Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules
2003; Elsevier BV; Volume: 162; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63915-6
ISSN1525-2191
AutoresHitoshi Nochi, Naoko Aoki, Kensuke Oikawa, Mitsuru Yanai, Yumi Takiyama, Yoshiaki Atsuta, Hiroya Kobayashi, Keisuke Sato, Masatoshi Tateno, Takeo Matsuno, Makoto Katagiri, Zhou Xing, Shoji Kimura,
Tópico(s)Immune Cell Function and Interaction
ResumoDAP12 (also known as KARAP) is a novel ITAM-bearing transmembrane adapter molecule that is expressed on the cell surface of natural killer cells, monocytes, dendritic cells, and macrophages. Several myeloid cell-specific DAP12-associating receptors, such as TREM receptor family, SIRP-β1, and MDL-1 have been identified. The in vivo function of DAP12 and its associating molecules in inflammation has remained primarily unknown. To investigate DAP12 signaling during chronic inflammation, we constructed two adenoviral gene transfer vectors to express FLAG/DAP12 (Ad-FDAP12) and the extracellular domain of mouse TREM-1 and the Fc portion of human IgG1 (Ad-TREM-1 Ig), respectively, and observed their modulatory activities in a mouse model of hepatic granulomatous inflammation elicited by zymosan A. Mice were injected with zymosan A intravenously and 24 hours after zymosan A, they were injected with Ad-FDAP12 or Ad-TREM-1 Ig. Zymosan A-induced hepatic granuloma formation peaked at day 7 and markedly declined by day 10. Although adenoviral-mediated DAP12 gene transfer did not enhance granuloma formation by day 7, it sustained and enhanced granuloma formation beyond day 7. However, an anti-FLAG monoclonal antibody used to potentiate the signaling of adenoviral-derived DAP12, enhanced granuloma formation at day 7. In sharp contrast to the effect by Ad-FDAP12, transgene expression in the liver of soluble form of extracellular domain of TREM-1 as an antagonist of DAP12 signaling, remarkably inhibited zymosan A-induced granuloma formation at all time points examined. Our findings thus suggest that both DAP12 and TREM-1 are involved in the development of granulomatous responses in the liver. DAP12 (also known as KARAP) is a novel ITAM-bearing transmembrane adapter molecule that is expressed on the cell surface of natural killer cells, monocytes, dendritic cells, and macrophages. Several myeloid cell-specific DAP12-associating receptors, such as TREM receptor family, SIRP-β1, and MDL-1 have been identified. The in vivo function of DAP12 and its associating molecules in inflammation has remained primarily unknown. To investigate DAP12 signaling during chronic inflammation, we constructed two adenoviral gene transfer vectors to express FLAG/DAP12 (Ad-FDAP12) and the extracellular domain of mouse TREM-1 and the Fc portion of human IgG1 (Ad-TREM-1 Ig), respectively, and observed their modulatory activities in a mouse model of hepatic granulomatous inflammation elicited by zymosan A. Mice were injected with zymosan A intravenously and 24 hours after zymosan A, they were injected with Ad-FDAP12 or Ad-TREM-1 Ig. Zymosan A-induced hepatic granuloma formation peaked at day 7 and markedly declined by day 10. Although adenoviral-mediated DAP12 gene transfer did not enhance granuloma formation by day 7, it sustained and enhanced granuloma formation beyond day 7. However, an anti-FLAG monoclonal antibody used to potentiate the signaling of adenoviral-derived DAP12, enhanced granuloma formation at day 7. In sharp contrast to the effect by Ad-FDAP12, transgene expression in the liver of soluble form of extracellular domain of TREM-1 as an antagonist of DAP12 signaling, remarkably inhibited zymosan A-induced granuloma formation at all time points examined. Our findings thus suggest that both DAP12 and TREM-1 are involved in the development of granulomatous responses in the liver. DAP12 (KARAP) is a novel immunoreceptor tyrosine-based activation motif (ITAM)-bearing transmembrane adapter molecule.1Olcese L Cambiaggi A Semenzato G Bottino C Moretta A Vivier E Human killer cell activatory receptors for MHC class I molecules are included in a multimeric complex expressed by natural killer cells.J Immunol. 