Portal de Periódicos da CAPES

CD11c+ Cells Are Required to Prevent Progression from Local Acute Lung Injury to Multiple Organ Failure and Death

2009; Elsevier BV; Volume: 176; Issue: 1 Linguagem: Inglês

10.2353/ajpath.2010.081027

1525-2191

Jami E. Milam, John R. Erb‐Downward, Gwo-Hsiao Chen, Marcin F. Osuchowski, Roderick A. McDonald, Stephen W. Chensue, Galen B. Toews, Gary B. Huffnagle, Michal A. Olszewski,

Immunotherapy and Immune Responses

To investigate the role of CD11c+ cells in endotoxin-induced acute lung injury, wild-type or CD11c-diphtheria toxin receptor transgenic mice were treated with intraperitoneal diphtheria toxin (5 ng/g b.wt.) in the presence or absence of intratracheal lipopolysaccharide (51 μg). Lipopolysaccharide treatment resulted in 100% mortality in CD11c-depleted animals but not in control animals. Analysis of local lung tissue revealed no differences in acute lung injury severity; however, analysis of distal tissues revealed severe damage and necrosis to multiple organs (liver, spleen, and kidneys) in CD11c-diphtheria toxin receptor mice but not in wild-type mice. In addition, dramatic increases in systemic levels of liver enzymes (alanine aminotransferase, 657 U/L, aspartate aminotransferase, 1401 U/L), blood urea (53 mg/dl), and 8-iso-prostaglandin F2α, a marker of oxidative stress (350 pg/ml), were observed. These data demonstrate that CD11c+ cells play a critical role in protecting the organs from systemic injury caused by a pulmonary endotoxin challenge. To investigate the role of CD11c+ cells in endotoxin-induced acute lung injury, wild-type or CD11c-diphtheria toxin receptor transgenic mice were treated with intraperitoneal diphtheria toxin (5 ng/g b.wt.) in the presence or absence of intratracheal lipopolysaccharide (51 μg). Lipopolysaccharide treatment resulted in 100% mortality in CD11c-depleted animals but not in control animals. Analysis of local lung tissue revealed no differences in acute lung injury severity; however, analysis of distal tissues revealed severe damage and necrosis to multiple organs (liver, spleen, and kidneys) in CD11c-diphtheria toxin receptor mice but not in wild-type mice. In addition, dramatic increases in systemic levels of liver enzymes (alanine aminotransferase, 657 U/L, aspartate aminotransferase, 1401 U/L), blood urea (53 mg/dl), and 8-iso-prostaglandin F2α, a marker of oxidative stress (350 pg/ml), were observed. These data demonstrate that CD11c+ cells play a critical role in protecting the organs from systemic injury caused by a pulmonary endotoxin challenge. Acute lung injury (ALI) and acute respiratory distress syndrome are conditions that are characterized by diffuse alveolar injury, subsequent impairment of arterial oxygenation associated with protein leakage into the alveolar space, and high mortality rates.1Ware LB Matthay MA The acute respiratory distress syndrome.N Engl J Med. 2000; 342: 1334-1349Crossref PubMed Scopus (4483) Google Scholar, 2Ware LB Pathophysiology of acute lung injury and the acute respiratory distress syndrome.Semin Respir Crit Care Med. 2006; 27: 337-349Crossref PubMed Scopus (399) Google Scholar, 3Reutershan J Ley K Bench-to-bedside review: acute respiratory distress syndrome-how neutrophils migrate into the lung.Crit Care. 2004; 8: 453-461Crossref PubMed Scopus (149) Google Scholar Interestingly, despite the severe limitation in gas exchange, the mortality associated with this syndrome is not due to hypoxia but rather to a progressive failure in multiple organ systems (frequently referred to as multiple organ dysfunction syndrome [MODS]).2Ware LB Pathophysiology of acute lung injury and the acute respiratory distress syndrome.Semin Respir Crit Care Med. 2006; 27: 337-349Crossref PubMed Scopus (399) Google Scholar The majority of injuries precipitating ALI are septic in nature, either due to systemic septicemia or a local pneumonitis that leads to the development of a systemic inflammatory condition.4Frutos-Vivar F Ferguson ND Esteban A Epidemiology of acute lung injury and acute respiratory distress syndrome.Semin Respir Crit Care Med. 2006; 27: 327-336Crossref PubMed Scopus (56) Google Scholar, 5Bhatia M Moochhala S Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome.J Pathol. 2004; 202: 145-156Crossref PubMed Scopus (958) Google Scholar Many proinflammatory mediators have been implicated in septic inflammation and MODS including nitric oxide, cytokines, complement factor C5a, and eicosanoids.2Ware LB Pathophysiology of acute lung injury and the acute respiratory distress syndrome.Semin Respir Crit Care Med. 2006; 27: 337-349Crossref PubMed Scopus (399) Google Scholar, 5Bhatia M Moochhala S Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome.J Pathol. 