protein was isolated from human plasma, and amino acid sequence analysis led to the screening of a macrophage cDNA library where a positive cDNA clone was isolated. The cDNA encoded a 441-amino acid protein, which contained a secretion signal sequence. Tew et al. (12Tew D.G. Southan C. Rice S.Q. Lawrence M.P. Li H. Boyd H.F. Moores K. Gloger I.S. Macphee C.H. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 591-599Crossref PubMed Scopus (159) Google Scholar) have purified and cloned a cDNA for the human plasma PAF-acetylhydrolase and demonstrated it to be a glycosylated protein ranging in size from 43 to 67 kDa. In addition to the extracellular enzyme, the molecular characterization of two intracellular PAF-acetylhydrolases has been reported. Bovine brain PAF-acetylhydrolase, isoform 1b, is a heterotrimeric enzyme composed of 29-, 30-, and 45-kDa subunits (13Hattori M. Arai H. Inoue K. J. Biol. Chem. 1993; 268: 18748-18753Abstract Full Text PDF PubMed Google Scholar, 14Hattori M. Adachi H. Aoki J. Tsujimoto M. Arai H. Inoue K. J Biol Chem. 1995; 270: 31345-31352Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Moreover, the cDNA sequences for a bovine and a human intracellular PAF-acetylhydrolase, isoform II, were published recently (15Hattori K. Adachi H. Matsuzawa A. Yamamoto K. Tsujimoto M. Aoki J. Hattori M. Arai H. Inoue K. J. Biol. Chem. 1996; 271: 33032-33038Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The intracellular isoform II exhibited 43% amino acid identity to the human plasma PAF-acetylhydrolase. The source of serum PAF-acetylhydrolase is not known, but a likely source is the liver since the liver secretes several plasma proteins in abundance, including lipoproteins. Cultured hepatocytes and the human hepatoma cell line, Hep G2, secrete PAF-acetylhydrolase into the culture media (16Satoh K. Imaizumi T. Yoshida H. Takamatsu S. Metab. Clin. Exp. 1993; 42: 672-677Abstract Full Text PDF PubMed Scopus (11) Google Scholar, 17Svetlov S.I. Howard K.M. Miwa M. Flickinger B.D. Olson M.S. Arch. Biochem. Biophys. 1996; 327: 113-122Crossref PubMed Scopus (24) Google Scholar, 18Tarbet E.B. Stafforini D.M. Elstad M.R. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1991; 266: 16667-16673Abstract Full Text PDF PubMed Google Scholar). Also, macrophages secrete large amounts of PAF-acetylhydrolase (19Stafforini D.M. Elstad M.R. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1990; 265: 9682-9687Abstract Full Text PDF PubMed Google Scholar, 20Narahara H. Frenkel R.A. Johnston J.M. Arch. Biochem. Biophys. 1993; 301: 275-281Crossref PubMed Scopus (47) Google Scholar), but whether macrophages contribute to the level of the circulating plasma enzyme has not been proven. Northern blot analyses of different human tissues demonstrated the presence of plasma-type acetylhydrolase mRNA in thymus and tonsil, tissues which contain macrophages in abundance (11Tjoelker L.W. Wilder C. Eberhardt C. Stafforini D.M. Dietsch G. Schimpf B. Hooper S. Le Trong H. Cousens L.S. Zimmerman G.A. et al.Nature. 1995; 374: 549-553Crossref PubMed Scopus (485) Google Scholar). Even though hepatocytes secrete the plasma-type PAF-acetylhydrolase in culture, Tjoelkeret al. (11Tjoelker L.W. Wilder C. Eberhardt C. Stafforini D.M. Dietsch G. Schimpf B. Hooper S. Le Trong H. Cousens L.S. Zimmerman G.A. et al.Nature. 