1997; 158: 5083-5086PubMed Google Scholar, 2Lanier LL Corliss BC Wu J Leong C Phillips JH Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells.Nature. 1998; 391: 703-707Crossref PubMed Scopus (738) Google Scholar It is expressed on the cell surface of natural killer cells and associated noncovalently with the activating types of killer immunoglobulin-like cell receptors (KARs).2Lanier LL Corliss BC Wu J Leong C Phillips JH Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells.Nature. 1998; 391: 703-707Crossref PubMed Scopus (738) Google Scholar, 3Lanier LL Corliss BC Wu J Phillips JH Association of DAP12 with activating CD94/NKG2C NK cell receptors.Immunity. 1998; 8: 693-701Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 4Tomasello E Olcese L Vely F Geourgeon C Blery M Moqrich A Gautheret D Djabali M Mattei MG Vivier E Gene structure, expression pattern, and biological activity of mouse killer cell activating receptor-associated protein (KARAP)/DAP-12.J Biol Chem. 1998; 273: 34115-34119Crossref PubMed Scopus (136) Google Scholar, 5Smith KM Wu J Bakker AB Phillips JH Lanier LL Ly-49D and Ly-49H associate with mouse DAP12 and form activating receptors.J Immunol. 1998; 161: 7-10PubMed Google Scholar Although the expression of KARs is restricted to natural killer and T-cell subsets,6Lanier LL NK cell receptors.Annu Rev Immunol. 1998; 16: 359-393Crossref PubMed Scopus (1470) Google Scholar DAP12 is expressed in a wide variety of cell types, including peripheral blood granulocytes, monocytes, macrophages, and dendritic cells.2Lanier LL Corliss BC Wu J Leong C Phillips JH Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells.Nature. 1998; 391: 703-707Crossref PubMed Scopus (738) Google Scholar, 4Tomasello E Olcese L Vely F Geourgeon C Blery M Moqrich A Gautheret D Djabali M Mattei MG Vivier E Gene structure, expression pattern, and biological activity of mouse killer cell activating receptor-associated protein (KARAP)/DAP-12.J Biol Chem. 1998; 273: 34115-34119Crossref PubMed Scopus (136) Google Scholar, 7Cantoni C Bottino C Vitale M Pessino A Augugliaro R Malaspina A Parolini S Moretta L Moretta A Biassoni R NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily.J Exp Med. 1999; 189: 787-795Crossref PubMed Scopus (376) Google Scholar Several myeloid cell-specific DAP12-associating receptors have been identified.8Bakker AB Baker E Sutherland GR Phillips JH Lanier LL Myeloid DAP12-associating lectin (MDL)-1 is a cell surface receptor involved in the activation of myeloid cells.Proc Natl Acad Sci USA. 1999; 96: 9792-9796Crossref PubMed Scopus (177) Google Scholar, 9Bouchon A Dietrich J Colonna M Inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes.J Immunol. 2000; 164: 4991-4995PubMed Google Scholar, 10Dietrich J Cella M Seiffert M Buhring HJ Colonna M Signal-regulatory protein beta 1 is a DAP12-associated activating receptor expressed in myeloid cells.J Immunol. 2000; 164: 9-12PubMed Google Scholar These receptors include the C-type lectin superfamily and Ig superfamily; the former corresponds to the myeloid DAP12-associating lectin 1 (MDL-1)8Bakker AB Baker E Sutherland GR Phillips JH Lanier LL Myeloid DAP12-associating lectin (MDL)-1 is a cell surface receptor involved in the activation of myeloid cells.Proc Natl Acad Sci USA. 1999; 96: 9792-9796Crossref PubMed Scopus (177) Google Scholar and the latter includes triggering receptor expressed on myeloid cells (TREM)-1, TREM-2, and TREM-39Bouchon A Dietrich J Colonna M Inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes.J Immunol. 2000; 164: 4991-4995PubMed Google Scholar, 11Daws MR Lanier LL Seaman WE Ryan JC Cloning and characterization of a novel mouse myeloid DAP12-associated receptor family.Eur J Immunol. 2001; 31: 783-791Crossref PubMed Scopus (146) Google Scholar, 12Chung DH Seaman WE Daws MR Characterization of TREM-3, an activating receptor on mouse macrophages: definition of a family of single Ig domain receptors on mouse chromosome 17.Eur J Immunol. 2002; 32: 59-66Crossref PubMed Scopus (83) Google Scholar and signal regulatory protein β1 (SIRP-β1)10Dietrich J Cella M Seiffert M Buhring HJ Colonna M Signal-regulatory protein beta 1 is a DAP12-associated activating receptor expressed in myeloid cells.J Immunol. 