CD8+ T Cells Promote Inflammation and Apoptosis in the Liver after Sepsis
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.061099
ISSN1525-2191
AutoresDoreen E. Wesche‐Soldato, Chun‐Shiang Chung, Stephen H. Gregory, Thais P. Salazar‐Mather, Carol A. Ayala, Alfred Ayala,
Tópico(s)Immune Cell Function and Interaction
ResumoAlthough studies blocking the Fas pathway indicate it can decrease organ damage while improving septic (cecal ligation and puncture, CLP) mouse survival, little is known about how Fas-Fas ligand (FasL) interactions mediate this protection at the tissue level. Here, we report that although Fas expression on splenocytes and hepatocytes is up-regulated by CLP and is inhibited by in vivo short interfering RNA, FasL as well as the frequency of CD8+ T cells are differentially altered by sepsis in the spleen (no change in FasL, decreased percentage of CD8+ and CD4+ T cells) versus the liver (increased FasL expression on CD8+ T cells and increase in percentage/number). Adoptive transfer of CLP FasL+/+ versus FasL−/− mouse liver CD8+ T cells to severe combined immunodeficient or RAG1−/− recipient mice indicated that these cells could induce inflammation. The FasL-mediated cytotoxic capacity of these septic mouse liver CD8+ T cells was shown by their ability to damage directly cultured hepatocytes. Finally, although CD8−/− mice exhibited a reduction in both CLP-induced liver active caspase-3 staining and blood interleukin-6 levels, only FasL−/− (but not CD8−/−) protected the septic mouse spleen from increasing apoptosis. Thus, although truncating Fas-FasL signaling ameliorates many untoward effects of sepsis, the pathological mode of action is distinct at the tissue level. Although studies blocking the Fas pathway indicate it can decrease organ damage while improving septic (cecal ligation and puncture, CLP) mouse survival, little is known about how Fas-Fas ligand (FasL) interactions mediate this protection at the tissue level. Here, we report that although Fas expression on splenocytes and hepatocytes is up-regulated by CLP and is inhibited by in vivo short interfering RNA, FasL as well as the frequency of CD8+ T cells are differentially altered by sepsis in the spleen (no change in FasL, decreased percentage of CD8+ and CD4+ T cells) versus the liver (increased FasL expression on CD8+ T cells and increase in percentage/number). Adoptive transfer of CLP FasL+/+ versus FasL−/− mouse liver CD8+ T cells to severe combined immunodeficient or RAG1−/− recipient mice indicated that these cells could induce inflammation. The FasL-mediated cytotoxic capacity of these septic mouse liver CD8+ T cells was shown by their ability to damage directly cultured hepatocytes. Finally, although CD8−/− mice exhibited a reduction in both CLP-induced liver active caspase-3 staining and blood interleukin-6 levels, only FasL−/− (but not CD8−/−) protected the septic mouse spleen from increasing apoptosis. Thus, although truncating Fas-FasL signaling ameliorates many untoward effects of sepsis, the pathological mode of action is distinct at the tissue level. Sepsis is a major cause of morbidity and mortality in patients in the intensive care unit, with approximately a third of the 750,000 annually reported cases resulting in death.1Angus DC Linde-Zwirble WT Lidicker J Clermont G Carcillo J Pinsky MR Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care.Crit Care Med. 2001; 29: 1303-1310Crossref PubMed Scopus (6592) Google Scholar At the present time, treatment is supportive, and because most molecular-based treatments have failed clinically, there is an urgent need for a better understanding of the pathology of sepsis and its resultant organ failure. With respect to organ damage and mortality associated with sepsis in mouse models, our laboratory and others have reported that it is at least in part attributable to the activation of the Fas-FasL signaling pathway and not TLR4.2Chung CS Xu YX Wang W Chaudry IH Ayala A Is Fas ligand or endotoxin responsible for mucosal lymphocyte apoptosis in sepsis?.Arch Surg. 1998; 133: 1213-1220Crossref PubMed Scopus (75) Google Scholar Studies that have blocked Fas signaling, including Fas fusion protein (FasFP)3Chung CS Song GY Lomas J Simms HH Chaudry IH Ayala A Inhibition of Fas/Fas ligand signaling improves septic survival: differential effects on macrophage apoptotic and functional capacity.J Leukoc Biol. 2003; 74: 344-351Crossref PubMed Scopus (101) Google Scholar, 4Chung CS Yang SL Song GY Lomas J Wang P Simms HH Chaudry IH Ayala A Inhibition of Fas signaling prevents hepatic injury and improves organ blood flow during sepsis.Surgery. 