1995; 374: 549-553Crossref PubMed Scopus (485) Google Scholar) found no detectable level of PAF-acetylhydrolase RNA in normal human liver. It is quite possible that the abundance of PAF-acetylhydrolase mRNA is so low as to be undetectable in whole liver total RNA. Alternatively, as a mechanism to avoid PAF-induced pathophysiology, PAF-acetylhydrolase expression may achieve detectable levels only in response to a need for greater degradation of increased PAF levels following tissue injury. Previous work in our laboratory has demonstrated that the isolated perfused rat liver responds to PAF with significant effects on both vascular resistance and glucose output (21Shukla S.D. Buxton D.B. Olson M.S. Hanahan D.J. J. Biol. Chem. 1983; 258: 10212-10214Abstract Full Text PDF PubMed Google Scholar, 22Buxton D.B. Shukla S.D. Hanahan D.J. Olson M.S. J. Biol. Chem. 1984; 259: 1468-1471Abstract Full Text PDF PubMed Google Scholar, 23Buxton D.B. Fisher R.A. Hanahan D.J. Olson M.S. J. Biol. Chem. 1986; 261: 644-649Abstract Full Text PDF PubMed Google Scholar). We have demonstrated that the liver can produce PAF in response to particulate (24Buxton D.B. Hanahan D.J. Olson M.S. J. Biol. Chem. 1984; 259: 13758-13761Abstract Full Text PDF PubMed Google Scholar, 25Buxton D.B. Fisher R.A. Briseno D.L. Hanahan D.J. Olson M.S. Biochem. J. 1987; 243: 493-498Crossref PubMed Scopus (51) Google Scholar) or endotoxin stimulation and that the Kupffer cells were the site of PAF synthesis (26Chao W. Siafaka-Kapadai A. Hanahan D.J. Olson M.S. Biochem. J. 1989; 261: 77-81Crossref PubMed Scopus (8) Google Scholar, 27Chao W. Siafaka-Kapadai A. Olson M.S. Hanahan D.J. Biochem. J. 1989; 257: 823-829Crossref PubMed Scopus (40) Google Scholar). Also, we specifically demonstrated the presence of PAF receptors (28Chao W. Liu H. DeBuysere M. Hanahan D.J. Olson M.S. J. Biol. Chem. 1989; 264: 13591-13598Abstract Full Text PDF PubMed Google Scholar) and PAF receptor RNA (17Svetlov S.I. Howard K.M. Miwa M. Flickinger B.D. Olson M.S. Arch. Biochem. Biophys. 1996; 327: 113-122Crossref PubMed Scopus (24) Google Scholar) in Kupffer cells. We have extended our research to intact animal models and have demonstrated that PAF accumulates in the intact liver exposed to various types of injury, including ischemia-reperfusion (29Zhou W. McCollum M.O. Levine B.A. Olson M.S. Hepatology. 1992; 16: 1236-1240Crossref PubMed Scopus (93) Google Scholar), obstructive jaundice (30Zhou W. Chao W. Levine B.A. Olson M.S. Am. J. Physiol. 1992; 263: G587-G592PubMed Google Scholar), and endotoxin exposure (31Olson M.S. Kitten A.M. Eakes A.T. Howard K.M. Miller J.E. Mustafa S.B. Shimazu T. Liver Innervation. John Libbey & Co., Ltd., London1996: 75-85Google Scholar). It may be appropriate to assume that increased PAF levels signal an increase in the level of PAF-acetylhydrolase for PAF degradation. For this reason, we have investigated whether plasma-type PAF-acetylhydrolase expression in the liver is regulated in response to endotoxic challenge in the intact rat. A diverse set of pathophysiological responses accompanies exposure to lipopolysaccharide (LPS) including the induction of endotoxic shock and activation of the immune system and the complement cascade (32Reatz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1076) Google Scholar). Several biological responses to LPS are thought to be mediated by the release of proinflammatory substances such as cytokines and lipid mediators. Using a liver-focused model of endotoxemia, we have demonstrated for the first time the presence of plasma-type PAF-acetylhydrolase mRNA in rat liver. The expression in the liver was cell type-specific and was limited to Kupffer cells. After endotoxin exposure, the expression of PAF-acetylhydrolase mRNA increased 20-fold. Concurrent with the increase in liver plasma-type PAF-acetylhydrolase mRNA, there was a 2-fold increase in the circulating plasma PAF-acetylhydrolase activity. Collagenase (type IV from Clostridium histolyticum), protease E (type XIV from Streptomyces griseus) and bovine serum albumin (fraction V, essentially fatty acid-free) were purchased from Sigma. Metrizamide (2-(3-acetamido-5-N-methylacetamido-2,4,6-triiodobenz-amido)-2-deoxy-d-glucose) was obtained from Nyegaard and Co. (Oslo, Norway). The rat cDNA homologue of the human plasma-type PAF-acetylhydrolase was kindly provided by ICOS (Bothell, WA). Unless specifically stated otherwise, any reference