2000; 164: 9-12PubMed Google Scholar (Figure 1A). The role of DAP12 and its associating receptors in inflammatory and immune responses still remains to be understood.13Aoki N Kimura S Xing Z Role of DAP12 in innate and adaptive immune responses.Curr Pharm Des. 2003; 9: 7-10Crossref PubMed Scopus (22) Google Scholar More recently, Bouchon and colleagues14Bouchon A Facchetti F Weigand MA Colonna M TREM-1 amplifies inflammation and is a crucial mediator of septic shock.Nature. 2001; 410: 1103-1107Crossref PubMed Scopus (830) Google Scholar demonstrated that blockade of TREM-1 protects mice against lipopolysaccharide (LPS)-induced shock and suggested a critical function of TREM-1 in acute inflammatory responses to bacteria. Furthermore, Sjolin and colleagues15Sjolin H Tomasello E Mousavi-Jazi M Bartolazzi A Karre K Vivier E Cerboni C Pivotal role of KARAP/DAP12 adaptor molecule in the natural killer cell-mediated resistance to murine cytomegalovirus infection.J Exp Med. 2002; 195: 825-834Crossref PubMed Scopus (96) Google Scholar observed that functional DAP12-deficient mice suffered weakened host defense against murine CMV infection. These observations suggest that DAP12 signaling may play a critical regulatory role in immune responses during infection and inflammation.Previously, we reported that signaling through the DAP12 ITAM motif was very important for terminal differentiation of the murine M1 leukemia cell line.16Aoki N Kimura S Takiyama Y Atsuta Y Abe A Sato K Katagiri M The role of the DAP12 signal in mouse myeloid differentiation.J Immunol. 2000; 165: 3790-3796Crossref PubMed Scopus (36) Google Scholar, 17Aoki N Kimura S Oikawa K Nochi H Atsuta Y Kobayashi H Sato K Katagiri M DAP12 ITAM motif regulates differentiation and apoptosis in M1 leukemia cells.Biochem Biophys Res Commun. 2002; 291: 296-304Crossref PubMed Scopus (9) Google Scholar We observed the vigorous morphological change of M1 cells to macrophages including giant cell formation after stimulation through DAP12. However, the role of DAP12 in the macrophage differentiation and activation during inflammation in vivo has not yet been established.To study the role of DAP12/TREM-1 signaling during chronic inflammation, we constructed two adenoviral gene vectors (Figure 1B): Ad-FDAP12 (allowing increased expression of FLAG-DAP12) and Ad-TREM-1 Ig (allowing expression of an antagonist of the DAP12-signaling pathway-soluble form of extracellular domain of TREM-1), and investigated their respective modulatory effect in a mouse model of zymosan A-induced hepatic granuloma.18Wynn AA Miyakawa K Miyata E Dranoff G Takeya M Takahashi K Role of granulocyte/macrophage colony-stimulating factor in zymocel-induced hepatic granuloma formation.Am J Pathol. 2001; 158: 131-145Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Zymosan A (zymosan, zymocel, β-glucans), which is composed of β-1,3 polyglucose, causes very strong stimulation of macrophages, neutrophils, and natural killer cells.19Xia Y Vetvicka V Yan J Hanikyrova M Mayadas T Ross GD The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells.J Immunol. 1999; 162: 2281-2290PubMed Google Scholar We hypothesized that DAP12 signaling could enhance granulomatous responses of monocytes/macrophages whereas TREM-1 Ig will suppress inflammatory response via its antagonistic effect on DAP12 signaling in vivo (Figure 1A). In this study, we demonstrated that zymosan A-induced granuloma formation was sustained and enhanced at later times by adenoviral-mediated DAP12 gene transfer. In contrast, gene transfer of a DAP12-signaling inhibitor, extracellular domain of TREM-1 markedly inhibited granuloma formation. Our results suggest that the DAP12-signaling pathway plays an important role in chronic inflammation and granuloma formation.Materials and MethodsMice, Cells, and Culture ConditionsC57BL/6NCrj female mice were purchased from Sankyo Labo Service Corp. (Sapporo, Japan). Experimental mice were used at 7 to 8 weeks of age. The murine myeloblastic leukemic cell line, M1, was obtained from Riken Gene Bank (Wakou, Japan). Cells were cultured in RPMI 1640 medium (Nissui Seiyaku Co., Tokyo, Japan) supplemented with 10% fetal calf serum and 5 × 10−5 mol/L 2-mercaptoethanol.AntibodiesRabbit anti-mouse DAP12 polyclonal antibody was generated by immunizing a rabbit (Japanese White) with GST-mouse DAP12 cytoplasmic domain fusion protein as described previously.16Aoki N Kimura S Takiyama Y Atsuta Y Abe A Sato K Katagiri M The role of the DAP12 signal in mouse myeloid differentiation.J Immunol. 