2004; 202: 145-156Crossref PubMed Scopus (958) Google Scholar However, to date, trials targeting many of these mediators have been unsuccessful,6Remick DG Cytokine therapeutics for the treatment of sepsis: why has nothing worked?.Curr Pharm Des. 2003; 9: 75-82Crossref PubMed Scopus (109) Google Scholar generating interest in interventions directed to the cellular rather than humoral components of the inflammatory response in these syndromes. Alveolar macrophages and dendritic cells (DCs) are CD11c+ antigen-presenting cells, and the main pathogen-sensing cells of the innate immune system in the lungs.7van Rijt LS Jung S Kleinjan A Vos N Willart M Duez C Hoogsteden HC Lambrecht BN In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma.J Exp Med. 2005; 201: 981-991Crossref PubMed Scopus (527) Google Scholar, 8Shortman K Naik SH Steady-state and inflammatory dendritic-cell development.Nat Rev Immunol. 2007; 7: 19-30Crossref PubMed Scopus (961) Google Scholar, 9Matsuo M Nagata Y Sato E Atanackovic D Valmori D Chen YT Ritter G Mellman I Old LJ Gnjatic S IFN-γ enables cross-presentation of exogenous protein antigen in human Langerhans cells by potentiating maturation.Proc Natl Acad Sci USA. 2004; 101: 14467-14472Crossref PubMed Scopus (33) Google Scholar, 10Fishwick D Barber C Beckett P Bradshaw LM Rawbone R Curran AD Immunologic response to inhaled endotoxin: changes in peripheral cell surface markers in normal individuals.J Occup Environ Med. 2004; 46: 467-472Crossref PubMed Scopus (4) Google Scholar These cells rapidly detect and respond to microbial products that are deposited in the lungs.10Fishwick D Barber C Beckett P Bradshaw LM Rawbone R Curran AD Immunologic response to inhaled endotoxin: changes in peripheral cell surface markers in normal individuals.J Occup Environ Med. 2004; 46: 467-472Crossref PubMed Scopus (4) Google Scholar Because of this ability to respond rapidly, these cells are of particular interest. Their responses that can lead to the clearance of the harmful stimuli also can lead to the generation of inflammatory mediators that can promote more profound lung injury.5Bhatia M Moochhala S Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome.J Pathol. 2004; 202: 145-156Crossref PubMed Scopus (958) Google Scholar, 7van Rijt LS Jung S Kleinjan A Vos N Willart M Duez C Hoogsteden HC Lambrecht BN In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma.J Exp Med. 2005; 201: 981-991Crossref PubMed Scopus (527) Google Scholar, 8Shortman K Naik SH Steady-state and inflammatory dendritic-cell development.Nat Rev Immunol. 2007; 7: 19-30Crossref PubMed Scopus (961) Google Scholar, 11Osterholzer JJ Milam JE Chen GH Toews GB Huffnagle GB Olszewski MA Role of dendritic cells and alveolar macrophages in regulating early host defense against pulmonary infection with Cryptococcus neoformans.Infect Immun. 2009; 77: 3749-3758Crossref PubMed Scopus (84) Google Scholar, 12Guo RF Ward PA Role of oxidants in lung injury during sepsis.Antioxid Redox Signal. 2007; 9: 1991-2002Crossref PubMed Scopus (195) Google Scholar With the use of CD11c-diphtheria toxin receptor (DTR) mice, transgenic mice that express the diphtheria toxin receptor on CD11c+ cells, in the current work we assessed the role of CD11c+ cells in controlling a nonlethal endotoxin-induced model of ALI. The CD11c-DTR mice are a robust, well established model that we have previously used to study the role of DCs in Cryptococcus neoformans infections.7van Rijt LS Jung S Kleinjan A Vos N Willart M Duez C Hoogsteden HC Lambrecht BN In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma.J Exp Med. 2005; 201: 981-991Crossref PubMed Scopus (527) Google Scholar, 11Osterholzer JJ Milam JE Chen GH Toews GB Huffnagle GB Olszewski MA Role of dendritic cells and alveolar macrophages in regulating early host defense against pulmonary infection with Cryptococcus neoformans.Infect Immun. 2009; 77: 3749-3758Crossref PubMed Scopus (84) Google Scholar, 13Shaw CA Starnbach MN Stimulation of CD8+ T cells following diphtheria toxin-mediated antigen delivery into dendritic cells.Infect Immun. 2006; 74: 1001-1008Crossref PubMed Scopus (13) Google Scholar, 14Scumpia PO McAuliffe PF O'Malley KA Ungaro R Uchida T Matsumoto T Remick DG Clare-Salzler MJ Moldawer LL Efron PA CD11c+ dendritic cells are required for survival in murine polymicrobial sepsis.J Immunol. 2005; 175: 3282-3286PubMed Google Scholar, 15Probst HC Tschannen K Odermatt B Schwendener R Zinkernagel RM Van Den Broek M Histological analysis of CD11c-DTR/GFP mice after in vivo depletion of dendritic cells.Clin Exp Immunol. 