2001; 130: 339-345Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar and FasL−/− mice,2Chung CS Xu YX Wang W Chaudry IH Ayala A Is Fas ligand or endotoxin responsible for mucosal lymphocyte apoptosis in sepsis?.Arch Surg. 1998; 133: 1213-1220Crossref PubMed Scopus (75) Google Scholar have shown a reduction of liver damage and improved survival after sepsis. Most recently, we used Fas and caspase 8 short interfering RNA (siRNA) in vivo after sepsis. Both of these, given as a hydrodynamic, posttreatment bolus injection after cecal ligation and puncture (CLP), decreased apoptosis in the liver and the spleen, decreased indicators of liver damage, and reduced mortality after sepsis by 50%.5Wesche-Soldato DE Chung CS Lomas-Neira JL Doughty LA Gregory SH Ayala A In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice.Blood. 2005; 106: 2295-2301Crossref PubMed Scopus (164) Google Scholar Although in these studies we observed that the suppression of Fas and caspase 8 signal was maintained in the whole liver and spleen up until day 10 after injection, however, the mode of action of this siRNA treatment remains unknown at a cellular level of action. Clearly, any cell that takes up Fas siRNA or is affected by it, even after CLP, would be considered a potential target and therefore may be involved in septic morbidity. Sepsis induces extensive apoptosis of lymphocytes, and this has been suggested to contribute to immunosuppression and mortality.2Chung CS Xu YX Wang W Chaudry IH Ayala A Is Fas ligand or endotoxin responsible for mucosal lymphocyte apoptosis in sepsis?.Arch Surg. 1998; 133: 1213-1220Crossref PubMed Scopus (75) Google Scholar, 5Wesche-Soldato DE Chung CS Lomas-Neira JL Doughty LA Gregory SH Ayala A In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice.Blood. 2005; 106: 2295-2301Crossref PubMed Scopus (164) Google Scholar, 6Hotchkiss RS Tinsley KW Swanson PE Chang KC Cobb JP Buchman TG Korsmeyer SJ Karl IE Prevention of lymphocyte cell death in sepsis improves survival in mice.Proc Natl Acad Sci USA. 1999; 96: 14541-14546Crossref PubMed Scopus (402) Google Scholar, 7Ayala A Xu YX Chung CS Chaudry IH Does Fas ligand or endotoxin contribute to thymic apoptosis during polymicrobial sepsis?.Shock. 1999; 11: 211-217Crossref PubMed Scopus (35) Google Scholar, 8Hotchkiss RS Chang KC Swanson PE Tinsley KW Hui JJ Klender P Xanthoudakis S Roy S Black C Grimm E Aspiotis R Han Y Nicholson DW Karl IE Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte.Nat Immun. 2001; 1: 496-501Crossref Scopus (465) Google Scholar, 9Ayala A Chaudry IH Immune dysfunction in murine polymicrobial sepsis: mediators, macrophages, lymphocytes and apoptosis.Shock. 1996; 6: S27-S38Crossref PubMed Scopus (169) Google Scholar, 10Iwata A Stevenson VM Minard A Tasch M Tupper J Lagasse E Weissman I Harlan JM Winn RK Over-expression of Bcl-2 provides protection in septic mice by a trans effect.J Immunol. 2003; 171: 3136-3141PubMed Google Scholar, 11Hotchkiss RS Swanson PE Knudson CM Chang KC Cobb JP Osborne DF Zollner KM Buchman TG Korsmeyer SJ Karl IE Overexpression of Bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis.J Immunol. 1999; 162: 4148-4156PubMed Google Scholar, 12Chung CS Wang W Chaudry IH Ayala A Increased apoptosis in lamina propria B cells during polymicrobial sepsis is FasL but not endotoxin mediated.Am J Physiol. 2001; 280: G812-G818Google Scholar, 13Hiramatsu M Hotchkiss RS Karl IE Buchman TG Cecal ligation and puncture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin and TNF-independent pathway.Shock. 