Deletion of the murine scavenger receptor CD68
2011; Elsevier BV; Volume: 52; Issue: 8 Linguagem: Inglês
10.1194/jlr.m015412
ISSN1539-7262
AutoresLi Song, Carolyn Lee, Christian Schindler,
Tópico(s)Immune cells in cancer
ResumoScavenger receptors (ScRs) are a structurally unrelated family of receptors with the ability to bind modified low density lipoprotein (LDL) as well as a broad range of polyanionic ligands. CD68, whose expression is restricted to mononuclear phagocytes, is a unique ScR family member, owing to its lysosome associated membrane protein (LAMP)-like domain and predominant endosomal distribution. Knockout (ko) mice were generated to directly evaluate the role murine CD68 may play in oxidized LDL (Ox-LDL) uptake. However, CD68−/− macrophages took up Ox-LDL robustly. Likewise, no defects were observed in the ability of CD68−/− mononuclear phagocytes to take up or mount an effective innate response against a number of microbes. Curiously, CD68−/− mononuclear phagocytes exhibited a trend toward enhanced antigen presentation to CD4+ T-cells, raising the possibility that CD68 may function either to negatively regulate antigen uptake, loading, or major histocompatibility complex class II (MHC-II) trafficking. Scavenger receptors (ScRs) are a structurally unrelated family of receptors with the ability to bind modified low density lipoprotein (LDL) as well as a broad range of polyanionic ligands. CD68, whose expression is restricted to mononuclear phagocytes, is a unique ScR family member, owing to its lysosome associated membrane protein (LAMP)-like domain and predominant endosomal distribution. Knockout (ko) mice were generated to directly evaluate the role murine CD68 may play in oxidized LDL (Ox-LDL) uptake. However, CD68−/− macrophages took up Ox-LDL robustly. Likewise, no defects were observed in the ability of CD68−/− mononuclear phagocytes to take up or mount an effective innate response against a number of microbes. Curiously, CD68−/− mononuclear phagocytes exhibited a trend toward enhanced antigen presentation to CD4+ T-cells, raising the possibility that CD68 may function either to negatively regulate antigen uptake, loading, or major histocompatibility complex class II (MHC-II) trafficking. CD68 and its murine homolog, macrosialin, is a highly glycosylated, ∼95-110 kDa member of the lysosome-associated membrane protein (LAMP)-1 family, which is widely expressed on mononuclear phagocytes (1Taylor P.R. Martinez-Pomares L. Stacey M. Lin H.H. Brown G.D. Gordon S. Macrophage receptors and immune recognition.Annu. Rev. Immunol. 2005; 23: 901-944Crossref PubMed Scopus (1002) Google Scholar–3Holness C.L. da Silva R.P. Fawcett J. Gordon S. Simmons D.L. Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family.J. Biol. Chem. 1993; 268: 9661-9666Abstract Full Text PDF PubMed Google Scholar). It was initially identified as the antigen recognized by monoclonal antibodies directed against human and murine macrophage epitopes (2Micklem K. Rigney E. Cordell J. Simmons D. Stross P. Turley H. Seed B. Mason D. A human macrophage-associated antigen (CD68) detected by six different monoclonal antibodies.Br. J. Haematol. 1989; 73: 6-11Crossref PubMed Scopus (135) Google Scholar, 3Holness C.L. da Silva R.P. Fawcett J. Gordon S. Simmons D.L. Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family.J. Biol. Chem. 1993; 268: 9661-9666Abstract Full Text PDF PubMed Google Scholar). Immunohistochemical studies revealed that CD68 expression was largely restricted to monocytes and tissue-specific macrophages, notably including those in the peritoneum, lungs, spleen, and liver, as well as Langerhans cells and microglia (1Taylor P.R. Martinez-Pomares L. Stacey M. Lin H.H. Brown G.D. Gordon S. Macrophage receptors and immune recognition.Annu. Rev. Immunol. 