Induction of Apoptosis by Crambene Protects Mice against Acute Pancreatitis via Anti-Inflammatory Pathways
2007; Elsevier BV; Volume: 170; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.061149
ISSN1525-2191
AutoresYang Cao, Sharmila Adhikari, Marie Véronique Clément, Matthew A. Wallig, Madhav Bhatia,
Tópico(s)Pancreatic and Hepatic Oncology Research
ResumoApoptosis is a teleologically beneficial form of cell death in acute pancreatitis. Our previous work has demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. However, little is known about how the induction of apoptosis reduces the severity of acute pancreatitis. Because the clearance of apoptotic cells might suppress inflammation and critically regulate immune responses, we postulate that clearance of apoptotic cells stimulates an anti-inflammatory response, which has a protective action against acute pancreatitis. To test this hypothesis, induction of apoptosis in acute pancreatitis in vivo and co-cultures of peritoneal resident macrophages with apoptotic acinar cells in vitro were used as experimental systems, testing expression of phagocytic receptors and levels of inflammatory mediators. Moreover, neutralizing anti-interleukin (IL)-10 monoclonal antibody (2.5 mg/kg) was used before the induction of apoptosis in acute pancreatitis, testing whether the protection from apoptosis induction would be removed. Our study showed that clearance of apoptotic acinar cells, which may occur essentially through the CD36-positive macrophage, stimulates the release of anti-inflammatory mediators like IL-10. IL-10 plays an important role in crambene-induced protection in acute pancreatitis. Thus, induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis via induction of anti-inflammatory pathways. Apoptosis is a teleologically beneficial form of cell death in acute pancreatitis. Our previous work has demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. However, little is known about how the induction of apoptosis reduces the severity of acute pancreatitis. Because the clearance of apoptotic cells might suppress inflammation and critically regulate immune responses, we postulate that clearance of apoptotic cells stimulates an anti-inflammatory response, which has a protective action against acute pancreatitis. To test this hypothesis, induction of apoptosis in acute pancreatitis in vivo and co-cultures of peritoneal resident macrophages with apoptotic acinar cells in vitro were used as experimental systems, testing expression of phagocytic receptors and levels of inflammatory mediators. Moreover, neutralizing anti-interleukin (IL)-10 monoclonal antibody (2.5 mg/kg) was used before the induction of apoptosis in acute pancreatitis, testing whether the protection from apoptosis induction would be removed. Our study showed that clearance of apoptotic acinar cells, which may occur essentially through the CD36-positive macrophage, stimulates the release of anti-inflammatory mediators like IL-10. IL-10 plays an important role in crambene-induced protection in acute pancreatitis. Thus, induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis via induction of anti-inflammatory pathways. Acute pancreatitis (AP) is a common disorder with potentially devastating consequences.1Bhatia M Neoptolemos JP Slavin J Inflammatory mediators as therapeutic targets in acute pancreatitis.Curr Opin Investig Drugs. 2001; 2: 496-501PubMed Google Scholar, 2Bhatia M Brady M Shokuhi S Christmas S Neoptolemos JP Slavin J Inflammatory mediators in acute pancreatitis.J Pathol. 2000; 190: 117-125Crossref PubMed Scopus (447) Google Scholar, 3Bhatia M Novel therapeutic targets for acute pancreatitis and associated multiple organ dysfunction syndrome.Curr Drug Targets Inflamm Allergy. 2002; 1: 343-351Crossref PubMed Google Scholar, 4Bhatia M Moochhala S Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome.J Pathol. 2004; 202: 145-156Crossref PubMed Scopus (951) Google Scholar Several recent studies have shown that the severity of AP is inversely related to the extent of acinar cell apoptosis.5Bhatia M Apoptosis versus necrosis in acute pancreatitis.Am J Physiol. 