1997; 7: 247-253Crossref PubMed Scopus (179) Google Scholar, 14Hotchkiss RS Swanson PE Cobb JP Jacobson A Buchman TG Karl IE Apoptosis in lymphoid and parenchymal cells during sepsis: findings in normal and T and B cell deficient mice.Crit Care Med. 1997; 25: 1298-1307Crossref PubMed Scopus (296) Google Scholar To elucidate further the mechanism by which the Fas pathway contributes to the pathology of sepsis, we sought to determine what immune and nonimmune cell types express increased Fas death receptor after CLP, therefore, serving as potential targets of Fas siRNA administration. By the same token, we also sought to establish cell types that express FasL and their contribution to Fas-FasL signaling in the liver and spleen after sepsis. Here, we report that in accordance with previous work, Fas is up-regulated in the liver and the spleen after sepsis, particularly in hepatocytes and lymphocytes, respectively. Last, we demonstrate that Fas ligand-expressing CD8+ T cells contribute to apoptosis and proinflammation in the liver but not the spleen after sepsis. Male C57BL/6 mice, 6 to 8 weeks of age, were used as wild-type controls for all experiments. For adoptive transfer studies, male RAG1−/− mice (B6.129S7-Rag1tm/Mom/J) as well as male severe combined immunodeficient (SCID) mice (B6.CB17-Prkdcscid/SzJ) were used as recipients. Donors for adoptive transfer experiments included female green fluorescent protein (GFP) transgenic C57BL/6-TgN (ACTbEGFP)/Osb and male FasL−/− mice (B6Smn.C3-Faslgld/J). This strain was also used for apoptosis studies as well as CD8−/− (B6.129S2-Cd8atm/Mak/J). All mice were purchased from Jackson Laboratories, Bar Harbor, ME. The studies described here were performed in accordance with the National Institutes of Health and The Guide for the Care and Use of Laboratory Animals, and were approved by the Brown University and Rhode Island Hospital institutional animal care and use committees. The surgical procedure to generate sepsis was performed as previously described.15Ayala A Herdon CD Lehman DL Ayala CA Chaudry IH Differential induction of apoptosis in lymphoid tissues during sepsis: variation in onset, frequency, and the nature of the mediators.Blood. 1996; 87: 4261-4275Crossref PubMed Google Scholar C57BL/6 male mice were lightly anesthetized using isoflurane (Abbott Laboratories, North Chicago, IL). The abdomen was shaved and scrubbed with povidone-iodine (Betadine; Alcon Laboratories, Fort Worth, TX). A midline incision (1.5 to 2 cm) was made below the diaphragm. The cecum was isolated, ligated, punctured twice with a 22-gauge needle, and was gently compressed to extrude a small amount of cecal material. The cecum was returned to the abdomen, and the muscle and skin incisions were closed with 6-0 Ethilon suture material (Ethicon, Inc., Somerville, NJ). Before suturing the skin 2 to 3 drops of lidocaine (Abbott Laboratories) was administered to the wound for analgesia. The mice were subsequently resuscitated with 1.0 ml of lactated Ringer's solution subcutaneously. Sham controls were subjected to the same surgical laparotomy and cecal isolation, but the cecum was neither ligated nor punctured. Fas siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO). The target sequences used for Fas is as follows: 5′-AAGUGCAAGUGCAAACCAGAC-3′.16Song E Lee- S-K. Wang J Ince N Ouyang N Min J Chen J Shankar P Lieberman J RNA interference targeting Fas protects mice from fulminant hepatitis.Nat Med. 2003; 9: 347-351Crossref PubMed Scopus (1024) Google Scholar For the adoptive transfer studies, a group of SCID and RAG1−/− mice (four to five mice per group per strain) received 50 μg of naked Fas siRNA/mouse 24 hours before adoptive transfer of CD8+ T cells. Typically, 50 μg of siRNA was dissolved in 1.5 ml of phosphate-buffered saline (PBS) and was then injected rapidly (throughout a period of 5 seconds) into the tail vein. In apoptosis studies, mice received 50 μg of Fas siRNA 30 minutes after CLP as described previously.5Wesche-Soldato DE Chung CS Lomas-Neira JL Doughty LA Gregory SH Ayala A In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice.Blood. 2005; 106: 2295-2301Crossref PubMed Scopus (164) Google Scholar Briefly, liver nonparenchymal cells (NPCs) were isolated via perfusion of the liver with 0.05% collagenase and 5% fetal calf serum in Hanks' balanced salt solution.17Gordon S Taylor PR Monocyte and macrophage heterogeneity.Nat Rev Immunol. 