2005; 23: 901-944Crossref PubMed Scopus (1002) Google Scholar, 4Moore K.J. Freeman M.W. Scavenger receptors in atherosclerosis: beyond lipid uptake.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1702-1711Crossref PubMed Scopus (432) Google Scholar). Moreover, expression appeared to correlate with macrophage activation (5da Silva R.P. Gordon S. Phagocytosis stimulates alternative glycosylation of macrosialin (mouse CD68), a macrophage-specific endosomal protein.Biochem. J. 1999; 338: 687-694Crossref PubMed Scopus (78) Google Scholar, 6O'Reilly D. Quinn C.M. El-Shanawany T. Gordon S. Greaves D.R. Multiple Ets factors and interferon regulatory factor-4 modulate CD68 expression in a cell type-specific manner.J. Biol. Chem. 2003; 278: 21909-21919Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Consistent with these observations, the CD68 promoter has been exploited successfully to direct the expression of transgenes toward the macrophage lineage in vivo (7Gough P.J. Raines E.W. Gene therapy of apolipoprotein E-deficient mice using a novel macrophage-specific retroviral vector.Blood. 2003; 101: 485-491Crossref PubMed Scopus (59) Google Scholar–9Pan H. Mostoslavsky G. Eruslanov E. Kotton D.N. Kramnik I. Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages.J. Immunol. Methods. 2008; 329: 31-44Crossref PubMed Scopus (33) Google Scholar). The characteristic pattern of expression on macrophages and a robust ex vivo affinity for oxidized low-density lipoproteins (Ox-LDL) (10Ramprasad M.P. Fischer W. Witztum J.L. Sambrano G.R. Quehenberger O. Steinberg D. The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68.Proc. Natl. Acad. Sci. USA. 1995; 92: 9580-9584Crossref PubMed Scopus (298) Google Scholar) led to the classification of CD68 as a group D scavenger receptor (4Moore K.J. Freeman M.W. Scavenger receptors in atherosclerosis: beyond lipid uptake.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1702-1711Crossref PubMed Scopus (432) Google Scholar, 11Murphy J.E. Tedbury P.R. Homer-Vanniasinkam S. Walker J.H. Ponnambalam S. Biochemistry and cell biology of mammalian scavenger receptors.Atherosclerosis. 2005; 182: 1-15Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Consistent with its classification as a LAMP-1 family member, CD68 was found to predominately localize to late endosomal and lysosomal compartments with a modest level of cell surface expression (12Kurushima H. Ramprasad M. Kondratenko N. Foster D.M. Quehenberger O. Steinberg D. Surface expression and rapid internalization of macrosialin (mouse CD68) on elicited mouse peritoneal macrophages.J. Leukoc. Biol. 2000; 67: 104-108Crossref PubMed Scopus (64) Google Scholar). Its short cytoplasmic tail, ability to rapidly traffic from cell surface to endosomes, and its intrinsic ability to bind Ox-LDL and phosphatidylserine, were often cited as evidence that CD68 served as a scavenger receptor (3Holness C.L. da Silva R.P. Fawcett J. Gordon S. Simmons D.L. Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family.J. Biol. Chem. 1993; 268: 9661-9666Abstract Full Text PDF PubMed Google Scholar, 10Ramprasad M.P. Fischer W. Witztum J.L. Sambrano G.R. Quehenberger O. Steinberg D. The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68.Proc. Natl. Acad. Sci. USA. 1995; 92: 9580-9584Crossref PubMed Scopus (298) Google Scholar, 12Kurushima H. Ramprasad M. Kondratenko N. Foster D.M. Quehenberger O. Steinberg D. Surface expression and rapid internalization of macrosialin (mouse CD68) on elicited mouse peritoneal macrophages.J. Leukoc. Biol. 