2000; 165: 3790-3796Crossref PubMed Scopus (36) Google Scholar Rat anti-murine F4/80 monoclonal antibody (mAb) (CI:A3-1) was purchased from BMA Biomedicals (August, Switzerland). Mouse anti-FLAG mAb (M2) was purchased from Sigma (St. Louis, MO). Control mouse IgG1 was purchased from Chemicon International Inc. (Temecula, CA).Adenovirus Vectors and Zymosan A-Induced Hepatic Granuloma FormationA fusion protein consisting of the extracellular domain of mouse TREM-1 and the Fc portion of human IgG1 was referred to as TREM-1 Ig (the human Ig Fc molecule was used both to prolong the half-life of soluble TREM-1 molecule and to serve as a tag). Recombinant adenovirus containing LacZ (Ad-LacZ), FLAG-DAP12 (Ad-FDAP12), or TREM-1 Ig (Ad-TREM-1 Ig) was generated by the COS-TPC method20Miyake S Makimura M Kanegae Y Harada S Sato Y Takamori K Tokuda C Saito I Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (786) Google Scholar using the Adenovirus Expression Vector kit (Takara, Shiga, Japan) (see Figure 1B for details). These viral vectors were deleted of E1A and E1B, as well as the E3 region. Encoded cDNA was expressed under the control of the CAG promoter.21Niwa H Yamamura K Miyazaki J Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-200Crossref PubMed Scopus (4547) Google Scholar Mice were injected with 350 μl of phosphate-buffered saline (PBS) containing 350 μg of zymosan A from Saccharomyces cerevisiae (Nacaraitesque, Inc., Kyoto Japan) into right retro-orbital plexus. Twenty-four hours after zymosan A injection, mice were injected with 1 × 109 plaque-forming units of viral vector in 100 μl of PBS into the left retro-orbital plexus. Mice were killed by cervical dislocation under diethyl ether anesthesia at days 3, 5, 7, and 10 after zymosan A injection.Localization of Transgene Expression in the Liver by LacZ Histochemical StainingFor X-gal staining, mouse liver was washed with 20 ml of PBS and fixed with 20 ml of fixation reagent (1% formalin, 0.2% glutaraldehyde, 0.002% Nonidet P-40 in PBS) using liver perfusion. Then, the liver was removed and soaked in fixation reagent for 30 minutes with gentle shaking, followed by three washes: 1) PBS for 10 minutes, 2) 1% Triton X in PBS for 10 minutes, and 3) PBS for 10 minutes. X-gal staining was performed with staining reagent (0.5 mmol/L MgCl2, 5 mmol/L K4[Fe(CN)6], 5 mmol/L K3[Fe(CN)6], 0.05% X-gal in PBS) at 37°C for 2 days. Stained liver was observed with a stereoscope.Immunoprecipitation, Electrophoresis, and BlottingCells were lysed in lysis buffer (0.5% Triton X-100, 50 mmol/L Tris, pH 8.0, 140 mmol/L NaCl, 10 mmol/L ethylenediaminetetraacetic acid) containing the protease inhibitor cocktail Complete Mini (Roche, Mannheim, Germany). Lysates were clarified by centrifugation and immunoprecipitated with anti-DAP12 Ab bound to rProtein A Sepharose Fast Flow (Amersham Pharmacia Biotech AB, Uppsala Sweden) for 1 to 2 hours at 4°C. The resulting immunocomplexes were washed and run on 4 to 12% NuPage bis-Tris sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (Novex, San Diego, CA) under reducing conditions. Proteins were then blotted onto Immobilon-P (Millipore, Bedford, MA), blocked in 5% skim milk, and probed with rabbit anti-DAP12 Ab or anti-FLAG mAb (M2) (Sigma) followed by donkey anti-rabbit IgG-horseradish peroxidase (Amersham Pharmacia Biotech AB) or sheep anti-mouse IgG-horseradish peroxidase (Amersham Pharmacia Biotech AB). The ECL system (Amersham Pharmacia Biotech AB) was used for detection.Light Microscopy and ImmunohistochemistryLiver tissues fixed in 10% formaldehyde were embedded in paraffin. Paraffin sections were cut at 3-μm thick and slides were stained with hematoxylin and eosin (H&E) for light microscopy. Hepatic granulomas were defined as being composed of more than 10 cells according to a previous report.18Wynn AA Miyakawa K Miyata E Dranoff G Takeya M Takahashi K Role of granulocyte/macrophage colony-stimulating factor in zymocel-induced hepatic granuloma formation.Am J Pathol. 