2004; 286: G189-G196Google Scholar, 6Gukovskaya AS Perkins P Zaninovic V Sandoval D Rutherford R Fitzsimmons T Pandol SJ Poucell-Hatton S Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat.Gastroenterology. 1996; 110: 875-884Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Kaiser AM Saluja AK Sengupta A Saluja M Steer ML Relationship between severity, necrosis, and apoptosis in five models of experimental acute pancreatitis.Am J Physiol. 1995; 269: C1295-C1304PubMed Google Scholar Moreover, it has been demonstrated that induction of apoptosis reduces the severity of experimental pancreatitis, whereas inhibition of apoptosis worsens the disease.8Bhatia M Wallig MA Hofbauer B Lee HS Frossard JL Steer ML Saluja AK Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis.Biochem Biophys Res Commun. 1998; 246: 476-483Crossref PubMed Scopus (119) Google Scholar, 9Frossard JL Rubbia-Brandt L Wallig MA Benathan M Ott T Morel P Hadengue A Suter S Willecke K Chanson M Severe acute pancreatitis and reduced acinar cell apoptosis in the exocrine pancreas of mice deficient for the Cx32 gene.Gastroenterology. 2003; 124: 481-493Abstract Full Text PDF PubMed Scopus (62) Google Scholar These studies suggested that apoptosis is a teleologically beneficial form of cell death in AP. However, the mechanisms by which introduction of apoptosis protects against AP are not yet clear. Phagocytosis of apoptotic bodies is one of the characteristic morphological and biochemical features of apoptosis.10Savill J Dransfield I Gregory C Haslett C A blast from the past: clearance of apoptotic cells regulates immune responses.Nat Rev Immunol. 2002; 2: 965-975Crossref PubMed Scopus (1321) Google Scholar A series of receptors presenting on the surface of phagocytes have been identified to mediate the recognition and ingestion of apoptotic cells, such as phosphatidylserine receptor (PSR), scavenger receptor (CD36), and vitronectin receptor (integrin αvβ3), etc.10Savill J Dransfield I Gregory C Haslett C A blast from the past: clearance of apoptotic cells regulates immune responses.Nat Rev Immunol. 2002; 2: 965-975Crossref PubMed Scopus (1321) Google Scholar, 11de Almeida CJ Linden R Phagocytosis of apoptotic cells: a matter of balance.Cell Mol Life Sci. 2005; 62: 1532-1546Crossref PubMed Scopus (47) Google Scholar Besides clearance of apoptotic cells, phagocytosis regulates immune responses by up-regulating anti-inflammatory mediators with or without down-regulating proinflammatory cytokines.12Bzowska M Guzik K Barczyk K Ernst M Flad HD Pryjma J Increased IL-10 production during spontaneous apoptosis of monocytes.Eur J Immunol. 2002; 32: 2011-2020Crossref PubMed Scopus (41) Google Scholar For instance, the release of transforming growth factor (TGF)-β1 by macrophages may be mediated by PSR during the clearance of apoptotic cells,13Monks J Rosner D Geske FJ Lehman L Hanson L Neville MC Fadok VA Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release.Cell Death Differ. 2005; 12: 107-114Crossref PubMed Scopus (173) Google Scholar, 14Fadok VA Xue D Henson P If phosphatidylserine is the death knell, a new phosphatidylserine-specific receptor is the bellringer.Cell Death Differ. 2001; 8: 582-587Crossref PubMed Scopus (80) Google Scholar whereas interleukin (IL)-10 is secreted by macrophages engaging with apoptotic cells via annexin I-dependent mechanisms.15Ferlazzo V D'Agostino P Milano S Caruso R Feo S Cillari E Parente L Anti-inflammatory effects of annexin-1: stimulation of IL-10 release and inhibition of nitric oxide synthesis.Int Immunopharmacol. 2003; 3: 1363-1369Crossref PubMed Scopus (67) Google Scholar It is therefore reasonable to postulate that following apoptosis of pancreatic acinar cells, phagocytosis triggers an anti-inflammatory response, which in turn protects against AP. Although relatively few methods of inducing pancreatic acinar cell apoptosis have been identified,8Bhatia M Wallig MA Hofbauer B Lee HS Frossard JL Steer ML Saluja AK Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis.Biochem Biophys Res Commun. 