to PAF-acetylhydrolase refers to the plasma-type PAF-acetylhydrolase. Male Sprague-Dawley rats weighing between 225 and 300 g, fed a standard laboratory chow ad libitum, were anesthetized by an intramuscular injection of 0.35 ml of a xylazine/ketamine mixture. A 1-cm lower midline abdominal incision was made, and a single loop of intestine was removed from the abdomen. LPS (Escherichia coli serotype 0111:B4; 3 mg/kg) dissolved in a solution of 0.9% saline, 0.1% BSA was slowly infused through a 27-gauge needle into a distal mesenteric vein. In control rats, a solution of 0.9% saline, 0.1% BSA without LPS was infused into the mesenteric vein. The rats were allowed to awaken and food and water were offered ad libitum. At preset times (1 min and 3, 6, 12, 24, and 48 h), the rats were reanesthetized with an intraperitoneal injection of sodium pentobarbital. Groups of control and LPS-treated rats (n ≥ 3) were either used for collection of serum and whole liver or for the isolation of the hepatic cell populations. Whole blood was removed by inferior vena cava cannulation, allowed to clot, and then centrifuged to obtain serum. When whole liver was sampled, the liver was removed and freeze-clamped immediately in liquid nitrogen and stored at −80 °C. For ribonuclease protection experiments, a full-length rat plasma-type PAF-acetylhydrolase cDNA was modified to generate an appropriate antisense RNA probe. A 700-bpEcoRI fragment was removed from the 3′ end of the PAF-acetylhydrolase cDNA. The remaining cDNA plus vector was agarose gel-purified and religated with T4 DNA ligase. This deletion construct placed the T3 RNA polymerase promoter adjacent to the EcoRI site at nucleotide 1125. The 3′-truncated PAF-acetylhydrolase cDNA was linearized withClaI (nucleotide 950) and T3 RNA polymerase was used to create a 245-bp [α-32P]UTP labeled antisense RNA probe (MaxiScript; Ambion, Austin, TX). As an internal control, a 355-bp rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antisense RNA probe was generated using T3 RNA polymerase from the pTRI-GAPDH template (Ambion). Because of the extreme difference in mRNA abundance between GAPDH and PAF-acetylhydrolase, the specific activity of the GAPDH antisense RNA probe was reduced by greater than 1000-fold. Eighty micrograms of liver total RNA were hybridized in solution with both antisense RNA probes (RPAII Kit, Ambion). After ribonuclease digestion, the samples were separated on a denaturing 5% polyacrylamide, 8 m urea gel. Differences in the amount of PAF-acetylhydrolase and GAPDH mRNA were visualized using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Yeast tRNA was included as a negative control. Hepatocytes were isolated from collagenase-digested livers by low speed centrifugation. Endothelial cells and Kupffer cells were isolated from rat livers using a modification of the centrifugal elutriation procedure of Knook and Sleyster (33Knook D.L. Sleyster E.C. Exp. Cell Res. 1976; 99: 444-449Crossref PubMed Scopus (351) Google Scholar) as described previously (34Gandhi C.R. Stephenson K. Olson M.S. Biochem. J. 1992; 281: 485-492Crossref PubMed Scopus (68) Google Scholar). The viability of the liver cell preparations was greater than 95% as determined by trypan blue exclusion. All RNA isolation procedures were based on the method of Chomczynski and Sacchi (35Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (65696) Google Scholar). Briefly, 1 g of frozen liver was pulverized in liquid nitrogen and homogenized in 5 ml of TRIzolTM (Life Technologies, Inc., Gaithersburg, MD). After the addition of 1 ml of chloroform and phase separation, the RNA was precipitated with 2.5 ml of isopropyl alcohol. For RNA obtained from hepatic cell types, the freshly isolated hepatocytes, endothelial cells, and Kupffer cells were immediately homogenized in TRIzol after centrifugal elutriation. For cultured Kupffer