Type I Interferon Production by Tertiary Lymphoid Tissue Developing in Response to 2,6,10,14-Tetramethyl-Pentadecane (Pristane)
2006; Elsevier BV; Volume: 168; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.050125
ISSN1525-2191
AutoresDina C. Nacionales, Kindra M. Kelly, Pui Y. Lee, Haoyang Zhuang, Yi Li, Jason S. Weinstein, Eric S. Sobel, Yoshiki Kuroda, Jun Akaogi, Minoru Satoh, Westley H. Reeves,
Tópico(s)Immune Cell Function and Interaction
ResumoLymphoid neogenesis is associated with antibody-mediated autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. Although systemic lupus erythematosus is the prototypical B-cell-mediated autoimmune disease, the role of lymphoid neogenesis in its pathogenesis is unknown. Intraperitoneal injection of 2,6,10,14-tetramethyl-pentadecane (TMPD, pristane) or mineral oil causes lipogranuloma formation in mice, but only TMPD-treated mice develop lupus. We report that lipogranulomas are a form of lymphoid neogenesis. Immunoperoxidase staining of lipogranulomas revealed B cells, CD4+ T cells, and dendritic cells and in some cases organization into T- and B-cell zones. Lipogranulomas also expressed the lymphoid chemokines CCL21, CCL19, CXCL13, CXCL12, and CCL22. Expression of the type I interferon (IFN-I)-inducible genes Mx1, IRF7, IP-10, and ISG-15 was greatly increased in TMPD- versus mineral oil-induced lipogranulomas. Dendritic cells from TMPD lipogranulomas underwent activation/maturation with high CD86 and interleukin-12 expression. Magnetic bead depletion of dendritic cells markedly diminished IFN-inducible gene (Mx1) expression. We conclude that TMPD-induced lupus is associated with the formation of ectopic lymphoid tissue containing activated dendritic cells producing IFN-I and interleukin-12. In view of the increased IFN-I production in systemic lupus erythematosus, these studies suggest that IFN-I from ectopic lymphoid tissue could play a role in the pathogenesis of experimental lupus in mice. Lymphoid neogenesis is associated with antibody-mediated autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. Although systemic lupus erythematosus is the prototypical B-cell-mediated autoimmune disease, the role of lymphoid neogenesis in its pathogenesis is unknown. Intraperitoneal injection of 2,6,10,14-tetramethyl-pentadecane (TMPD, pristane) or mineral oil causes lipogranuloma formation in mice, but only TMPD-treated mice develop lupus. We report that lipogranulomas are a form of lymphoid neogenesis. Immunoperoxidase staining of lipogranulomas revealed B cells, CD4+ T cells, and dendritic cells and in some cases organization into T- and B-cell zones. Lipogranulomas also expressed the lymphoid chemokines CCL21, CCL19, CXCL13, CXCL12, and CCL22. Expression of the type I interferon (IFN-I)-inducible genes Mx1, IRF7, IP-10, and ISG-15 was greatly increased in TMPD- versus mineral oil-induced lipogranulomas. Dendritic cells from TMPD lipogranulomas underwent activation/maturation with high CD86 and interleukin-12 expression. Magnetic bead depletion of dendritic cells markedly diminished IFN-inducible gene (Mx1) expression. We conclude that TMPD-induced lupus is associated with the formation of ectopic lymphoid tissue containing activated dendritic cells producing IFN-I and interleukin-12. In view of the increased IFN-I production in systemic lupus erythematosus, these studies suggest that IFN-I from ectopic lymphoid tissue could play a role in the pathogenesis of experimental lupus in mice. Chronic inflammation can lead to the formation of ectopic lymphoid tissue, a process known as “lymphoid neogenesis.”1Kratz A Campos-Neto A Hanson MS Ruddle NH Chronic inflammation caused by lymphotoxin is lymphoid neogenesis.J Exp Med. 1996; 183: 1461-1472Crossref PubMed Scopus (348) Google Scholar The development of this tertiary lymphoid tissue partially recapitulates