2000; 67: 104-108Crossref PubMed Scopus (64) Google Scholar, 13Sambrano G.R. Steinberg D. Recognition of oxidatively damaged and apoptotic cells by an oxidized low density lipoprotein receptor on mouse peritoneal macrophages: role of membrane phosphatidylserine.Proc. Natl. Acad. Sci. USA. 1995; 92: 1396-1400Crossref PubMed Scopus (275) Google Scholar). Although initially characterized for their ability to direct lipid and apoptotic cell uptake, the ability of scavenger receptors (ScRs) to recognize polyanionic ligands has also afforded them an important role in the nonopsonic phagocytosis of numerous microbes (1Taylor P.R. Martinez-Pomares L. Stacey M. Lin H.H. Brown G.D. Gordon S. Macrophage receptors and immune recognition.Annu. Rev. Immunol. 2005; 23: 901-944Crossref PubMed Scopus (1002) Google Scholar, 4Moore K.J. Freeman M.W. Scavenger receptors in atherosclerosis: beyond lipid uptake.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1702-1711Crossref PubMed Scopus (432) Google Scholar). Consistent with this, a number of single and double murine ScR knockouts (ko) have validated their roles in these seemingly disparate inflammatory processes (1Taylor P.R. Martinez-Pomares L. Stacey M. Lin H.H. Brown G.D. Gordon S. Macrophage receptors and immune recognition.Annu. Rev. Immunol. 2005; 23: 901-944Crossref PubMed Scopus (1002) Google Scholar, 4Moore K.J. Freeman M.W. Scavenger receptors in atherosclerosis: beyond lipid uptake.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1702-1711Crossref PubMed Scopus (432) Google Scholar). Although CD68 shares a number of these features, more recent studies have called into question its role in the recognition and uptake of Ox-LDL (14de Beer M.C. Zhao Z. Webb N.R. van der Westhuyzen D.R. de Villiers W.J. Lack of a direct role for macrosialin in oxidized LDL metabolism.J. Lipid Res. 2003; 44: 674-685Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Rather, they raised the possibility that CD68 may regulate the response to ligands after internalization. To more carefully explore the potential role CD68 plays in the ability of macrophages to either take up or direct an innate response to Ox-LDL and microbial ligands, knockout mice were generated. Analysis of CD68−/− mononuclear phagocytes failed to support an important role in the uptake of either Ox-LDL, microbes, or an associated inflammatory response. However, the enhanced capacity of these knockout cells to present antigen to CD4+ T-cells raises the possibility that CD68 may negatively regulate major histocompatibility complex class II (MHC-II) activity. CD68 gene targeting vector was generated by PCR amplification (Expanded Long Template PCR System; Roche, Indianapolis, IN) of a 1.6 kb short arm and 6.5 kb long arm from C57Bl6/J mice genomic DNA, as illustrated in Fig. 2, with primers: 5′ T GC GGC CGC CCC TAT GCT GAA ACC CAA GTG TTC C 3′ and 5′ C TC TAG ACA TAT G AG AGA TGC TCA GAC CAG CTA GGC 3′ for the short arm and 5′ CTC GAG CGG CTC CCT GTG TGT CTG ATC TTG C 3′ and 5′ GCG GCC GC A GGA CAG GGG CTA CAC AGA AAC CC 3′ for the long arm (Not I, Xba I, Nde I, Xho I, and Not I endonuclease restriction sites are underlined and were included for cloning; New England Biolab, MA). A loxP-flanked neomycin cassette was amplified from pMC1neo Poly A plasmid (Stratagene, San Diego, CA) with primers: 5′ CTC GAG ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA TGA ACA AAC GAC CCA ACA CCC GTG C 3′ and 5′ GTC GAC ATA ACT TCG TAT AAT GTA TGC TAT ACG AAG TTA TGC AGT GTG GTT TTG CAA GAG GAA GC 3′ (Xho I and Sal I enoduclease restriction sites are underlined) and cloned, inverted, downstream from an rtTA cassette (15Malleret