2005; 141: 398-404Crossref PubMed Scopus (210) Google Scholar, 16Bennett CL Clausen BE DC ablation in mice: promises, pitfalls, and challenges.Trends Immunol. 2007; 28: 525-531Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar The results presented here demonstrate that the presence of CD11c+ cells is critical in preventing oxidative stress and the progression of mild ALI to a MODS-like syndrome, which rapidly results in death. All animal work for these studies was approved by the University of Michigan Committee on the Use and Care of Animals. The wild-type, BALB/c, and C57BL/6 mice and their transgenic counterparts, transgenic CD11c-DTR BALB/c and transgenic CD11c-DTR C57BL/6 mice, were housed in sterilized, filter-top microisolator cages. The mice were maintained by the Unit for Laboratory Animal Medicine at the University of Michigan (Ann Arbor, MI), with food and water provided ad libitum. The generation and screening of CD11c-DTR mice have been reported previously.17Jung S Unutmaz D Wong P Sano G De los Santos K Sparwasser T Wu S Vuthoori S Ko K Zavala F Pamer EG Littman DR Lang RA In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens.Immunity. 2002; 17: 211-220Abstract Full Text Full Text PDF PubMed Scopus (1442) Google Scholar The offspring mice were generated using standard mouse-cloning procedures and screened for the presence of transgene via Southern blotting. The CD11c-DTR mice were crossed five generations onto the C57BL/6 or the BALB/c backgrounds and subsequently bred in homozygous transgenic lines.17Jung S Unutmaz D Wong P Sano G De los Santos K Sparwasser T Wu S Vuthoori S Ko K Zavala F Pamer EG Littman DR Lang RA In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens.Immunity. 2002; 17: 211-220Abstract Full Text Full Text PDF PubMed Scopus (1442) Google Scholar Mice were injected i.p. with 5 ng/g b.wt. of diphtheria toxin (DT) (D-0564; Sigma-Aldrich, St. Louis, MO) in 0.25 ml of PBS or sterile PBS alone at day −1, as reported previously.17Jung S Unutmaz D Wong P Sano G De los Santos K Sparwasser T Wu S Vuthoori S Ko K Zavala F Pamer EG Littman DR Lang RA In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens.Immunity. 2002; 17: 211-220Abstract Full Text Full Text PDF PubMed Scopus (1442) Google Scholar, 18Saito M Iwawaki T Taya C Yonekawa H Noda M Inui Y Mekada E Kimata Y Tsuru A Kohno K Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice.Nat Biotechnol. 2001; 19: 746-750Crossref PubMed Scopus (361) Google Scholar The effectiveness of the depletion was assessed by flow cytometry. To assure that the endotoxin levels in DT preparations were negligible, DT aliquots were tested with the E-TOXATE (Limulus amebocyte lysate) kit (ET0200; Sigma-Aldrich), according to the manufacturer's protocols (data not shown). To induce an acute lung injury, mice were challenged with 51 μg of lipopolysaccharides (LPS) (L1519, Sigma-Aldrich) from Klebsiella via the intratracheal route, as shown previously.3Reutershan J Ley K Bench-to-bedside review: acute respiratory distress syndrome-how neutrophils migrate into the lung.Crit Care. 2004; 8: 453-461Crossref PubMed Scopus (149) Google Scholar, 19Beutler B Milsark IW Cerami AC Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin.Science. 1985; 229: 869-871Crossref PubMed Scopus (1880) Google Scholar In brief, mice were anesthetized by i.p. injection of ketamine (100 mg/kg, Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (6.8 mg/kg, Lloyd Laboratories, Shenandoah, IA) and restrained on a small surgical board. A small incision was made through the skin over the trachea, and the underlying tissue was separated. The 51 μg of LPS was dispensed into the trachea in 30 μl of sterile water via a 30-gauge needle attached to a 1-ml tuberculin syringe. The skin was closed with cyanoacrylate adhesive. The mice recovered from the procedure with minimal visible trauma. The lung leukocyte isolation was performed 1 day after LPS challenge; this time point is consistent with the peak leukocyte recruitment after the low-dose LPS challenge. Individual lungs were excised, minced, and enzymatically digested for 45 minutes in 15 ml of digestion buffer (RPMI 1640, 5% fetal calf serum, antibiotics, 1 mg/ml collagenase, and 30 μg/ml DNase). The tissues were further dispersed by drawing the suspension up and down 20 times through the bore of a 10-ml syringe. Collagenase solution was removed by centrifugation, and the erythrocytes in the cell pellet were lysed in NH4Cl buffer (0.83% NH4Cl, 0.1% KHCO3, and 0.037% Na2EDTA, pH 7.4) on ice. Leukocytes were washed with RPMI 1640, filtered through Nitex filters resuspended in complete media, and centrifuged for 30 minutes at 3000 × g in the presence