AGI Mar 39/3
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چکیده
Ikejima, Kenichi, Nobuyuki Enomoto, Vitor Seabra, Ayako Ikejima, David A. Brenner, and Ronald G. Thurman. Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells. Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G591–G598, 1999.—CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenasePronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPSinduced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-a (TNF-a) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-a mRNA in cells isolated via collagenasePronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.
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AGI Mar 39/3
JAMES R. GUM, JR.,1,2 JAMES W. HICKS,3 ANNE-MARIE GILLESPIE,4 ELAINE J. CARLSON,4 LAZLO KÖMÜVES,5 SATYAJIT KARNIK,1 JOE C. HONG,3 CHARLES J. EPSTEIN,4 AND YOUNG S. KIM1,3 1Gastrointestinal Research Laboratory, Department of Veterans Affairs Medical Center, San Francisco 94121; and Departments of 2Anatomy, 3Medicine, 4Pediatrics, and 5Dermatology, University of California at San Francisco, Calif...
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UTA ECKHARDT,1 ALICE SCHROEDER,1 BRUNO STIEGER,1 MATHIAS HÖCHLI,2 LUKAS LANDMANN,3 RONALD TYNES,4 PETER J. MEIER,1 AND BRUNO HAGENBUCH1 1Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, CH-8091 Zurich; 2Central Laboratory for Electron Microscopy, University of Zurich, CH-8028 Zurich; 3Department of Anatomy, University of Basel, CH-4000 Basel; and 4D...
متن کاملAGI Mar 39/3
Asfaha, Samuel, Cameron J. Bell, John L. Wallace, and Wallace K. MacNaughton. Prolonged colonic epithelial hyporesponsiveness after colitis: role of inducible nitric oxide synthase. Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G703–G710, 1999.—Colonic epithelial secretion is an important host defense mechanism. We examined whether a bout of colitis would produce long-lasting changes i...
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Gupta, Sanjeev, Pankaj Rajvanshi, Emma Aragona, Chang-Don Lee, Purnachandra R. Yerneni, and Robert D. Burk. Transplanted hepatocytes proliferate differently after CCl4 treatment and hepatocyte growth factor infusion. Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G629– G638, 1999.—To understand regulation of transplanted hepatocyte proliferation in the normal liver, we used genetically ...
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Darimont, Christian, Nathalie Gradoux, and Alain De Pover. Epidermal growth factor regulates fatty acid uptake and metabolism in Caco-2 cells. Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G606–G612, 1999.—Epidermal growth factor (EGF) has been reported to stimulate carbohydrate, amino acid, and electrolyte transport in the small intestine, but its effects on lipid transport are poorly...
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Wang, David Q.-H., Frank Lammert, David E. Cohen, Beverly Paigen, and Martin C. Carey. Cholic acid aids absorption, biliary secretion, and phase transitions of cholesterol in murine cholelithogenesis. Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G751–G760, 1999.—Cholic acid is a critical component of the lithogenic diet in mice. To determine its pathogenetic roles, we fed chow or 1% c...
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