1998; 246: 476-483Crossref PubMed Scopus (119) Google Scholar, 16Gukovskaya AS Gukovsky I Jung Y Mouria M Pandol SJ Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis.J Biol Chem. 2002; 277: 22595-22604Crossref PubMed Scopus (118) Google Scholar, 17Gerasimenko JV Gerasimenko OV Palejwala A Tepikin AV Petersen OH Watson AJ Menadione-induced apoptosis: roles of cytosolic Ca(2+) elevations and the mitochondrial permeability transition pore.J Cell Sci. 2002; 115: 485-497PubMed Google Scholar, 18Wallig MA Gould DH Fettman MJ Selective pancreato-toxicity in the rat induced by the naturally occurring plant nitrile 1-cyano-2-hydroxy-3-butene.Food Chem Toxicol. 1988; 26: 137-147Crossref PubMed Scopus (40) Google Scholar, 19Wallig MA Kore AM Crawshaw J Jeffery EH Separation of the toxic and glutathione-enhancing effects of the naturally occurring nitrile, cyanohydroxybutene.Fundam Appl Toxicol. 1992; 19: 598-606Crossref PubMed Scopus (32) Google Scholar crambene (1-cyano-2-hydroxy-3-butene; CHB), a stable nitrile hydrolysis product of the glucosinolate epi-progoitrin found in many cruciferous vegetables,20Nho CW Jeffery E Crambene, a bioactive nitrile derived from glucosinolate hydrolysis, acts via the antioxidant response element to upregulate quinone reductase alone or synergistically with indole-3-carbinol.Toxicol Appl Pharmacol. 2004; 198: 40-48Crossref PubMed Scopus (32) Google Scholar has been shown to induce extensive apoptosis in pancreatic acinar cells and to protect mice against experimental AP.8Bhatia M Wallig MA Hofbauer B Lee HS Frossard JL Steer ML Saluja AK Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis.Biochem Biophys Res Commun. 1998; 246: 476-483Crossref PubMed Scopus (119) Google Scholar The current study aimed to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against AP by crambene. All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication, 1996). Male Swiss mice (18 to 23 g) were used. Crambene was isolated from autolyzed crambe meal using immiscible solvent extraction followed by high-performance liquid chromatography.18Wallig MA Gould DH Fettman MJ Selective pancreato-toxicity in the rat induced by the naturally occurring plant nitrile 1-cyano-2-hydroxy-3-butene.Food Chem Toxicol. 1988; 26: 137-147Crossref PubMed Scopus (40) Google Scholar Caerulein was obtained from Bachem (Bubendorf, Switzerland). All other reagents were of the highest purity commercially available. Animals were randomly assigned to receive either saline or crambene at one dose of 70 mg/kg given via tail vein injection in a volume of 0.2 ml.8Bhatia M Wallig MA Hofbauer B Lee HS Frossard JL Steer ML Saluja AK Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis.Biochem Biophys Res Commun. 1998; 246: 476-483Crossref PubMed Scopus (119) Google Scholar After receiving crambene for 0, 12, 18, 24, 36, 48, 72, and 96 hours, samples of pancreas were collected for IL-10 and TGF-β1 assay. For the time course of caerulein administration studies, mice were randomly assigned to receive either saline or CHB as described. After 12 hours, animals were given hourly injections of caerulein (Cae; 50 μg/kg) for 0, 3, 6, or 10 hours. For IL-10-neutralizing studies, animals were divided randomly into five groups: 1) control group, mice were given hourly intraperitoneal injections of saline for 10 hours; 2) caerulein group (Cae), mice received 10 hourly intraperitoneal injections of caerulein (50 μg/kg)21Bhatia M Saluja AK Hofbauer B Frossard JL Lee HS Castagliuolo I Wang CC Gerard N Pothoulakis C Steer ML Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury.Proc Natl Acad Sci USA. 1998; 95: 4760-4765Crossref PubMed Scopus (264) Google Scholar, 22Bhatia M Brady M Zagorski J Christmas SE Campbell F Neoptolemos JP Slavin J Treatment with neutralising antibody against cytokine induced neutrophil chemoattractant (CINC) protects rats against acute pancreatitis associated lung injury.Gut. 2000; 47: 838-844Crossref PubMed Scopus (142) Google Scholar; 3) crambene + caerulein group (CHB + Cae), 12 hours after crambene administration as described, mice were injected with caerulein as in group 2; 4) anti-IL-10 + caerulein group [monoclonal antibody (mAb) + Cae], mice received anti-mouse IL-10 monoclonal antibody (R&D