G. Haditsch U. Genoux D. Jones M.W. Bliss T.V. Vanhoose A.M. Weitlauf C. Kandel E.R. Winder D.G. Mansuy I.M. Inducible and reversible enhancement of learning, memory, and long-term potentiation by genetic inhibition of calcineurin.Cell. 2001; 104: 675-686Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). This unit was then cloned immediately downstream and in frame with the CD68 initiation ATG located at the 3′ end of the short arm. The targeting construct was assembled in a vector provided by Dr. V. Lin that features a negative selectable diphtheria toxin (DT) gene (16Srinivas S. Watanabe T. Lin C.S. William C.M. Tanabe Y. Jessell T.M. Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.BMC Dev. Biol. 2001; 1: 4Crossref PubMed Scopus (2340) Google Scholar). The integrity of the targeting construct was confirmed by sequencing prior to electroporation into 129 R1 ES cells (17Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.Proc. Natl. Acad. Sci. USA. 1993; 90: 8424-8428Crossref PubMed Scopus (1992) Google Scholar). Neomycin resistant clones were evaluated by Southern blotting with a probe localized to the 5′ end of the short arm (see Fig. 2). Properly targeted CD68 knockout ES cells (2 of 192 neomycin-positive clones) were introduced into blastocyts to generate chimeric mice, as previously reported (18Park C. Li S. Cha E. Schindler C. Immune response in Stat2 knockout mice.Immunity. 2000; 13: 795-804Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Chimeric mice were either crossed with 129 mice or backcrossed seven generations to the C57Bl/6J background. Transmission of the knockout allele was determined by Southern blot and PCR. Columbia University review committees approved all relevant studies. Resident peritoneal macrophages were harvested by lavage with 10 ml RPMI 1640 (Invitrogen-GIBCO, Grand Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT) from freshly euthanized mice. Recovered cells were plated (1 × 106 cells/ml), and nonadherent cells were removed after 2 h by aspiration. Peritoneal-elicited cells (PEC; i.e., macrophages) were recovered by the same procedure four days after instillation of 1 ml of 4% thioglycollate (DIFCO; Voigt Global Dist., Inc, Lawrence, KS). Bone marrow-derived macrophages (BMMs) were prepared in RPMI 1640 supplemented with 10% FCS, penicillin/streptomycin (GIBCO), and 20% L-cell conditioned media, as previously reported (19Zhao W. Cha E.N. Lee C. Park C.Y. Schindler C. Stat2-dependent regulation of MHC class II expression.J. Immunol. 2007; 179: 463-471Crossref PubMed Scopus (38) Google Scholar). Bone marrow-derived dendritic cells (BMDC) were prepared similarly, except that they were grown in RPMI 1640 supplemented with a 1/30 dilution of GM-CSF conditioned media (from J558 cells), 5% FCS, pen/strep, and β-mercaptoethenol (GIBCO), as previously described (20Melillo J.A. Song L. Bhagat G. Blazquez A.B. Plumlee C.R. Lee C. Berin C. Reizis B. Schindler C. Dendritic cell (DC)-specific targeting reveals Stat3 as a negative regulator of DC function.J. Immunol. 2010; 184: 2638-2645Crossref PubMed Scopus (159) Google Scholar). Cells were fed gently with fresh media on days 3 and 5. On day 7, loosely adherent clustering dendritic cells (DC) were recovered (∼75% CD11c+) and replated for an additional two or three days prior to analysis. Phagocytic activity was measured by incubating macrophages with either heat-killed FITC-labeled S. aureus as previously described (21Chang D.H. Angelin-Duclos C. Calame K. BLIMP-1: trigger for differentiation of myeloid lineage.Nat. Immunol. 