2005; 5: 953-964Crossref PubMed Scopus (3786) Google Scholar The liver was then dissected and broken up, and hepatocytes were removed via slow centrifugation of 57 × g. Cells in the supernatant were then centrifuged and separated on a 30% Histodenz gradient (weight by volume) (Sigma, St. Louis, MO). NPCs in the top layer were then washed and used for flow cytometry phenotyping or further cell isolations. CD8+ T cells used for adoptive transfer experiments were isolated using MACS columns (Miltenyi Biotech, Auburn, CA). Typically, just under 107 cells were resuspended in degassed running buffer consisting of 0.5% bovine serum albumin and 2 mmol/L ethylenediaminetetraacetic acid in PBS. Biotin-antibody cocktail, including 1 μg of biotin-conjugated CD31 monoclonal antibody (BD Pharmingen, San Diego, CA)/106 cells and anti-biotin microbeads (Miltenyi Biotech) were added to remove non-CD8+ T cells. The suspension was run through a MACS column (Miltenyi Biotech), and the purified CD8+ T-cell population was collected. This population was determined to be 95% pure by flow cytometry staining for CD8 on a BD FACSArray (BD Biosciences, San Jose, CA). This population was also counted after sham, CLP, and CLP/Fas siRNA procedures to determine the number of CD8+ T cells in the liver. Viable hepatocyte populations were isolated using the two-step perfusion method as previously described.18Jiang X Gregory SH Wing EJ Immune CD8+ T lymphocytes lyse Listeria monocytogenes-infected hepatocytes by a classical MHC class I-restricted mechanism.J Immunol. 1997; 158: 287-293PubMed Google Scholar In brief, mice were anesthetized and subjected to laparotomy. The hepatic portal vein was cannulated, and the liver was perfused for 4 minutes with warm Hanks' balanced salt solution containing sodium bicarbonate, ethylenediaminetetraacetic acid, and HEPES using a peristaltic pump (Masterflex; Cole-Palmer, Vernon Hills, IL). The liver was then perfused with warm modified L-15 medium containing glucose and collagenase IV for 15 minutes. The liver was then gently broken up in the collagenase solution and filtered through a 100-μm cell strainer with RPMI 1640 medium + 5% fetal calf serum and centrifuged at 30 × g for 4 minutes. The hepatocytes were then resuspended, viability assessed by trypan blue exclusion, and were subsequently cultured in HEPES-buffered RPMI 1640 medium containing insulin, sodium pyruvate, and 10% fetal calf serum. To determine the effects of CD8+ T cells from septic and nonseptic donor mice, CD8+ T cells were isolated from the liver as described above and adoptively transferred into SCID or RAG1−/− mouse recipients, livers of which were subsequently examined for pathological changes. However, before these studies, we initially attempted to establish the nature of the homing of these isolated cells to certain tissues. To do this, 1.0 × 106 CD8+ T cells from the liver of GFP transgenic mice were isolated and injected via tail vein into C57BL/6 recipients. Twenty-four hours after injection, the liver, thymus, spleen, heart, and lung were harvested from the recipients and flash-frozen in OCT. Frozen tissues were sectioned on a cryostat (Leica, Wetzlar, Germany), stained with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA), and visualized using an Eclipse E400 fluorescence microscope (Nikon, Tokyo, Japan) to assess the presence of GFP cells. The presence of CD8+ T cells was measured as the index of the number of GFP cells versus the total number of 4,6-diamidino-2-phenylindole/nuclear-stained cells in a given field. To assess the functional capacity of CD8+ T cells to injure/damage and/or inflame the liver, male C57BL/6 mice underwent sham or CLP, and a group of FasL−/− mice underwent CLP. At this time, one group of SCID or RAG1−/− recipients received a pretreatment of 50 μg of Fas siRNA hydrodynamically. Twenty-four hours later, mice were sacrificed, livers harvested, and CD8+ T cells isolated as described above. CD8+ T cells (1.0 × 106) were then transferred to SCID and RAG1−/− recipients via tail vein injection. Twenty-four hours later, SCID and RAG1−/− recipients were sacrificed, and the liver and blood/plasma harvested for subsequent assays and histology. Histological changes in these sections were determined by a pathologist who was blinded to the treatment identity of the samples. For electron microscopic examination, the livers of the RAG1−/− recipient mice were first perfused with 0.15 