Systems, Inc.) intraperitoneally at one dose of 2.5 mg/kg, suitable for in vivo blocking21Bhatia M Saluja AK Hofbauer B Frossard JL Lee HS Castagliuolo I Wang CC Gerard N Pothoulakis C Steer ML Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury.Proc Natl Acad Sci USA. 1998; 95: 4760-4765Crossref PubMed Scopus (264) Google Scholar, 22Bhatia M Brady M Zagorski J Christmas SE Campbell F Neoptolemos JP Slavin J Treatment with neutralising antibody against cytokine induced neutrophil chemoattractant (CINC) protects rats against acute pancreatitis associated lung injury.Gut. 2000; 47: 838-844Crossref PubMed Scopus (142) Google Scholar; 12 hours afterward, mice were injected with caerulein as in group 2; and 5) anti-IL-10 + crambene + caerulein group (mAb + CHB + Cae), mice received crambene together with IL-10 antibody as described in groups 3 and 4, respectively; 12 hours afterward, mice were injected with caerulein as in group 2. One hour after the final caerulein injection in all experiments, animals were euthanized by an intraperitoneal injection of a lethal dose of pentobarbital. Blood and pancreatic tissue were rapidly harvested for the studies as described below. The severity of pancreatic injury induced by caerulein was evaluated by plasma amylase, pancreatic edema, and myeloperoxidase (MPO). Plasma amylase activity was measured as described.3Bhatia M Novel therapeutic targets for acute pancreatitis and associated multiple organ dysfunction syndrome.Curr Drug Targets Inflamm Allergy. 2002; 1: 343-351Crossref PubMed Google Scholar, 8Bhatia M Wallig MA Hofbauer B Lee HS Frossard JL Steer ML Saluja AK Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis.Biochem Biophys Res Commun. 1998; 246: 476-483Crossref PubMed Scopus (119) Google Scholar, 23Marshall JJ Iodice AP Whelan WJ A new serum alpha-amylase assay of high sensitivity.Clin Chim Acta. 1977; 76: 277-283Crossref PubMed Scopus (27) Google Scholar Pancreatic water content and MPO activity were determined as previously described.21Bhatia M Saluja AK Hofbauer B Frossard JL Lee HS Castagliuolo I Wang CC Gerard N Pothoulakis C Steer ML Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury.Proc Natl Acad Sci USA. 1998; 95: 4760-4765Crossref PubMed Scopus (264) Google Scholar, 22Bhatia M Brady M Zagorski J Christmas SE Campbell F Neoptolemos JP Slavin J Treatment with neutralising antibody against cytokine induced neutrophil chemoattractant (CINC) protects rats against acute pancreatitis associated lung injury.Gut. 2000; 47: 838-844Crossref PubMed Scopus (142) Google Scholar, 24Frossard JL Pastor CM Hadengue A Effect of hyperthermia on NF-kappaB binding activity in cerulein-induced acute pancreatitis.Am J Physiol. 2001; 280: G1157-G1162Google Scholar Ten randomly chosen microscopic fields were examined for each pancreatic sample, and the extent of acinar cell injury/necrosis was expressed as the percentage of the total acinar tissue that was occupied by areas meeting the criteria for injury/necrosis as previously described.21Bhatia M Saluja AK Hofbauer B Frossard JL Lee HS Castagliuolo I Wang CC Gerard N Pothoulakis C Steer ML Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury.Proc Natl Acad Sci USA. 1998; 95: 4760-4765Crossref PubMed Scopus (264) Google Scholar, 22Bhatia M Brady M Zagorski J Christmas SE Campbell F Neoptolemos JP Slavin J Treatment with neutralising antibody against cytokine induced neutrophil chemoattractant (CINC) protects rats against acute pancreatitis associated lung injury.Gut. 2000; 47: 838-844Crossref PubMed Scopus (142) Google Scholar, 25Bhatia M Slavin J Cao Y Basbaum AI Neoptolemos JP Preprotachykinin-A gene deletion protects mice against acute pancreatitis and associated lung injury.Am J Physiol. 