of 20% Percoll (Sigma-Aldrich) to separate leukocytes from cell debris and epithelial cells. The isolated leukocytes were re-pelleted and resuspended in complete medium. Total live lung leukocyte numbers were enumerated in the presence of trypan blue using a hemocytometer. After euthanasia, tracheas were exposed and cannulated with polyethylene tubing (PE50). Catheters were secured with surgical silk and mounted on a 23-gauge needle/tuberculin syringe. The lungs were lavaged twice with 0.75 ml of ice-cold PBS. The fluid, recovered by aspiration with gentle chest massage (∼0.5 ml total), was spun at 1000 rpm for 10 minutes, and the supernatant was transferred to a new tube and stored at −20°C for further analysis. Protein levels in the bronchoalveolar lavage fluid were measured using the Bradford assay (Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, CA). Isolated lung cells (105) in complete medium were cytospun onto glass slides (Shandon Cytospin, Pittsburgh, PA). The slides were fixed for 2 minutes during a one-step, methanol-based Wright-Giemsa stain (Harleco, EM Diagnostics, Gibbstown, NJ), followed by steps 2 and 3 of the Diff-Quik whole-blood stain kit (Diff-Quik, Baxter Scientific, Miami, FL). Neutrophils, mononuclear cells, and eosinophils were visually counted under a microscope from randomly chosen high-power fields, for a total of 200 cells. The percentage of a specific leukocyte subset was multiplied by the total number of leukocytes to calculate the absolute number of the subset in the sample. Total RNA from whole lung samples harvested on day 3 after LPS challenge was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's directions. The purified RNA was subsequently reverse-transcribed, and DNA was amplified using a SuperScript One-Step RT-PCR kit (Invitrogen). The following murine oligonucleotide primers were used for RT-PCR analysis: tumor necrosis factor (TNF)-α, 5′-CCTGTAGCCCACGTCGTAGC-3′ (sense) and 5′-AGCAATGACTCCAAAGTAGACC-3′ (antisense); interleukin (IL)-6, 5′-GACAAAGCCAGAGTCCTTCAGAGAG-3′ (sense) and 5′-CTAGGTTTGCCGAGTAGATCTC-3′ (antisense); interferon (IFN)-γ, 5′-TCTACCTCAGACTCTTTGAAGTCT-3′ (sense) and 5′-CAGCGACTCCTTTTCCGCTT-3′ (antisense); IL-10, 5′-CTATGCTGCCTGCTCTTACTG-3′ (sense) and 5′-GATGGCCTTGTAGACACCTTGG-3′ (antisense); granulocyte-macrophage colony-stimulating factor (GM-CSF), 5′-GTGGCTGCAGAATTTACTTTTCCTGGGCATTGTG-3′ (sense) and 5′-TTGCATTCAAAGGGGATATCAGTCAGAAAG-3′ (antisense), IL-23p19, 5′-GTTCCAAGATCCTTCGAAGCCTC-3′ (sense) and 5′-GCCTGGGCTCACTTTTTCTGATG-3′ (antisense); MIP-2, 5′-GCTGGCCACCAACCACCAGG-3′ (sense) and 5′-AGCGAGGCACATCAGGTACG-3′ (antisense); KC, 5′-TGAGCTGCGCTGTCAGTGCCT-3′ (sense) and 5′-AGAAGCCAGCGTTCACCAGA-3′ (antisense); and β-actin, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ (sense) and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (antisense). The reverse transcription was conducted at 45°C for 45 minutes and the RT enzyme was denatured at 94°C for 2 minutes. The PCR steps were cycled 25 or 30 times (denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and elongation at 68°C for 1 minute). The final elongation step was performed at 72°C for 7 minutes. The β-actin (housekeeping gene) was used as the mRNA loading control. Samples were separated by electrophoresis in a 2% agarose gel. Bands were visualized by ethidium bromide/UV luminescence and photographed. To establish the rate of CD11c+ cell depletion, a lung cell suspension (5 × 105) was Fc-blocked with anti-murine CD16/CD32, rat IgG2b and labeled with either a labeled isotype control or with the following anti-mouse monoclonal antibodies/fluorescent dye conjugates: CD11c-fluorescein isothiocyanate (553801, BD Biosciences, San Jose, CA); and Gr-1-PE (553128, BD Biosciences). Cells were incubated for 30 minutes on ice, centrifuged, and washed with staining buffer, fixed in 2.5% paraformaldehyde in buffered saline, and analyzed by flow cytometry. The FL1 (CD11c) versus FL2 (Gr-1) scatter plots were analyzed, following initial selection of intact leukocytes using forward versus side scatter plots. This previously published gating strategy for defining CD11c+ DCs and CD11c+ alveolar macrophages in lung leukocyte isolates was used to quantify the percentage of CD11c+ cells and to confirm their depletion.20de Heer HJ Hammad H Soullie T Hijdra D Vos N Willart MA Hoogsteden HC Lambrecht BN Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen.J Exp Med. 2004; 200: 89-98Crossref PubMed Scopus (668) Google Scholar Isotype control antibodies were used to subtract the background. On day 2, post-LPS challenge mice were euthanized, and blood was collected via orbital bleeding and instantly