2001; 158: 131-145Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar In an immunohistochemical study, after deparaffinization and inhibition of the endogenous peroxidase activity, the sections were stained by Histofine SAB-PO Kit (Nichirei, Tokyo, Japan) using the antibodies mentioned above. Hematoxylin was used for nuclear staining.Assays for Differentiation by DAP12 SignalingTo immobilize antibodies, the SonicSeal slide wells (Nalge Nunc International Corp., Naperville, IL.) were incubated with anti-FLAG mAb (M2) (20 μg/ml in PBS) overnight at 4°C and washed with culture medium twice. M1 cells were incubated with LPS (10 μg/ml) from Escherichia coli serotype 0111:B4 (Sigma) overnight (15 to 17 hours) and washed with culture medium. Then, the cells were infected with a multiplicity of infection of 25 of Ad-LacZ and Ad-FDAP12 for 1 hour, and were transferred to anti-FLAG mAb (M2)-coated SonicSeal slide wells. The cells were cultured for 3 days and were stained with H&E.Flow Cytometry for Quantitation of Macrophage DifferentiationCultured M1 cells (1 × 106) were blocked with 50% goat serum for 1 hour at 4°C and then incubated with saturating amounts of fluorescein isothiocyanate-conjugated rat anti-mouse Mac-1 (M1/70) (PharMingen, San Diego, CA) and phycoerythrin-conjugated anti-mouse MHC Class II (M5/114.15.2) (PharMingen) for 30 minutes in staining buffer (PBS, 1% fetal calf serum, 0.1% sodium azide) at 4°C. Dead cells were gated out using 2 μg/ml of propidium iodide at the last step of staining. The fluorescence intensity was analyzed by FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA).Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) AnalysisTotal RNA was prepared using Sepasol-RNA I (Nacaraitesque, Inc.) according to the manufacturer's protocol. cDNA was prepared from 1 μg of total RNA by using the first-strand cDNA synthesis kit for RT-PCR (AMV) (Roche). Primers used were as follows: FLAG-DAP12 forward (5′-GCG AAT TCC GCG TCA TGG CCT TAC CAG TGA-3′), FLAG-DAP12 reverse (5′-ACC CTG TGG ATC TGT ATT-3′), TREM-1 forward (5′-CGG AAT TCG AGC TTG AAG GAT GAG GAA GGC-3′), TREM-1 reverse (5′-AAT CCA GAG TCT GTC ACT TGA AGG TCA GTC-3′), β-actin forward (5′-ACC CAC ACT GTG CCC ATG TA-3′), β-actin reverse (5′-CGG AAC CGC TCA TTG CC-3′). PCR was performed under the conditions of 1 minute at 94°C, 30 cycles (5 seconds at 94°C, 30 seconds at 60°C, 90 seconds at 72°C), 7 minutes at 72°C.Isolation of F4/80-Positive CellsWe performed collagenase perfusion using a buffer (140 mmol/L NaCl, 10 mmol/L HEPES, 5 mmol/L, CaCl2, 2H2O) including 400 U/ml of collagenase type 4 (Sigma). Hepatocytes were removed by centrifugation at 50 × g.22Klocker U Schultz U Schaller H Protzer U Endotoxin stimulates liver macrophages to release mediators that inhibit an early step in hepadnavirus replication.J Virol. 2000; 74: 5525-5533Crossref PubMed Scopus (24) Google Scholar Then cells containing Kupffer cells and monocytes were collected and labeled with F4/80 followed by goat anti-rat IgG microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). We purified F4/80-positive cells using the MACS system (Miltenyi Biotec).Statistical AnalysisThe results were analyzed using one-way analysis of variance, post hoc test, and the Mann-Whitney U-test. All data in this study are expressed as the mean ± SD and P < 0.05 is considered significant.ResultsStimulation of DAP12 Facilitated Macrophage Differentiation in Ad-FDAP12-Infected M1 CellsMouse leukemic M1 cells infected with Ad-FDAP12 could transport FLAG-DAP12 to the cell surface during LPS stimulation because of the concomitant expression of associate molecule of DAP12 (data not shown). Without the associated molecule, the surface expression of DAP12 was not detectable in our system using flow cytometry analysis (data not shown). To investigate the effect of Ad-FDAP12 gene transfer on macrophage differentiation, we