2003; 284: G830-G836Google Scholar, 26Bhatia M Wong FL Fu D Lau HY Moochhala SM Moore PK Role of hydrogen sulfide in acute pancreatitis and associated lung injury.FASEB J. 2005; 19: 623-625Crossref PubMed Scopus (264) Google Scholar Those criteria were defined as either 1) the presence of acinar cell ghosts or 2) vacuolization and swelling of acinar cells, both of which had been associated with an inflammatory reaction. Pancreatic homogenates were assayed for IL-10, TGF-β1, IL-1β, TNF-α, and MCP-1 using a sandwich enzyme-linked immunosorbent assay according to the manufacturer's instructions (Duoset kit; R&D Systems, Minneapolis, MN). Total RNA was extracted from pancreas with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The primers were synthesized by Proligo (Singapore). The sequences of CD36 was 5′-ATGACGTGGCAAAGAACAGCAGC-3′ (sense) and 5′-GCAACAAACATCACCACTCCAATCC-3′ (antisense). The reaction mixture was first heated to 95°C for 3 minutes and followed by 35 cycles of amplifications, consisting of 95°C for 30 seconds, 68°C for 30 seconds, and 72°C for 30 seconds. PCR amplification was performed in MyCycler (Bio-Rad, Hercules, CA). PCR products were analyzed on 1.5% (w/v) agarose gels containing 0.5 μg/ml ethidium bromide. Individual pancreata from mice were homogenized in ice-cold lysis buffer containing a cocktail of protease inhibitors. After removing nuclei and cell debris, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (50 μg/lane) and electrophoretically transferred to nitrocellulose membranes. Nonspecific binding was blocked by 5% nonfat dry milk. The blots were then incubated overnight with goat anti-mouse CD36 polyclonal antibody (R&D Systems) at 1:2000 dilution, followed by a donkey anti-goat horseradish peroxidase-conjugated secondary antibody (R&D Systems) at 1:1000 dilution in the buffer containing 2.5% nonfat dry milk. The blots were developed for visualization using enhanced chemiluminescence detection kit (Pierce, Rockford, IL). The terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling reaction was performed with the ApopTag Plus Fluorescein In situ Apoptosis Detection kit (Chemicon International, Temecula, CA) in accordance with the manufacturer's protocol. Images were acquired on a Carl Zeiss fluorescence microscope (Carl Zeiss Inc., Thornwood, NY) using band-pass filters designed to detect fluorescein isothiocyanate (FITC). Cryosections were prepared. CD36 was visualized after incubation with a goat anti-mouse primary anti-body (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:50 dilution and then followed by rabbit anti-goat peroxidase-labeled polymer-conjugated secondary antibody (Chemicon) at 1:500 dilution in goat serum. Immunostained sections were counterstained with hematoxylin. For double labeling, tissue sections were incubated with goat anti-mouse CD36 polyclonal (Santa Cruz Biotechnology) and rabbit anti-activated caspase-3 polyclonal (Sigma-Aldrich, St. Louis, MO) or anti-F4/80 (Santa Cruz Biotechnology) antibodies. Sections were then incubated with appropriate secondary antibodies (Alexa Fluor-488 chicken anti-rabbit IgG for anti-activated caspase-3 or anti-F4/80 and Alexa Fluor-568 rabbit anti-goat IgG for anti-CD36). Images were acquired on a Carl Zeiss fluorescent microscope using band-pass filters designed to detect FITC and rhodamine, respectively. RPMI 1640 medium (Invitrogen) was supplemented with containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mmol/L glutamine (Invitrogen). Another supplement added to the medium was 10% fetal bovine serum (FBS) (HyClone, Logan, UT), heat-inactivated for 30 minutes at 56°C. The solution is subsequently referred to as 10% FBS. Closed peritoneal lavage was performed on anesthetized mice by injection of 10 ml of ice-cold 10% FBS, followed by gravity drainage through a 1.1-mm diameter needle. The peritoneal lavage was kept on ice until seeding, during which time the cells were washed once and taken up in 4 ml of ice-cold 10% FBS. A 20-μl sample was taken for counting the total number of cells by hemacytometer. Peritoneal lavage cells were seeded at 7.5 × 104 in 10% FBS in each 1.8 cm2 well of a four-well Lab-Tek glass chamber slide (NUNC A/S, Roskilde, Denmark). Cells were allowed to settle and adhere for 1.5 hours at 37°C in an incubator, after which nonadherent cells were removed by washing with 10% FBS using a standardized protocol of vigorous manual washing via a sterilized Pasteur pipette. FBS (0.9 ml, 10%) was refilled per well, and cells were maintained at 37°C for an additional 1.5 hours. Preparations