cells, 1 ml of TRIzol was added per 60-mm plate at the appropriate times. For Northern blots, 10 μg of total RNA was loaded on a 0.8% agarose, 2.2 m formaldehyde MOPS gel and electrophoresed at 70 V for 4 h. The separated RNA was transferred to a Magna nylon membrane (Micron Separations Inc., Westborough, MA) and hybridized with a 32P-labeled PAF-acetylhydrolase cDNA prepared by random priming (36Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (18513) Google Scholar). Hybridizations were performed at high stringency (50% formamide, 1 m NaCl, 10% dextran sulfate, 50 mm Tris, pH 7.5, 0.1% sodium pyrophosphate, and 0.2% Denhardt's solution) at 42 °C for 16 h and the membranes were washed twice in 2 × SSC, 1% SDS at 65 °C for 20 min and once in 0.1 × SSC, 0.1% SDS at room temperature for 15 min. After hybridization with the PAF-acetylhydrolase cDNA, the membranes were stripped and rehybridized with a32P-end-labeled oligonucleotide complimentary to 18 S RNA to verify sample loading and the integrity of the RNA. Northern blots were visualized and quantitated using the PhosphorImager (Molecular Dynamics). Rat neutrophils were depleted by an injection of vinblastine sulfate as described previously (30Zhou W. Chao W. Levine B.A. Olson M.S. Am. J. Physiol. 1992; 263: G587-G592PubMed Google Scholar). Briefly, vinblastine sulfate (0.75 mg/kg) was dissolved in physiological saline and injected i.v. into a tail vein 4 days prior to exposure to LPS. LPS was administered as described above and Kupffer cells were isolated on day 5 following vinblastine treatment, a time at which the vinblastine sulfate has induced maximal neutropenia (37Lemanske R.F. Guthman D.A. Oertel H. Barr L. Kaliner M. J. Immunol. 1983; 130: 2837-2842PubMed Google Scholar). Rat serum was diluted 1/10 with phosphate-buffered saline containing 0.1% BSA. The diluted serum (50 μl) was incubated with 40 μl of 1.25 × 10−3m unlabeled PAF (50 nmol) and 10 μl of 1 × 10−7m [3H]acetyl-PAF in a glass tube at 37 °C for 10 min. The reaction was stopped by cooling on ice. Forty microliters of a 7% BSA solution were added to the reaction mixture and incubated for 5 min at 0 °C. Sixty microliters of a 30% trichloroacetic acid solution were added, and the mixture was incubated for an additional 10 min at 0 °C. To separate the denatured protein, the reaction mixture was centrifuged for 5 min at 3800 ×g. 100 μl of the supernatant were mixed with 10 ml of scintillation mixture, and the amount of liberated radioactive acetate was counted in a liquid scintillation counter. The control values of released acetic acid were obtained for both free serum and serum heated for 10 min in boiling water (38Miwa M. Miyake T. Yamanaka T. Sugatani J. Suzuki Y. Sakata S. Araki Y. Matsumoto M. J. Clin. Invest. 1988; 82: 1983-1991Crossref PubMed Scopus (284) Google Scholar). After enzymatic digestion of the rat liver and centrifugal elutriation of Kupffer cells, isolated Kupffer cells were maintained at 37 °C in RPMI 1640 culture medium (Life Technologies, Inc.), supplemented with 25 mmol/liter HEPES, l-glutamine, 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT), 112 units/ml penicillin, and 112 units/ml streptomycin in 60-mm tissue culture dishes. Cells were plated at a density of 10 million cells per 60-mm culture dish. All cells were incubated in 90% air and 10% CO2. On the second day of culture, the RPMI medium was changed. Under pathophysiological conditions, endotoxin exposure can occur through increased absorption of endotoxin from the gastrointestinal tract leading to systemic endotoxemia. The liver is critical in protecting the systemic circulation from gut-derived LPS. Increased hepatic absorption of LPS from the gastrointestinal tract has been associated with instances of chemical induced liver injury (39Grun M. Liehr H. Rasenack U. Acta Hepato-Gastroenterol. 