2000; 1: 169-176Crossref PubMed Scopus (130) Google Scholar); or LDL (Intracel Resources, Frederick, MD) that had been moderately oxidized with 40 μM CuSO4 as previously reported (22Hendriks W.L. van der Boom H. van Vark L.C. Havekes L.M. Lipoprotein lipase stimulates the binding and uptake of moderately oxidized low-density lipoprotein by J774 macrophages.Biochem. J. 1996; 314: 563-568Crossref PubMed Scopus (49) Google Scholar). Lipid uptake was measured by flow cytometry of live cells by gating on DiI (1,1′-dioetadeeyl-3,3,3′,3′-tetramethylindocarboeyanine perchlorate; Sigma-Aldrich, St. Louis, MO) labeled Ox-LDL, or in fixed PECs by staining with oil red O, as previously reported (22Hendriks W.L. van der Boom H. van Vark L.C. Havekes L.M. Lipoprotein lipase stimulates the binding and uptake of moderately oxidized low-density lipoprotein by J774 macrophages.Biochem. J. 1996; 314: 563-568Crossref PubMed Scopus (49) Google Scholar, 23Song L. Schindler C. IL-6 and the acute phase response in murine atherosclerosis.Atherosclerosis. 2004; 177: 43-51Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). PECs were also stained with antibodies specific for CD68 (1:100, 2 h; MAC1957, FA11; Serotec, Oxford, UK) and LAMP-1 (Southern Biotech, Birmingham, AL) after samples had been fixed (4% paraformaldehyde, methanol; 20 min) and blocked (2% goat serum) as previously reported (24Song L. Bhattacharya S. Yunus A.A. Lima C.D. Schindler C. Stat1 and SUMO modification.Blood. 2006; 108: 3237-3244Crossref PubMed Scopus (36) Google Scholar, 25Bhattacharya S. Schindler C. Regulation of Stat3 nuclear export.J. Clin. Invest. 2003; 111: 553-559Crossref PubMed Scopus (116) Google Scholar). Antibodies were detected with anti-rat IgG-FITC (1:100, 1 h; STAR69, Serotec) or anti-rat IgG-Cy3 (1:500, 1 h; Jackson ImmunoResearch, West Grove, PA) or anti-rat IgG-HRP (Vector Labs, Burlingame, CA) and visualized with a DAB substrate kit from Vectastain (Vector Labs) (23Song L. Schindler C. IL-6 and the acute phase response in murine atherosclerosis.Atherosclerosis. 2004; 177: 43-51Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Images were captured with a Nikon Eclipse TE300 microscope on a Spot camera (Sterling Heights, MI) and analyzed with OpenLabs (Waltham, MA) software. The ability of macrophages to generate nitric oxide (NO) in response to treatment with lipopolysaccharide (LPS) (100 ng/ml; from E. coli serotype 055:B5; Sigma-Aldrich, St. Louis, MO) and/or IFN-γ (50 U/ml; murine; PBL, Piscataway, NJ) was carried out as previously reported (24Song L. Bhattacharya S. Yunus A.A. Lima C.D. Schindler C. Stat1 and SUMO modification.Blood. 2006; 108: 3237-3244Crossref PubMed Scopus (36) Google Scholar). Growth of Legionella pneumophila, a facultative intracellular bacterium, was evaluated by infecting BMMs (0.5 × 106 per well in a 12-well plate, in triplicate) with 3,000 colony-forming units (CFU) of postlogarithmic growth bacteria, as recently reported (26Plumlee C.R. Lee C. Beg A.A. Decker T. Shuman H.A. Schindler C. Interferons direct an effective innate response to Legionella pneumophila infection.J. Biol. Chem. 2009; 284: 30058-30066Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The capacity of macrophages to promote viral growth was evaluated by infecting BMMs with vesicular stomatitis virus (VSV; Indiana strain) at multiple multiplicities of infections (MOI) and titering the supernatant for plaque-forming units (PFU) on Vero cells, as previously reported (24Song L. Bhattacharya S. Yunus A.A. Lima C.D. Schindler C. Stat1 and SUMO modification.Blood. 