mol/L sodium cacodylate buffer for 2 minutes at a rate of 3 ml/minute using a peristaltic pump (Cole-Palmer Masterflex). The liver was subsequently perfusion-fixed with 2.5% glutaraldehyde in 0.15 mol/L sodium cacodylate buffer for 10 minutes, also at a rate of 3 ml/minute. The tissue was excised and immediately submersed in fixative at 4°C and prepared into 1-mm3 blocks. After overnight fixation, the tissue was washed with buffer and postfixed in 1% OsO4 (osmium tetroxide) in 0.15 mol/L sodium cacodylate buffer for 1 hour at 4°C. After fixation, samples were rinsed in buffer and dehydrated in a graded acetone series and embedded in Spurr's epoxy resin. Semithin sections (1 μm) were prepared using a Reichert Ultracut S microtome (Leica) then stained with methylene blue-azure II and evaluated for areas of interest. Ultrathin sections (50 to 60 nm) were prepared, retrieved onto 300-mesh copper grids, and contrasted with uranyl acetate and lead citrate. Sections were examined using a Morgagni 268 transmission electron microscope, and images were collected with an AMT Advantage 542 charge-coupled device camera system (FEI Company, Hillsboro, OR). For adoptive transfer studies, the plasma of recipient mice was harvested 24 hours after injection to analyze circulating levels of IL-6 via a sandwich method enzyme-linked immunosorbent assay kit (BD Pharmingen), performed according to the manufacturer's instructions (n = 4 to 5 per group). Likewise, septic mouse liver Mig and IP-10 levels were also determined by Quantikine enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN). Protein lysates of mouse liver NPCs and spleen were run on 12% Tris-glycine gels (Invitrogen, Carlsbad, CA). Blotting procedures, chemiluminescent detection, and densitometric analysis were performed as previously described by our laboratory.19Grutkoski PS Chen Y Chung CS Ayala A Sepsis-induced SOCS-3 expression is immunologically restricted to phagocytes.J Leukoc Biol. 2003; 74: 916-922Crossref PubMed Scopus (27) Google Scholar Membranes were probed with Fas ligand rabbit anti-rat polyclonal antibody (Chemicon, Temecula, CA) and bands were detected at 32 and 40 kd. For annexin V staining, 1.0 × 106 cells were washed twice with cold PBS and resuspended in 1× annexin binding buffer (Abcam, Cambridge, MA). Cells (1.0 × 105) were then stained with annexin V-PE (BD Pharmingen) and 7-aminoantinomycin D (Serotec, Raleigh, NC) and incubated for 15 minutes at room temperature in the dark. Cells were then analyzed by flow cytometry. For anti-active caspase 3 staining, paraffin slides underwent epitope retrieval using citrate buffer and serum-blocked using a 2% fetal calf serum solution. Sections were incubated with anti-active caspase 3 antibody (BD Pharmingen) for 1 hour and then blocked with a peroxidase-blocking solution. Sections were then incubated with anti-rabbit horseradish peroxidase secondary antibody. After washing slides were incubated with diaminobenzidine substrate solution and counterstained with Mayer's hematoxylin. As described previously by Chung and colleagues,20Chung CS Song GY Moldawer LL Chaudry IH Ayala A Neither Fas ligand nor endotoxin is responsible for inducible phagocyte apoptosis during sepsis/peritonitis.J Surg Res. 2000; 91: 147-153Abstract Full Text PDF PubMed Scopus (22) Google Scholar livers from C57BL/6 and CD8−/− septic mice as well CD8−/− mice posttreated with Fas siRNA were homogenized in the presence of lysis buffer containing dithiothreitol. Reaction buffer (×2) containing dithiothreitol and AFC-DEVD (caspase 3) (Biomol, Plymouth Meeting, PA) was added to 400 μg of liver lysate. After a 1-hour incubation at 37°C, samples were read on a fluorescent plate reader (FLx800; Bio-Tek Inc., Winooski, VT) at excitation 400 nm and emission 505 nm, and the extent of AFC release was reported in arbitrary fluorescent units. Hepatocytes from naïve, sham, 4-hour CLP, as well as Fas siRNA 24-hour pretreated 4-hour CLP C57BL/6 mice were isolated as described. Hepatocytes were plated at a concentration of 2.0 × 104/well in a 96-well plate. CD8+ T cells were added as effector cells at ratios of 1:1, 1:2, 1:5, and 1:10. Hepatocytes were also plated alone (as background control) and with 1% Triton X-100 (as a maximum release