pretreated M1 cells with LPS to induce DAP12-associating molecules on the cell surface overnight before infection with Ad-FDAP12 and stimulation by immobilized anti-FLAG mAb. In contrast to Ad-LacZ, Ad-FDAP12-infected M1 cells showed morphologicalchanges suggestive of macrophage differentiation on stimulation via DAP12 (Figure 2A). To verify macrophage differentiation, we also performed fluorescence-activated cell sorting analysis by using mAbs against murine MHC class II and macrophage surface molecule Mac-1. Indeed, Ad-FDAP12-infected cell population contained at least 100% more macrophages that expressed bright Mac-1 or both Mac-1 and MHC class II (28%) whereas the control cell population contained ∼14% of macrophages (Figure 2B).Figure 2Morphological change and cell-surface phenotypic analysis of Ad-FDAP12-infected M1 cells stimulated with immobilized anti-FLAG mAb. A: Ad-LacZ-infected cells were used as a control. After pretreatment with LPS (10 μg/ml) for 15 to 17 hours, M1 cells were infected by adenoviral vector. Cells were cultured on the sonic seal slide wells coated with anti-FLAG mAb for 3 days without LPS. Cells were stained with H&E and observed under the microscope. B: After pretreatment with LPS (10 μg/ml) for 15 to 17 hours, M1 cells were infected by adenoviral vectors and cultured on the dish coated with anti-FLAG mAb for 3 days without LPS. Cells were collected and stained with fluorescein isothiocyanate-conjugated anti-Mac-1 and phycoerythrin-conjugated anti-MHC class II monoclonal antibodies. Samples were analyzed by flow cytometry. Original magnification, ×200 (A).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Hepatic Adenoviral-Mediated Gene TransferBecause signaling through DAP12 may affect monocyte differentiation and activation (Figure 2),16Aoki N Kimura S Takiyama Y Atsuta Y Abe A Sato K Katagiri M The role of the DAP12 signal in mouse myeloid differentiation.J Immunol. 2000; 165: 3790-3796Crossref PubMed Scopus (36) Google Scholar, 17Aoki N Kimura S Oikawa K Nochi H Atsuta Y Kobayashi H Sato K Katagiri M DAP12 ITAM motif regulates differentiation and apoptosis in M1 leukemia cells.Biochem Biophys Res Commun. 2002; 291: 296-304Crossref PubMed Scopus (9) Google Scholar, 23Bouchon A Hernandez-Munain C Cella M Colonna M A DAP12-mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendritic cells.J Exp Med. 2001; 194: 1111-1122Crossref PubMed Scopus (345) Google Scholar, 24Gingras MC Lapillonne H Margolin JF TREM-1, MDL-1, and DAP12 expression is associated with a mature stage of myeloid development.Mol Immunol. 2001; 38: 817-824Crossref Scopus (107) Google Scholar we set out to investigate the role of DAP12 during inflammation in vivo using adenoviral vectors and zymosan A-induced hepatic granuloma formation system. Additionally, we also examined the effect of Ad-TREM-1 Ig that would block the signal thorough TREM-1/DAP12 in monocytes and neutrophils (Figure 1A).14Bouchon A Facchetti F Weigand MA Colonna M TREM-1 amplifies inflammation and is a crucial mediator of septic shock.Nature. 2001; 410: 1103-1107Crossref PubMed Scopus (830) Google Scholar We first investigated transgene expression in the liver after the delivery of an adenoviral vector expressing a marker gene coding for LacZ via the retro-orbital plexus route. This route of gene transfer was previously shown to lead to transgene expression predominantly in the liver.25Mitchell M Jerebtsova M Batshaw ML Newman K Ye X Long-term gene transfer to mouse fetuses with recombinant adenovirus and adeno-associated virus (AAV) vectors.Gene Ther. 2000; 23: 1986-1992Crossref Scopus (54) Google Scholar Two days after intravenous gene transfer, the mouse liver injected with Ad-LacZ showed abundant LacZ staining (Figure 3). In contrast, the control mouse had little LacZ staining. LacZ expression in the liver lasted for at least 10 days after gene transfer (data not shown). These results suggest that transgene can be significantly expressed in the liver after intravenous adenoviral gene transfer.Figure 3Ad-LacZ transgene expression in the liver after intravenous gene transfer by retro-orbital plexus injection. Mice were injected with or without 1 × 109 plaque-forming units of Ad-LacZ into left retro-orbital plexus. Two days after injection, the mice were killed and the livers were analyzed by X-gal staining. Stained liver was observed with a stereoscope.