2006; 108: 3237-3244Crossref PubMed Scopus (36) Google Scholar). Fluorescence activated cell sorting (FACS) analysis was carried out on cells harvested from euthanized mice. Broncho-alveolar lavage (BAL) fluid was recovered in three sequential 1 ml (PBS) lung lavages, as reported (27Jewell N.A. Cline T. Mertz S.E. Smirnov S.V. Flano E. Schindler C. Grieves J.L. Durbin R.K. Kotenko S.V. Durbin J.E. Lambda interferon is the predominant interferon induced by influenza A virus infection in vivo.J. Virol. 2010; 84: 11515-11522Crossref PubMed Scopus (203) Google Scholar, 28Martin F.J. Gomez M.I. Wetzel D.M. Memmi G. O'Seaghdha M. Soong G. Schindler C. Prince A. Staphylococcus aureus activates type I IFN signaling in mice and humans through the Xr repeated sequences of protein A.J. Clin. Invest. 2009; 119: 1931-1939PubMed Google Scholar). Bone marrow (BM) and peritoneal macrophages were prepared as described above. Additional leukocytes were harvested from femurs (BM cells) and spleen (splenocytes), or blood. Single-cell suspensions were RBC-depleted (Sigma), resuspended in RPMI 1640/10% FCS, and then counted and/or stained for FACS. Adherent cells (BMMs) were treated for 5 min with trypsin/EDTA (Sigma-Aldrich), whereas BMDCs were collected by gentle pipetting prior to staining for FACS. Cells were stained with anti-CD11b (M1/70), F4/80 (BM8), Gr-1 (RB6-8C5), CD8a (53-6.7), CD206 (48-2069) from eBioscience (San Diego, CA) or anti-CD11c (HL3), CD3e (145-2C11), CD4 (RM4-5), I-Ab (MHC-II; AF6-120.1), CD86 (GL1), NK1.1 (PK136) from BD Pharmingen (San Diego, CA) or anti-CD68 (MAC1957; FA11) and CD206 (MR5D3) from Serotec (Oxford, UK), after treatment with Fc-blocker (anti-CD16/CD32, 2.4G2; BD-Pharmingen). Cells were fixed and stained with CD68 antibodies as per manufacturer (Serotec). Data were acquired on a FACS Calibur or FACS LSR-II (Beckton Dickinson, Franklin Lakes, NJ) and analyzed by FlowJo software (TreeStar, Ashland, OR). Cultured macrophages or DCs were treated with LPS (100 or 1,000 ng/ml; Sigma), IFN-γ (50 U/ml; murine; PBL, Piscataway, NJ), interleukin (IL)-4 (10 ng/ml; Peprotech, Rocky Hill, NJ), zymosan (100 µg/ml; Sigma) or silicates (0.4 mg/ml; see below). Supernatants were collected after 24 h and evaluated by ELISA for tumor necrosis factor (TNF)-α and IL-6 production (eBioscience) or IL-1β production (Pierce, Rockford, IL). Quartz (Min-U-Sil 5) was a gift from US Silica (Berkeley Springs, WV) and featured >98% purity, an average size of 1.83 µm (range 1.70-1.89 µm), and no amorphous coating (29Porter D.W. Millecchia L.L. Willard P. Robinson V.A. Ramsey D. McLaurin J. Khan A. Brumbaugh K. Beighley C.M. Teass A. et al.Nitric oxide and reactive oxygen species production causes progressive damage in rats after cessation of silica inhalation.Toxicol. Sci. 2006; 90: 188-197Crossref PubMed Scopus (64) Google Scholar, 30Cassel S.L. Eisenbarth S.C. Iyer S.S. Sadler J.J. Colegio O.R. Tephly L.A. Carter A.B. Rothman P.B. Flavell R.A. Sutterwala F.S. The Nalp3 inflammasome is essential for the development of silicosis.Proc. Natl. Acad. Sci. USA. 2008; 105: 9035-9040Crossref PubMed Scopus (691) Google Scholar). Tridymite (99% purity, 0.5-1 µm, no amorphous coating) was a generous gift from B. Pernis (31Pernis B. Paronetto F. Adjuvant effect of silica (tridymite) on antibody production.Proc. Soc. Exp. Biol. Med. 1962; 110: 390-392Crossref PubMed Scopus (75) Google Scholar, 32Pernis B. Silica and the immune system.Acta Biomed. 