1977; 24: 64-81PubMed Google Scholar), partial hepatectomy (40Mochida S. Ohta Y. Ogata I. Fujiwara K. J. Hepatol. 1992; 16: 266-272Abstract Full Text PDF PubMed Scopus (23) Google Scholar), and intestinal ischemia/reperfusion (41Gathiram P. Wells M.T. Raidoo D. Brock-Utne J.G. Gaffin S.L. Circ. Shock. 1989; 27: 103-109PubMed Google Scholar, 42Zhi-Yong S. Dong Y.L. Wang X.H. J. Trauma. 1992; 32: 148-153Crossref PubMed Scopus (93) Google Scholar, 43Olofsson P. Nylander G. Olsson P. Acta Chir. Scand. 1985; 151: 635-639PubMed Google Scholar). The infusion of LPS directly into a rat mesenteric vein was employed as a liver-focused model of endotoxemia. The effects of this type of endotoxin exposure on plasma-type PAF-acetylhydrolase expression in whole liver and isolated hepatic cells and on PAF-acetylhydrolase activity in circulating blood were investigated at times ranging from immediately after LPS exposure to 48 h later. We assayed whole liver for the presence of plasma-type PAF-acetylhydrolase mRNA in both saline- and LPS-infused rats. A ribonuclease protection assay was employed to investigate liver PAF-acetylhydrolase expression at various times after endotoxin exposure (Fig. 1). Both a 245-bp antisense PAF-acetylhydrolase RNA and a 355-bp antisense GAPDH RNA were hybridized in solution with 80 μg of total liver RNA. After RNase digestion, the protected fragments were distinguished by their change in mobility, and the band intensity reflected the amount of target mRNA in the samples. In control rat liver, a 175-bp PAF-acetylhydrolase-protected fragment was barely detectable in the samples. However, by 12 h after LPS administration a slight increase in PAF-acetylhydrolase mRNA was observed. At 24 h after endotoxin exposure, there occurred a 20-fold increase in the amount of PAF-acetylhydrolase mRNA present in the livers of rats receiving LPS relative to control livers. A control reaction (Fig. 1,probe alone) containing the two antisense RNA fragments without added RNases was processed in parallel to demonstrate the integrity of the full-length probes and to illustrate the difference in the specific activity of the two probes. The difference in specific activity of the GAPDH and PAF-acetylhydrolase probes was a result of the antisense probe synthesis reaction (see “Experimental Procedures”). A relatively constant 316-bp GAPDH-protected fragment was detected in all samples, indicating equal RNA loading in all lanes. A smaller protected PAF-acetylhydrolase fragment also was observed in the samples from LPS-exposed liver at 12 and 24 h. This minor fragment likely resulted from excess RNase digestion at the RNA ends. To determine the cell type responsible for the increase in PAF-acetylhydrolase expression, hepatocytes, Kupffer cells, and endothelial cells were isolated from endotoxin-exposed livers 24 h after treatment. Total RNA was obtained from these cells immediately after completion of the isolation process. Northern blot analyses of these RNA samples (Fig. 2) indicated that Kupffer cells and endothelial cells but not hepatocytes contained a 1.8-kb transcript which hybridized at high stringency to the full-length PAF-acetylhydrolase cDNA. Freshly isolated sinusoidal endothelial cells (not cultured) contain approximately 10% contamination by Kupffer cells (data not shown). The amount of signal detected in the endothelial cell RNA is consistent with a 10% contamination by Kupffer cells; therefore, we concluded that Kupffer cells were the primary cell type responsible for the elevated PAF-acetylhydrolase expression detected in whole liver 24 h after LPS exposure. A larger 4.4-kb band was determined to be nonspecific binding to the 28 S RNA. After hybridization of the Northern blot with the PAF-acetylhydrolase cDNA, the membrane was stripped and reprobed with a 32P-labeled oligonucleotide complimentary to 18 S RNA. The 18 S oligonucleotide hybridized with relatively equal intensity