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Prolongation of Zymosan A-Induced Hepatic Granuloma Formation by Ad-FDAP12 and Inhibition by Ad-TREM-1 IgBecause application of high-titer adenoviral vectors in mice may result in severe liver injury26Gao GP Yang Y Wilson JM Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy.J Virol. 1996; 70: 8934-8943Crossref PubMed Google Scholar, 27Lieber A He CY Meuse L Schowalter D Kirillova I Winther B Kay MA The role of Kupffer cell activation and viral gene expression in early liver toxicity after Infusion of recombinant adenovirus vectors.J Virol. 1997; 71: 8798-8807Crossref PubMed Google Scholar, 28Lieber A He CY Meuse L Himeda C Wilson C Kay MA Inhibition of NF-kB activation in combination with Bcl-2 expression allows for persistence of first-generation adenovirus vectors in the mouse liver.J Virol. 1998; 72: 9267-9277Crossref PubMed Google Scholar and thus confound granuloma formation by zymosan, we determined appropriate doses of zymosan A and adenoviral vectors in trial experiments. The peak of zymosan A-induced hepatic granuloma formation was observed at 7 days after a single injection with 350 μg of zymosan A and it vanished by 11 days after the administration (data not shown). Thus, this dose of zymosan was chosen for our following experiments (in contrast to larger doses often used in other studies18Wynn AA Miyakawa K Miyata E Dranoff G Takeya M Takahashi K Role of granulocyte/macrophage colony-stimulating factor in zymocel-induced hepatic granuloma formation.Am J Pathol. 2001; 158: 131-145Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) to keep a relatively short course of granulomatous responses that would allow the observation of the effect of adenoviral-mediated transgene expression after a single delivery of viral vector (a dose of 1 × 109 plaque-forming units of adenoviral vector was chosen).To investigate the role of DAP12 and TREM-1 in zymosan A-induced granuloma formation, we administered the control vector (Ad-LacZ), Ad-FDAP12, or Ad-TREM-1 Ig to groups of mice at day 1 after zymosan A injection. Under lower magnifications of light microscopy, at days 3 and 5, the number of granulomas was similar between control and Ad-FDAP12 groups whereas the number of granulomas was slightly less in Ad-TREM-1 Ig-treated mice (Figure 4, Figure 5). By day 7, although the number of granulomas in the control and Ad-FDAP12 groups markedly increased and remained similar, the number of granulomas in Ad-TREM-1 Ig-treated mice was minimal (Figure 6) (the size of granuloma in Ad-TREM-1 Ig group also tended to be smaller; see Figure 9 for more details). By day 10, granulomas in the control group primarily disappeared, close to the constant low level of granuloma formation in Ad-TREM-1 Ig-treated mice (Figure 7). In sharp contrast, the number of granulomas in Ad-FDAP12-treated mice continued to increase (Figure 7). The number of granulomas was also enumerated at four different time points in three groups of mice (Figure 8) and the results were in line with morphological observations. Under higher magnification of light microscopy, the discrete granuloma structure and kinetic influx of inflammatory cells were revealed (Figure 9). At day 3, the major cell types were polymorphonuclear leukocytes and monocytes/macrophages, regardless of treatment given (Figure 9; A, E, I). By day 5, the main cell type within the granuloma was mononuclear cells including macrophages and lymphocytes (Figure 9; B, F, J). At day 7, many macrophage-derived epithelioid cells were seen, suggestive of mature granuloma formation (Figure 9; C, G, K). By day 10, many cells in the control group underwent apoptosis (Figure 9D), which was associated with diminishing granuloma whereas many epithelioid cells were still seen in the granuloma of Ad-FDAP12-treated mice (Figure 9H).Figure 4Zymosan A-induced hepatic granu
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