2005; 76: 38-44PubMed Google Scholar). Silicates, which were baked at 250°C for 16 h prior to suspension in PBS, were sonicated prior to stimulation and negative for endotoxin contamination (Limulus assay; Cambrex Bio Science, Walkersville, MD). An amount of 3 × 105 ovalbumin (OVA)-specific CD4+ T-cells (anti-CD4 MACS beads; Miltenyi Biotech, Auburn, CA) from OT-II TCR transgenic spleens were cultured for four days with 3 × 104 BMDCs, which had previously been loaded for 4 h with increasing concentrations of OVA (Worthington Biochemical Corporation, Lakewood, NJ) or OVA peptide 326-332 (New England Peptide, Inc., Gardner, MA). T-cell proliferation was measured by 3H-thymidine incorporation (14 h, 37°C; Perkin Elmer Life Sciences, Shelton, CT), as previously reported (20Melillo J.A. Song L. Bhagat G. Blazquez A.B. Plumlee C.R. Lee C. Berin C. Reizis B. Schindler C. Dendritic cell (DC)-specific targeting reveals Stat3 as a negative regulator of DC function.J. Immunol. 2010; 184: 2638-2645Crossref PubMed Scopus (159) Google Scholar). DNA or RNA was recovered from FACS-purified leukocyte populations with Trizol (Invitrogen). RNA was reverse transcribed with SuperScript™ II (Invitrogen), as previously reported (19Zhao W. Cha E.N. Lee C. Park C.Y. Schindler C. Stat2-dependent regulation of MHC class II expression.J. Immunol. 2007; 179: 463-471Crossref PubMed Scopus (38) Google Scholar, 24Song L. Bhattacharya S. Yunus A.A. Lima C.D. Schindler C. Stat1 and SUMO modification.Blood. 2006; 108: 3237-3244Crossref PubMed Scopus (36) Google Scholar). DNA and cDNA were either PCR amplified by standard or quantitative methods (ABI Prism 7700 with SYBR green master mix; Applied Biosystems, Foster City, CA). Gene expression was normalized to a β-actin or 18S rRNA control. Ct values and standard curves were generated by plotting log of DNA concentration versus Ct values from 1:5 serial dilutions with SDS1.9.1 software (Applied Biosystems). Primers were annealed at 60C and run 40 cycles. Primers sets included: Arginase-1 (forward) 5′ aga cca cag tct ggc agt tg, (reverse) 5′ cca ccc aaa tga cac ata gg; 18S (forward) 5′ tca aga acg aaa gtc gga gg 3′, (reverse) 5′ gga cat cta agg gca tca ca 3′; eif4a1 (forward) 5′ gga agg cgt cat cga gag taa ctg, (reverse) 5′ ccc aat gca ggc atg aca aag agg; mupd.1 (forward) 5′ gcg ctg ttc ctg aca ctc cag acc, (reverse) 5′ cca gta gaa gag gac ctg ggc agc. Although predominately restricted to endosomes, CD68 expression has been largely associated with myeloid cells from the macrophage lineage (1Taylor P.R. Martinez-Pomares L. Stacey M. Lin H.H. Brown G.D. Gordon S. Macrophage receptors and immune recognition.Annu. Rev. Immunol. 2005; 23: 901-944Crossref PubMed Scopus (1002) Google Scholar). Consistent with previous reports, CD68 staining was strongest after membrane permeablization, and it was largely localized to LAMP1-positive vesicles (supplementary Fig. I) (1Taylor P.R. Martinez-Pomares L. Stacey M. Lin H.H. Brown G.D. Gordon S. Macrophage receptors and immune recognition.Annu. Rev. Immunol. 2005; 23: 901-944Crossref PubMed Scopus (1002) Google Scholar). When primary murine myeloid cells were evaluated, strong CD68 expression was observed in several macrophage lineages, including alveolar, splenic, BM, and peritoneal macrophages, as well as BM culture-derived macrophages (see Fig. 1) (1Taylor P.R. Martinez-Pomares L. Stacey M. Lin H.H. Brown G.D. Gordon S. Macrophage receptors and immune recognition.Annu. Rev. Immunol. 2005; 23: 901-944Crossref PubMed Scopus (1002) Google Scholar, 33Landsman L. Jung S. Lung macrophages serve as obligatory intermediate between blood monocytes and alveolar macrophages.J. Immunol. 