routinely consisted of >99% mononuclear cells, as verified by Turk's staining. Just before the interaction stage, the cells were again rinsed with 10% FBS using a Pasteur pipette. Pancreata from the same mouse in which isolation of peritoneal macrophages was performed were infused with buffer A (140 mmol/L NaCl, 4.7 mmol/L KCl, 1.13 mmol/L MgCl2, 1 mmol/L CaCl2, 10 mmol/L glucose, and 10 mmol/L HEPES, pH 7.2) containing 200 IU/ml collagenase using a 29G syringe, minced with sharp-tip surgical scissors until a fine suspension was achieved, and incubated for 10 minutes at 37°C in a shaking water bath. The digested tissue was passed through buffer A containing 50 mg/ml bovine serum albumin and washed twice with buffer A for further experiments. Cell viability was determined by trypan blue exclusion. Viability of the cells was greater than 95%. The prepared acini were distributed into microcentrifuge tubes (around 1 × 105 to 1 × 106 pancreatic acinar cells per tube) containing buffer A using a micropipette. Crambene was added into these tubes with the varying working concentration of 2 mmol/L. Acini were incubated with or without crambene at 37°C in a shaker water bath for 3 hours. After 3 hours of incubation, the cells were washed three times in buffer A and taken up in 1 ml of 10% FBS. Samples of 20 μl were taken for analysis of apoptosis. Apoptosis was determined by annexin V-FITC/propidium iodide staining detection as previous described.27Cao Y Adhikari S Ang AD Clement MV Wallig M Bhatia M Crambene induces pancreatic acinar cell apoptosis via the activation of mitochondrial pathway.Am J Physiol. 2006; 291: G95-G101Crossref Scopus (53) Google Scholar Freshly washed apoptotic acinar cells (0.9 ml) were gently added into wells containing macrophages from the same mouse, after which the whole-chamber slides were placed in a 37°C incubator. Cells were allowed to interact for 1.5 hours. Afterward, culture supernatant was collected for the IL-10 assay as described above. The remaining nonphagocytosed acinar cells were removed by washing with 10% FBS using a standardized protocol of vigorous manual washing via a sterilized Pasteur pipette. After taking off the chamber structure part with a scalpel, slides were fixed with 3.7% formaldehyde for 10 minutes followed by extensive washing with PBS. Slides were then blocked with 2% FBS for 45 minutes. Areas for macrophage seeding alone were incubated with rabbit anti-mouse F4/80 antibodies (Santa Cruz Biotechnology) at 1:100 dilution in 2% FBS and goat anti-mouse CD36 polyclonal antibodies (Santa Cruz Biotechnology) at 1:200 dilution in 2% FBS. Slides were washed and then incubated with the appropriate secondary antibodies (Alexa Fluor-488 chicken anti-rabbit IgG for anti-activated anti-F4/80 and Alexa Fluor-568 rabbit anti-goat IgG for anti-CD36, all at 1:200 dilution in 2% FBS) for 30 minutes at room temperature. Slides were allowed to air dry for at least 15 minutes and were mounted with fluorescent mounting medium (Immersol 518F; Zeiss, Wetzlar, Germany). Images were acquired on a Carl Zeiss fluo rescent microscope using band-pass filters designed to detect FITC and rhodamine. Areas for macrophage seeding with pancreatic acinar cells were incubated with goat anti-mouse CD36 polyclonal antibody (Santa Cruz Biotechnology) at 1:200 dilution in 2% FBS followed by incubation with Alexa Fluor-568 rabbit anti-goat IgG for anti-CD36 at 1:200 dilution in 2% FBS for 30 minutes at room temperature. Following washing, slides were then incubated with 10 μg/ml Hoechst dye for 10 minutes according to Bonifacino et al.28Bonifacino JS Dasso M Lippincott-Schwartz J Harford JB Yamada KM Current Protocols in Cell Biology. John K. Wiley & Sons, Philadelphia2002Google Scholar After washing, slides were allowed to air dry for at least 15 minutes and mounted with fluorescent mounting medium (Zeiss). Images acquired on a Carl Zeiss fluorescent microscope using band-pass filters designed to detect 4,6-diamidino-2-phenylindole and rhodamine. Statistical analysis of the data was accomplished using one-way analysis of variance (analysis of variance). The data reported in this