centrifuged to obtain plasma. Plasma was diluted 1:1 with 15% KOH for 1 hour at 40°C. The free lipids were extracted from the samples twice using the 1:3 dilution of ethyl acetate and dried down under grade 5 nitrogen. Samples were then resuspended in electroimmunoassay buffer, and the 8-iso-prostaglandin (PG) F2α levels were measured by an 8-iso-PG F2α-specific electroimmunoassay (Cayman Chemical, Ann Arbor, MI) according the manufacturer's protocols. On day 2, post-LPS challenge mice were euthanized, and blood was collected via orbital bleeding into sero-separator mini tubes and allowed to clot at room temperature. Serum was separated via centrifugation and assayed using diagnostic tests. Activities of alanine aminotransferase and aspartate aminotransferase and levels of blood urea nitrogen and creatine were assayed and evaluated. All diagnostic tests were performed in the University of Michigan's Unit for Laboratory Animal Medicine Animal Diagnostic Laboratory. Dead animals underwent autopsy to evaluate gross pathology. To evaluate the lesions, organs sampled for histological examination were fixed by submersion in 10% neutral buffered formalin and subsequently dehydrated/processed for paraffin embedding. The 5-μm sections were cut from paraffin blocks, de-paraffinized, stained with H&E and examined by light microscopy. For comparative histopathological analysis, mice from parallel groups were euthanized at 3 days postinfection, lungs were inflated with formalin and processed for histology as described above. Other organs and tissues for comparative histopathological analysis were processed as described above. Although all experiments were performed in both C57BL/6 and BALB/c strains, the results presented were calculated on the basis of data obtained from the C57BL/6 mice, unless noted otherwise. Survival data were analyzed by the Kaplan-Maier (% of surviving animals) method, and comparison of multiple survival curves was performed by log-rank analysis. All remaining quantitative data are reported as means ± SE. Statistical significance was determined using one-way analysis of variance and the unpaired two-tailed Student's t-test corrected for multiple comparisons, when appropriate; P < 0.05 was considered statistically significant. All analyses were performed using the Prism 3.0 program for Windows (GraphPad Prism, San Diego, CA). To test the efficacy of our experimental model, the extent of CD11c+ cell depletion in the CD11c-DTR mice was evaluated after DT treatment. Lung leukocytes were isolated and analyzed by flow cytometry (Figure 1). The myeloid DC and lung alveolar macrophages were selected based on previously identified gate positions in Gr-1 versus CD11c+ scatter plots.20de Heer HJ Hammad H Soullie T Hijdra D Vos N Willart MA Hoogsteden HC Lambrecht BN Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen.J Exp Med. 2004; 200: 89-98Crossref PubMed Scopus (668) Google Scholar Before DT treatment both wild-type and CD11c-DTR mice had similar numbers of CD11c+ cells in the lungs (2.0 ± 0.5 and 2.3 ± 0.2 × 106/lung, respectively). After the DT treatment, the lung population of CD11c+ cells in CD11c-DTR mice decreased by 94% (down to 0.08 ± 0.06 × 106/lung) but remained unaltered (2.1 ± 0.2 × 106/lung) in the wild-type mice. Our results showed that the DT-induced depletion of CD11c+ cells in CD11c-DTR mice is highly effective, producing a relevant experimental model. Wild-type and CD11c-DTR mice, which had received DT or PBS on day −1, were given an intratracheal inoculation of LPS or sterile PBS on day 0 and monitored. CD11c-DTR mice that had been given both DT and LPS very rapidly began to display symptoms of illness (hunched posture and lethargy), and by day 2 50% of the mice had died. None of these animals survived beyond day 3 (Figure 2). In contrast, 100% survival of wild-type mice was observed for all of the treatment groups past the end of the 10-day observation period. In the CD11c-DTR groups not given DT and LPS, 100% of the CD11c-DTR mice that received LPS alone and 80% of the CD11c-DTR mice that received DT alone survived. The 20% mortality seen in the CD11c-DRT mice treated with DT alone was unexpected but reproducible. The DT was tested for LPS contamination using the E-TOXATE (Limulus amebocyte lysate) kit, but levels were found to be below the limit of detection of the assay. Previous studies have shown that the portal circulation carries a significant load of bacterial products derived from the gut microbiota into the liver.21Lunz 3rd, JG Specht SM Murase N Isse K Demetris AJ Gut-derived commensal bacterial products inhibit liver dendritic cell maturation by stimulating hepatic interleukin-6/signal transducer and activator of transcription 3 activity.Hepatology. 