2007; 179: 3488-3494Crossref PubMed Scopus (202) Google Scholar). Likewise, robust CD68 expression was observed in several DC populations, including pulmonary- and splenic-CD8α+ DCs and BM culture-derived DCs, but not in CD8α- DCs (Fig. 1). As anticipated, CD68 was not detected on B220+ or CD3+ lymphocytes (not shown). To explore the potential role CD68 plays in ligand uptake, its gene was targeted for deletion by a recombination vector featuring two homology arms and a neomycin cassette (see Fig. 2). Chimeric mice with a targeted allele were then either crossed with 129 or C57Bl/6 mice and then interbred to homozygosity. Progeny CD68 knockout (CD68−/−) mice developed with normal Mendelian genetics in both backgrounds (e.g., Fig. 2B; data not shown); they exhibited no obvious phenotypes, including no evidence of organomegally and no significant perturbations of defined leukocyte populations in either the blood or spleen (Tables 1 and 2; supplementary Fig. II). Western blotting, immunohistochemical studies, and FACS analysis confirmed an absence of CD68 expression in macrophages from CD68 knockout mice (Fig. 2C–E). CD68+/− (i.e., heterozygous) mice exhibited a corresponding partial decrease in CD68 expression (Fig. 2C). Given the relatively high density of genes at the CD68 locus, it was important to confirm that the expression of both eif4a1 and mpdu.1 (upstream and downstream flanking genes; see Fig. 2) was not affected by the introduction of a neomycin cassette into the CD68 locus (supplementary Fig. III). In summary, these studies revealed that homozygous deletion of the CD68 gene was not associated with any significant developmental or homeostatic defects.TABLE 1Leukocyte subtypes in CD68−/− bloodB220CD3CD11b ; Gr-1CD11b ; Gr-1CD11b ; Gr-1wt44.9 ± 3.927.2 ± 3.94.4 ± 2.36.8 ± 0.42.3 ± 0.1het41.4 ± 3.826.8 ± 4.83.9 ± 0.67.5 ± 0.62.2 ± 0.1ko44.1 ± 5.520.4 ± 1.78.1 ± 3.26.3 ± 0.82.4 ± 0.5Peripheral blood from three-month-old matched wild-type (wt), heterozygous (het), and CD68−/− (ko) mice that were backcrossed to C57Bl/6J mice six times, was stained for B220 (B lymphocytes), CD3 (T lymphocytes), CD11b (macrophages), and Gr-1 (granulocytes/monocytes)and analyzed by FACS. Mean and standard deviation of the percentage of positive cells are shown. Open table in a new tab TABLE 2Leukocyte distribution in CD68−/− spleenB220CD3CD11bCD11cwt61.1 ± 1.725.1 ± 3.24.3 ± 3.92.3 ± 1.8het58.3 ± 5.025.4 ± 2.44.3 ± 2.32.4 ± 1.0ko59.8 ± 1.725.5 ± 2.73.8 ± 1.21.8 ± 0.7Splenocytes from three-month-old matched wild-type (wt), heterozygous (het), and CD68−/− (ko) mice (as in Table 1) were stained for B220 (B lymphocytes), CD3 (T lymphocytes), CD11b (macrophages), and CD11c (dendritic cells) and analyzed by FACS. Mean and standard deviation of the percentage of positive cells are shown. Open table in a new tab Peripheral blood from three-month-old matched wild-type (wt), heterozygous (het), and CD68−/− (ko) mice that were backcrossed to C57Bl/6J mice six times, was stained for B220 (B lymphocytes), CD3 (T lymphocytes), CD11b (macrophages), and Gr-1 (granulocytes/monocytes)and analyzed by FACS. Mean and standard deviation of the percentage of positive cells are shown. Splenocytes from three-month-old matched wild-type (wt), heterozygous (het), and CD68−/− (ko) mice (as in Table 1) were stained for B220 (B lymphocytes), CD3 (T lymphocytes), CD11b (macrophages), and CD11c (dendritic cells) and analyzed by FACS. Mean and standard deviation of the percentage of positive cells are shown. Shortly after its initial characterization (3Holness C.L. da Silva R.P. Fawcett J. Gordon S. Simmons D.L. Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family.J. Biol. Chem. 1993; 268: 9661-9666Abstract Full Text PDF PubMed Google Scholar, 34Rabinowitz S.S. Gordon S. Macrosialin, a macrophage-restricted membrane sialoprotein differentially glycosylated in response to inflammatory stimuli.J. Exp. Med. 1991; 174: 827-836Crossref PubMed Scopus (196) Google Scholar), CD68 was identified as
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