control). Cultures were left overnight and were subsequently analyzed to establish the percent cytotoxicity as measured by release of lactate dehydrogenase (LDH). LDH release was assessed as described in the manufacturer's instructions (BioVision, Mountain View, CA). Percent cytotoxicity was calculated as: (test sample − background)/(maximum release − background) × 100. Sham, CLP, and CLP/siRNA mice treated via hydrodynamic delivery, as well as naïve control mice were euthanized at 24 hours. The liver was perfused and NPCs isolated as described above. Kupffer cells were enriched by adherence on plastic. Nonadherent cells were obtained from the wash eluent, concentrated/washed by centrifugation, viability established by trypan blue exclusion, resuspended in PBS at 1.0 × 106 cells/ml, incubated with a nonspecific Fc blocker (BD Pharmingen), and stained for CD4, CD8, and Fas (BD Pharmingen) as previously described in our laboratory.21Rhee RJ Carlton S Lomas JL Lane C Brossay L Cioffi WG Ayala A Inhibition of CD1d activation suppresses septic mortality: a role for NK-T cells in septic immune dysfunction.J Surg Res. 2003; 115: 74-81Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar The spleen was also harvested and the splenocytes processed in a comparable manner for staining for CD4 and CD8 expression. All cells were read (5000 events) on a BD FACSArray (BD Biosciences). Blood from recipient mice treated with sham, wild-type CLP, or FasL−/− CLP CD8+ T cells was collected 24 hours prior in a syringe containing 2 U of heparin, transferred to a microtube, and centrifuged at 10,000 × g for 10 minutes at 4°C. Plasma samples were stored at −80°C until assayed. Plasma aspartate aminotransferase and alanine aminotransferase levels were determined using a kit (Biotron Diagnostics, Hemet, CA), according to the manufacturer's instructions. The data are presented as a mean and SE of the mean for each group. Differences in percentile (ie, apoptotic index percentage, Fas expression) data (following standard data transformation) and changes in IL-6 levels were considered to be significant at P < 0.05, as determined using the one-way analysis of variance. Differences between multiple groups were established using Tukey's test or the Student-Newman-Keuls multiple comparison. Although we have previously shown that in vivo Fas siRNA administration maintains suppression of Fas protein in the liver and spleen up to 10 days, while concomitantly improving survival after CLP, it was not clear which cell types were in fact the target of this treatment.5Wesche-Soldato DE Chung CS Lomas-Neira JL Doughty LA Gregory SH Ayala A In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice.Blood. 2005; 106: 2295-2301Crossref PubMed Scopus (164) Google Scholar This information is not only critical to understanding the nature of the siRNA's therapeutic effect but also should point to critical pathological events in the development of organ injury in this model of sepsis. Previous studies have shown that T lymphocytes, particularly CD4+, CD8+, as well as B220+ B cells were lost in the spleen after CLP.2Chung CS Xu YX Wang W Chaudry IH Ayala A Is Fas ligand or endotoxin responsible for mucosal lymphocyte apoptosis in sepsis?.Arch Surg. 1998; 133: 1213-1220Crossref PubMed Scopus (75) Google Scholar, 5Wesche-Soldato DE Chung CS Lomas-Neira JL Doughty LA Gregory SH Ayala A In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice.Blood. 2005; 106: 2295-2301Crossref PubMed Scopus (164) Google Scholar, 6Hotchkiss RS Tinsley KW Swanson PE Chang KC Cobb JP Buchman TG Korsmeyer SJ Karl IE Prevention of lymphocyte cell death in sepsis improves survival in mice.Proc Natl Acad Sci USA. 1999; 96: 14541-14546Crossref PubMed Scopus (402) Google Scholar, 7Ayala A Xu YX Chung CS Chaudry IH Does Fas ligand or endotoxin contribute to thymic apoptosis during polymicrobial sepsis?.Shock. 1999; 11: 211-217Crossref PubMed Scopus (35) Google Scholar, 8Hotchkiss RS Chang KC Swanson PE Tinsley KW Hui JJ Klender P Xanthoudakis S Roy S Black C Grimm E Aspiotis R Han Y Nicholson DW Karl IE Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte.Nat Immun. 2001; 1: 496-501Crossref Scopus (465) Google Scholar, 9Ayala A Chaudry IH Immune dysfunction in murine polymicrobial sepsis: mediators, macrophages, lymphocytes and apoptosis.Shock. 