2007; 46: 1946-1959Crossref PubMed Scopus (52) Google Scholar It is likely that the hypersensitivity to LPS that these CD11c-DTR exhibit after DT treatment extends to microbiota-derived bacterial products as well. To exclude the possibility of a strain-specific phenomenon, a similar experiment was performed using CD11c-DTR mice generated on the BALB/c background. Consistently, combined administration of DT + LPS in BALB/c CD11c-DTR mice produced 100% lethality with a slightly accelerated time frame: 100% morality of BALB/c CD11c-DTR by day 2 versus 50% in C57BL/6 (data not shown). These results show that mice depleted of CD11c cells become extremely vulnerable to LPS challenge and that this effect was independent of their genetic backgrounds. To determine whether the 100% mortality of CD11c+-depleted ALI mice was associated with an exaggerated inflammatory response in the lungs, we quantified two major hallmarks of inflammatory response. First, pulmonary leukocyte populations were analyzed in the wild-type and CD11c-DTR mice at the peak of the inflammatory response after LPS (day 1). Total and differential leukocyte counts were performed in enzymatically dispersed lungs of mice treated with or without LPS and with or without DT. In all LPS-treated mice the total numbers of eosinophils, neutrophils, and mononuclear cells were higher than those in the saline group. However, there was no significant difference in the composition of the cellular influx of wild-type and CD11c-DTR mice (Figure 3, A–D). This result indicates that the depletion of CD11c+ cells did not alter either the magnitude or character of the inflammatory response in the lungs exposed to the LPS challenge. Second, to determine whether the CD11c+ cell depletion affected the LPS-induced protein leak into the alveolar space, total protein concentration in the bronchoalveolar lavage fluid was measured in the wild-type and CD11c-DTR mice. No significant difference in protein concentration was observed in either wild-type or CD11c-DTR mice treated with DT only compared with their saline-treated controls (data not shown). Significant increases in the concentration of protein in the bronchoalveolar lavage fluid of LPS-treated mice were observed; however, the magnitude of the protein increase was similar in both wild-type and CD11c-DTR, whether or not DT had been administered (Figure 4). The histological analysis of lungs at day 3 post-LPS displayed characteristics of acute lung injury, ranging from the moderate (multifocal acute neutrophilic bronchopneumonia) to the severe (widespread acute neutrophilic bronchopneumonia [data not shown]). Again, whether the mice had been treated with DT made no difference in the histological findings. These results show that the CD11c depletion did not result in the enhanced alveolar-capillary protein leak, differences in leukocyte subsets at the peak of inflammation, or increased histological evidence of lung injury. Together, these data demonstrate that despite a marked difference in mortality between CD11c-depleted mice and nondepleted mice, there was no difference in the severity of lung injury or degree of local response. Induction of cytokines and chemokines during ALI can either contribute to tissue injury or play a protective role in the lungs.5Bhatia M Moochhala S Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome.J Pathol. 2004; 202: 145-156Crossref PubMed Scopus (958) Google Scholar In addition, as soluble mediators, cytokines and chemokines have systemic effects and contribute to the systemic response resulting from local injury. Our next objective was to determine whether CD11c+ cell depletion affected pulmonary cytokine responses after the LPS challenge. Cytokines exhibiting either injurious (IFN-γ, TNF-α, IL-6, IL-23p19, MIP-2, and KC) or protective (GM-CSF and IL-10) properties in ALI were analyzed (Figure 5). The LPS inhalation alone caused a similar increase of mRNA expression for all cytokines analyzed, regardless of the strain (data not shown). However, the depletion of CD11c+ cells did not have a uniform effect on LPS-induced cytokine expression in the lungs of transgenic (Tg) mice. Whereas the expression of proinflammatory IFN-γ and TNF-α cytokines was reduced in comparison with that in the wild-type mice, the expression of proinflammatory KC and MIP-2 chemokines was notably up-regulated. A similar level of increase was apparent for anti-inflammatory cytokine (IL-10 and IL-23p19), but no evident change was observed in the expression of GM-CSF and IL-6 between wild-type and Tg mice administered DT + LPS. Thus, CD11c+ cell depletion caused some changes in pulmonary cytokine responses; however, these changes were not consistent with a massive inflammatory response and/or increased lung injury in CD11c+-depleted mice challenged with LPS. Widespread cell injury associated with membrane lipid peroxidation and oxidative stress is implicated in the pathogenesis of ALI and the progression to MODS. To evaluate the magnitude of systemic oxidative stress, a marker of lipid peroxidation, 8-iso-PGF2α was measured in plasma 3 days after LPS challenge. After the DT + LPS treatment, the circulating level of 8-iso-PGF2α in the CD11c-DTR mice increased to 340 pg/ml and was threefold greater than the 110 pg/ml measured in the wild-type mice (Figure 6). This finding indicates that the depletion of CD11c+ cells in ALI mice resulted in a dramatic increase of systemic oxidative stress and injury. We evaluated the effect of CD11c+ cell depletion on liver pathology in mice with ALI. No macro- or micropathological changes were noted in livers of wild-type animals, regardless of the treatment (Figure 7, A, C, E, and G). In contrast, extensive liver pathology was evident in CD11c-DTR mice treated with DT + LPS (Figure 7F). Macroscopically all livers from the CD11c-DTR DT + LPS mice were very pale and bruised (not shown), consistent with extensive necrosis. The microscopic examination revealed severe damage of hepatic tissue, featuring centrilobular vacuolar fatty change, congestion, and extensive necrosis of hepatocytes and epithelium (Figure 7, B, D, F, and H). Additional micropathology included widespread hepatocellular microlipidosis with periportal lymph node nodules containing scattered necrotic cells and granulocyte infiltrates. The effect of CD11c+ cell depletion on kidney pathology was also evaluated. No macro- or micropathological changes were noted in kidneys of wild-type animals, regardless of the treatment (Figure 8, A, D, G, J, C, F, I, and L). In contrast, microscopic examination of the tubular compartment in CD11c-DTR mice treated with DT + LPS revealed the presence of numerous necrotic epithelial cells and tubular dilatation (Figure 8, B, E, H, and K), consistent with acute tubular necrosis. This extensive organ damage and necrotic changes observed in the CD11c-DTR DT + LPS group corresponded with the dramatic elevation of circulating liver enzymes (Figure 9, A and B) and significant increases in blood urea nitrogen (Figure 9C). The same treatment (DT + LPS) in the wild-type mice and LPS alone in either strain did not cause elevation in any of these markers. These data indicate that a mild lung injury in the absence of CD11c+ cells causes mice to rapidly progress to a MODS-like condition consisting of massive liver damage and necrosis and kidney failure and ultimately leads to the death of the animal. Although many advances have been made in the understanding of ALI, the mechanisms that prevent the escalation of the injury and subsequent progression to MODS are not understood.1Ware LB Matthay MA The acute respiratory distress syndrome.N Engl J Med. 2000; 342: 1334-1349Crossref PubMed Scopus (4483) Google Scholar In the work presented we have used a model of self-resolving ALI, induced by local pulmonary LPS challenge, and CD11c-DTR transgenic mice to study the involvement of CD11c+ cells in this process. This study has shown for the first time that the presence of CD11c+ cells during local ALI is required to prevent its progression to MODS. Depletion of CD11c+ cells rendered mice dramatically more susceptible to progression from ALI to systemic distress and death. Interestingly, the ALI itself was not more severe in mice that had been depleted of their CD11c+ cells, but LPS-treated CD11c+ depleted mice displayed symptoms consistent with a MODS-like syndrome: systemic oxidative stress as well as soluble markers of severe liver and kidney damage.22Mizock BA The multiple organ dysfunction syndrome.Dis Mon. 2009; 55: 476-526Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Histological analysis of these tissues supported these findings, revealing pronounced organ damage and tissue necrosis. Together these data indicate that CD11c+ cells are critical for controlling the progression of ALI to multiple organ dysfunction. Although the role of CD11c+ cells in ALI has not been extensively studied, there have been studies on the role of CD11c+ cells in multimicrobial sepsis,14Scumpia PO McAuliffe PF O'Malley KA Ungaro R Uchida T Matsumoto T Remick DG Clare-Salzler MJ Moldawer LL Efron PA CD11c+ dendritic cells are required for survival in murine polymicrobial sepsis.J Immunol. 