to all three hepatic cell types. Kupffer cells isolated from livers of rats exposed to LPS for 24 h showed a pronounced increase in expression of PAF-acetylhydrolase. To determine the time course of PAF-acetylhydrolase RNA induction, rats were exposed to LPS as before. At 3, 6, 12, 24, and 48 h after exposure, Kupffer cells were isolated and examined for the presence of PAF-acetylhydrolase transcripts. Northern blot analyses (Fig.3) demonstrated a small increase in RNA levels at 6 h after LPS administration. PAF-acetylhydrolase message levels appeared to maximize at 24 h. At 48 h following LPS exposure, PAF-acetylhydrolase transcripts remained elevated at 80% of the level seen at 24 h. The time course of PAF-acetylhydrolase induction in Kupffer cells isolated from LPS-exposed livers, agreed with the time course of PAF-acetylhydrolase mRNA observed in the analysis of whole liver. The previous experiments demonstrated an increase in PAF-acetylhydrolase mRNA in response to LPS administration and that the Kupffer cell was responsible for the increase in RNA detected. To exclude the possibility that an infiltration of neutrophils and mononuclear cells into the liver was responsible for the increase in PAF-acetylhydrolase mRNA, Kupffer cells were isolated from LPS-exposed neutropenic rats. Vinblastine sulfate (0.75 mg/kg) was injected 4 days prior to LPS infusion. Twenty-four hours after LPS infusion, Kupffer cells were isolated and compared with LPS-infused rats that had not received vinblastine sulfate. A Northern blot comparison (Fig.4) of total RNA from these Kupffer cells demonstrated no change in the amount of PAF-acetylhydrolase induction when corrected for RNA loading. The predominant cellular source(s) of the plasma PAF-acetylhydrolase in vivohas not been determined. To investigate whether the increase in PAF-acetylhydrolase expression in endotoxin-challenged livers could result in elevated serum PAF-acetylhydrolase levels, we assayed rat serum at 1 min and 6, 12, and 24 h after saline or LPS infusion. No change in plasma PAF-acetylhydrolase activity was detected at 1 min and 12 h. A small but statistically significant increase in PAF-acetylhydrolase activity was detected at 6 h. Furthermore, a 2-fold increase in plasma PAF-acetylhydrolase activity was detected at 24 h following LPS exposure (Fig.5). In one rat assayed 48 h after LPS administration, the serum PAF-acetylhydrolase activity remained elevated (data not shown). To investigate the mechanism(s) responsible for the LPS-induced increase in acetylhydrolase expression, we intended to employ in vitro models. In initial experiments, we observed pronounced PAF-acetylhydrolase expression in cultured Kupffer cells from untreated rats (data not shown). To characterize this observation, Kupffer cells were isolated from untreated rats and the PAF-acetylhydrolase mRNA was assayed at different times following the establishment of the Kupffer cells in culture. Freshly isolated Kupffer cells expressed barely detectable levels of PAF-acetylhydrolase. PAF-acetylhydrolase RNA increased as early as 5.5 h after the Kupffer cells were plated (Fig.6). Within 19 h of plating, Kupffer cell PAF-acetylhydrolase mRNA was fully induced. This level of mRNA was sustained throughout the duration of the culture interval even when extended to 7 days. When cultured Kupffer cells were treated with LPS no further increase in PAF-acetylhydrolase mRNA was detected. In fact, LPS decreased PAF-acetylhydrolase RNA in cultured Kupffer cells (data not shown). Ribonuclease protection assays revealed the presence of plasma-type PAF-acetylhydrolase mRNA in rat liver. In untreated rat liver, PAF-acetylhydrolase RNA was barely detectable in 80 μg of total liver RNA. Based on the difference in the specific activity of the ribonuclease protection probes, PAF-acetylhydrola
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