1996; 6: S27-S38Crossref PubMed Scopus (169) Google Scholar, 10Iwata A Stevenson VM Minard A Tasch M Tupper J Lagasse E Weissman I Harlan JM Winn RK Over-expression of Bcl-2 provides protection in septic mice by a trans effect.J Immunol. 2003; 171: 3136-3141PubMed Google Scholar, 11Hotchkiss RS Swanson PE Knudson CM Chang KC Cobb JP Osborne DF Zollner KM Buchman TG Korsmeyer SJ Karl IE Overexpression of Bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis.J Immunol. 1999; 162: 4148-4156PubMed Google Scholar, 12Chung CS Wang W Chaudry IH Ayala A Increased apoptosis in lamina propria B cells during polymicrobial sepsis is FasL but not endotoxin mediated.Am J Physiol. 2001; 280: G812-G818Google Scholar, 13Hiramatsu M Hotchkiss RS Karl IE Buchman TG Cecal ligation and puncture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin and TNF-independent pathway.Shock. 1997; 7: 247-253Crossref PubMed Scopus (179) Google Scholar, 14Hotchkiss RS Swanson PE Cobb JP Jacobson A Buchman TG Karl IE Apoptosis in lymphoid and parenchymal cells during sepsis: findings in normal and T and B cell deficient mice.Crit Care Med. 1997; 25: 1298-1307Crossref PubMed Scopus (296) Google Scholar In accordance with this, we have found these cells types were indeed lost in the spleen after CLP (Figure 1A). With Fas siRNA treatment, however, these populations were rescued after CLP. In the liver, on the other hand, there seemed to be an increase in the percentage of CD4+ and CD8+ cells after CLP that was diminished to sham levels after Fas siRNA treatment (Figure 1A). Numbers of these cells followed the same trend, indicating the change in percentage was not attributable to influx or loss of other cell types in the population. The number of CD4+ or CD8+ cells from the spleen extrapolated from the flow cytometry experiments is shown in Figure 1B, top graph. This was also supported by the observation that there was a twofold increase in the total number of CD8+ T cells isolated from the liver after CLP using Miltenyi columns. This was also decreased after Fas siRNA treatment (Figure 1B, bottom graph). Of note, the influx of liver T cells was not associated with a change in Mig or IP-10 levels because no difference was seen in septic or sham mouse liver expression (data not shown). Previously, we have shown that Fas receptor is up-regulated in the liver and the spleen after CLP.5Wesche-Soldato DE Chung CS Lomas-Neira JL Doughty LA Gregory SH Ayala A In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice.Blood. 2005; 106: 2295-2301Crossref PubMed Scopus (164) Google Scholar With respect to FasL, we analyzed expression in the liver NPC fraction as well as in the spleen via Western blot. Although FasL increased on liver NPCs after CLP, it did not seem to change in the spleen (Figure 2A). To elucidate further what cell type in the liver NPC fraction was expressing FasL, CD8+ T cells were isolated, and as shown in Figure 2B, CD8+ T cells were found to express more FasL after CLP when compared with sham. Densitometric representation of these changes in FasL is shown in Figure 2C. Because we found that CD8+ T cells appeared to increase by almost twofold in the liver after CLP and were expressing increased FasL, we speculated, based on the work of Sherwood and colleagues,22Sherwood ER Lin CY Tao W Hartmann CA Dujan JE French AJ Varma TK B2 microglobulin knockout mice are resistant to lethal intra-abdominal sepsis.Am J Respir Crit Care Med. 2003; 167: 1641-1649Crossref PubMed Scopus (43) Google Scholar, 23Sherwood ER Enoh VT Murphy E Lin CY Mice depleted of CD8+ T and NK cells are resistant to injury caused by cecal ligation and puncture.Lab Invest. 2004; 84: 1655-1665Crossref PubMed Scopus (66) Google Scholar that these cells might have the capacity to promote localized tissue injury/inflammation and/or damage to the liver. In an attempt to test this hypothesis, we established an adoptive transfer system that would allow us to determine the effect of CD8+ T cells from septic and nonseptic mice in immunodeficient recipients (SCID and RAG1−/−; the absence of functional recipient lymphoid cells should allow assessment of donor lymp
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