article represent mean ± SEM from multiple determinations in three or more different experiments. Differences in the observed results were considered significant if P < 0.05. As shown in Figure 1, caerulein treatment results in a time-related increase in severity of AP. Prophylactic administration of crambene attenuates the severity of AP prominently in mice given 10 hourly caerulein injections. However, the protective effect of crambene in terms of hyperamylasemia attenuation starts in mice receiving six hourly caerulein injections, ie, 18 hours after crambene treatment. Because the clearance of apoptotic cells occurs from 18 hours after crambene administration as suggested by our previous work,8Bhatia M Wallig MA Hofbauer B Lee HS Frossard JL Steer ML Saluja AK Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis.Biochem Biophys Res Commun. 1998; 246: 476-483Crossref PubMed Scopus (119) Google Scholar the current data indicate that the hyperamylasemia attenuation effect of crambene is temporally coincident with clearance of apoptotic cells. In other words, phagocytosis may stimulate a protective effect on AP. To display the phagocytosis in AP in vivo, we investigated pancreatic expression of key phagocytic receptors such as PSR, integrin αvβ3, and CD36 under conditions that result in AP. The mRNA levels of these molecules were all detectable in normal pancreas. However, both PSR and integrin αvβ3 mRNA in caerulein-treated groups were lower than in their respective control groups, with no significant change in mice pretreated with crambene (Figure 2A). On the contrary, pancreatic CD36 mRNA showed a time-dependent elevation above the control. Moreover, pretreatment with crambene significantly up-regulated mRNA levels of CD36 in mice receiving six and 10 hourly caerulein injections compared with mice treated with caerulein alone (Figure 2); ie, the increase of pancreatic CD36 mRNA was temporally coincident with phagocytosis. Therefore, CD36 is likely to be the primary scavenger receptor involved in crambene-mediated phagocytosis in mouse AP. To identify the cells that express CD36, double immunofluorescence labeling was performed in pancreatic sections from mice pretreated with crambene followed by 10 hourly injections of caerulein, using antibodies against CD36 and F4/80. F4/80 is a well-known macrophage-specific marker present on the surface of most macrophages.29Khazen W M'Bika JP Tomkiewicz C Benelli C Chany C Achour A Forest C Expression of macrophage-selective markers in human and rodent adipocytes.FEBS Lett. 2005; 579: 5631-5634Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 30Austyn JM Gordon S F4/80, a monoclonal antibody directed specifically against the mouse macrophage.Eur J Immunol. 1981; 11: 805-815Crossref PubMed Scopus (1283) Google Scholar As indicated in the top panel of Figure 3, CD36-stained cells are co-localized with F4/80-marked cells. However, not all F4/80-positive cells were labeled with CD36 (Figure 3, bottom panel). These data suggest that macrophages in the pancreas are the major cells responsible for the clearance of apoptotic pancreatic acinar cells. To investigate the locational relationship between phagocytic cells and apoptotic cells, we used terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling staining as well as double immunofluorescent staining. As shown in Figure 4, prophylactic administration of crambene results in a significant increase of in situ labeling of cleaved DNA, an indicator of apoptosis. In Figure 5, merged, activated caspase-3-labeled cells (left) were observed close to CD36-stained cells (right). This indicates that macrophages act as the phagocytic cells, acting through the surface receptor CD36.Figure 5Double immunofluorescence labeling of CD36 and activated caspase-3 in pancreatic sections. Pancreatic cryosections from mice with caerulein-induced pancreatitis (10 hourly injections) pretreated with crambene were examined for the expression of CD36 (red) and activated caspase-3 (green) by immunofluorescent microscopy. The merged images (center) indicate that CD36-stained cel
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