normal lymph node development, which depends in part on lymphotoxin α as well as B-cell (CXCL13) and T-cell (CCL21 and CCL19) attractant chemokines.2Drayton DL Ying X Lee J Lesslauer W Ruddle NH Ectopic LT alpha beta directs lymphoid organ neogenesis with concomitant expression of peripheral node addressin and a HEV-restricted sulfotransferase.J Exp Med. 2003; 197: 1153-1163Crossref PubMed Scopus (196) Google Scholar, 3Luther SA Lopez T Bai W Hanahan D Cyster JG BLC expression in pancreatic islets causes B cell recruitment and lymphotoxin-dependent lymphoid neogenesis.Immunity. 2000; 12: 471-481Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 4Yoneyama H Matsuno K Zhang Y Murai M Itakura M Ishikawa S Hasegawa G Naito M Asakura H Matsushima K Regulation by chemokines of circulating dendritic cell precursors, and the formation of portal tract-associated lymphoid tissue, in a granulomatous liver disease.J Exp Med. 2001; 193: 35-49Crossref PubMed Scopus (189) Google Scholar, 5Hjelmstrom P Fjell J Nakagawa T Sacca R Cuff CA Ruddle NH Lymphoid tissue homing chemokines are expressed in chronic inflammation.Am J Pathol. 2000; 156: 1133-1138Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar Lymphoid neogenesis is associated with humoral autoimmunity in a variety of situations6Hjelmstrom P Lymphoid neogenesis: de novo formation of lymphoid tissue in chronic inflammation through expression of homing chemokines.J Leukoc Biol. 2001; 69: 331-339Crossref PubMed Scopus (209) Google Scholar: the thyroid gland in Hashimoto's thyroiditis, thymus in myasthenia gravis, nervous system in multiple sclerosis, salivary glands in Sjogren's syndrome, and synovium in rheumatoid arthritis.7Stott DI Hiepe F Hummel M Steinhauser G Berek C Antigen-driven clonal proliferation of B cells within the target tissue of an autoimmune disease. 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We have shown previously that chronic inflammation induced by the hydrocarbon oil 2,6,10,14-tetramethyl-pentadecane (TMPD) causes autoantibodies and clinical manifestations closely resembling systemic lupus erythematosus (SLE) in most nonautoimmune-prone mouse strains.11Satoh M Reeves WH Induction of lupus-associated autoantibodies in BALB/c mice by intraperitoneal injection of pristane.J Exp Med. 1994; 180: 2341-2346Crossref PubMed Scopus (248) Google Scholar, 12Satoh M Kumar A Kanwar YS Reeves WH Antinuclear antibody production and immune complex glomerulonephritis in BALB/c mice treated with pristane.Proc Natl Acad Sci USA. 1995; 92: 10934-10938Crossref PubMed Scopus (171) Google Scholar BALB/c mice injected intraperitoneally with TMPD develop high levels of the lupus-related autoantibodies against double-stranded DNA, Sm, RNP, and Su11Satoh M Reeves WH Induction of lupus-associated autoantibodies in BALB/c mice by intraperitoneal injection of pristane.J Exp Med. 1994; 180: 2341-2346Crossref PubMed Scopus (248) Google Scholar along with immune complex-mediated glomerulonephritis12Satoh M Kumar A Kanwar YS Reeves WH Antinuclear antibody production and immune complex glomerulonephritis in BALB/c mice treated with pristane.Proc Natl Acad Sci USA. 1995; 92: 10934-10938Crossref PubMed Scopus (171) Google Scholar and arthritis.13Potter M Wax JS Genetics of susceptibility to pristane-induced plasmacytomas in BALB/cAn: reduced susceptibility in BALB/cJ with a brief description of pristane-induced arthritis.J Immunol. 1981; 127: 1591-1595PubMed Google Scholar, 14Wooley PH Seibold JR Whalen JD Chapdelaine JM Pristane-induced arthritis. The immunologic and genetic features of an experimental murine model of autoimmune disease.Arthritis Rheum. 1989; 32: 1022-1030Crossref PubMed Scopus (117) Google Scholar, 15Bigazzi PE Murine lupus induced by tetramethylpentadecane: an animal model of systemic human autoimmunity.Clin Immunol. 