2005; 175: 3282-3286PubMed Google Scholar, 23Ding Y Chung CS Newton S Chen Y Carlton S Albina JE Ayala A Polymicrobial sepsis induces divergent effects on splenic and peritoneal dendritic cell function in mice.Shock. 2004; 22: 137-144Crossref PubMed Scopus (66) Google Scholar, 24Tinsley KW Grayson MH Swanson PE Drewry AM Chang KC Karl IE Hotchkiss RS Sepsis induces apoptosis and profound depletion of splenic interdigitating and follicular dendritic cells.J Immunol. 2003; 171: 909-914PubMed Google Scholar a syndrome that frequently results in ALI, as well as organ failure and death.1Ware LB Matthay MA The acute respiratory distress syndrome.N Engl J Med. 2000; 342: 1334-1349Crossref PubMed Scopus (4483) Google Scholar, 22Mizock BA The multiple organ dysfunction syndrome.Dis Mon. 2009; 55: 476-526Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 25Riedemann NC Guo RF Ward PA The enigma of sepsis.J Clin Invest. 2003; 112: 460-467Crossref PubMed Scopus (559) Google Scholar Here it has been observed that CD11c+ cells also play a critical role in the progression of sepsis. One such study, using a model of cecal ligation and puncture-induced sepsis and the CD11c-DTR mice, showed that depletion of CD11c+ cells similarly caused a significant increase in sepsis-associated mortality. Furthermore, this increase was eliminated on adoptive transfer of wild-type CD11c+ cells to a mouse that had been previously depleted of CD11c+ cells.14Scumpia PO McAuliffe PF O'Malley KA Ungaro R Uchida T Matsumoto T Remick DG Clare-Salzler MJ Moldawer LL Efron PA CD11c+ dendritic cells are required for survival in murine polymicrobial sepsis.J Immunol. 2005; 175: 3282-3286PubMed Google Scholar However, cecal ligation and puncture has a more severe impact on the systemic homeostasis, as it involves surgical manipulation to cause necrotizing lesions in a portion of intestine and septic insult that spans over extended periods of time. In contrast, the inhaled LPS model used in this study is viewed as a mild, temporary, and self-limited lung injury that is predominantly contained within the lungs. Our data demonstrate that even such limited injury is tightly controlled and that CD11c+ cells play the major role in its pulmonary containment. The mechanism by which the CD11c+ cells are preventing organ damage remains an open question. We considered whether tissue-specific CD11c+ DCs function to filter out dangerous bacterial products (eg, LPS), and thus systemic depletion of CD11c+ cells renders the organs vulnerable to damage. This mechanism could explain how CD11c+ cells protected mice from cecal ligation and puncture-induced sepsis; however, in the current study, neither the degree of injury (Figure 9) nor the amount of inflammation (Figures 5 and 9) is different in the lungs (both control and CD11c+-depleted) despite being the site of LPS treatment. One would expect that if the CD11c+ cells were directly protecting the tissue from damage, then greater damage would be visible at the site of LPS challenge in their absence. Alternatively, the mechanism by which the CD11c+ cells could be protecting the organs would be the production of a factor(s) necessary to facilitate proper immune functioning and that the loss of this signal results in a feed-forward inflammatory response. One line of evidence that supports this hypothesis is that transfection with an IL-10-expressing adenovirus caused the DCs to adopt a regulatory phenotype and when transferred to a cecal ligation and puncture-challenged animal conferred greater survival and resistance to sepsis.26Oberholzer C Oberholzer A Bahjat FR Minter RM Tannahill CL Abouhamze A LaFace D Hutchins B Clare-Salzler MJ Moldawer LL Targeted adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival in murine sepsis.Proc Natl Acad Sci USA. 2001; 98: 11503-11508Crossref PubMed Scopus (103) Google Scholar, 27Oberholzer A Oberholzer C Bahjat KS Ungaro R Tannahill CL Murday M Bahjat FR Abouhamze Z Tsai V LaFace D Hutchins B Moldawer LL Clare-Salzler MJ Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells.J Immunol. 2002; 168: 3412-3418PubMed Google Scholar Although our present study has not addressed this mechanism(s), it is clear that CD11c+ cells are required to prevent a self-resolving ALI from progressing to systemic organ dysfunction and ultimately death. Further studies are needed to address the mechanistic questions that cannot be answered by the current study. We thank Drs. Steffen Jung, Leonie S. van Rijt, and Bart N. Lambrecht for consultation of data analysis. We also thank Dr. John Osterholzer and Nicole Falkowski for their assistance in parts of experimental efforts and Drs. Rashida Horton and Kathryn Eaton for their help in pathological data analysis.