2005; 114: 97-99Crossref PubMed Scopus (5) Google Scholar In contrast, medicinal mineral oil (a complex hydrocarbon mixture) does not induce these autoantibodies nor does it cause renal or joint disease.16Satoh M Kuroda Y Yoshida H Behney KM Mizutani A Akaogi J Nacionales DC Lorenson TD Rosenbauer RJ Reeves WH Induction of lupus autoantibodies by adjuvants.J Autoimmun. 2003; 21: 1-9Crossref PubMed Scopus (86) Google Scholar In the present study, we show that TMPD-induced chronic inflammation leads to lymphoid neogenesis. Dendritic cells present in the tertiary lymphoid tissue produced high levels of type I interferons (IFN-I), which are implicated in the pathogenesis of SLE.17Baechler EC Batliwalla FM Karypis G Gaffney PM Ortmann WA Espe KJ Shark KB Grande WJ Hughes KM Kapur V Gregersen PK Behrens TW Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.Proc Natl Acad Sci USA. 2003; 100: 2610-2615Crossref PubMed Scopus (1750) Google Scholar, 18Bennett L Palucka AK Arce E Cantrell V Borvak J Banchereau J Pascual V Interferon and granulopoiesis signatures in systemic lupus erythematosus blood.J Exp Med. 2003; 197: 711-723Crossref PubMed Scopus (1603) Google Scholar In contrast, there was little IFN-I production in inflammatory tissues elicited by mineral oil injection. This study provides evidence for a link between chronic inflammation, lymphoid neogenesis, and the pathogenesis of SLE. Four-week-old female BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME) and housed in a conventional facility in barrier cages. At 3 months of age, they received a single intraperitoneal injection (0.5 ml) of either TMPD (Sigma-Aldrich, St. Louis, MO) or medicinal mineral oil (Harris Teeter, Mathews, NC). Peritoneal cells, granulomas, spleen, and blood were harvested 12 to 20 weeks later. These studies were approved by the Institutional Animal Care and Use Committee. The peritoneal cavity was lavaged with 5 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 10 mmol/L HEPES, glutamine, and penicillin/streptomycin (complete Dulbecco's modified Eagle's medium) plus 10 U/ml heparin. Cells were collected by centrifugation, and supernatants were frozen at −80°C until analysis. After lysing erythrocytes in 0.15 mol/L NH4Cl, 10 mmol/L KHCO3, and 2 mmol/L ethylenediaminetetraacetic acid, the peritoneal cells were resuspended in complete medium. Peritoneal CD11c+ dendritic cells, CD11b+ macrophages/monocytes, CD19+ B cells, and CD3+ T cells were positively selected using magnetic beads (Miltenyi Biotech, Auburn, CA) following the manufacturer's instructions. Cells were usually >95% positive for each population by flow cytometry. In some experiments, CD11c+ cells were depleted using anti-CD11c microbeads. Untreated and CD11c-depleted peritoneal cells were cultured in 12-well culture dishes either without stimulation or with lipopolysaccharide (LPS, from Salmonella minnesota, 100 ng or 10 μg/ml; Sigma-Aldrich), polyinosinic-polycytidylic acid (poly IC, 50 μg/ml; Sigma-Aldrich), or CpG oligodeoxyribonucleotide (ODN) no. 1826 (10 μg/ml; Coley Pharmaceutical Group Inc., Wellesley, MA). RNA was extracted 4 hours later using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). Individual and pooled lipogranulomas were picked from the peritoneal lining of mice treated with TMPD or mineral oil 12 to 20 weeks earlier, and RNA was extracted using TRIzol reagent. Single cell suspensions for flow cytometry were prepared by digesting pooled lipogranulomas with collagenase (2 mg/ml) and dispase (1 mg/ml) (Invitrogen Life Technologies) and shaking at 200 rpm at 37°C for 30 minutes. The cells were washed three times with phosphate-buffered saline (PBS) and 2 mmol/L ethylenediaminetetraacetic acid, filtered through a 70-μm pore-size cell strainer, and resuspended in flow cytometry buffer. In some experiments, RAW 264.7 cells (mouse monocyte-macrophage cell line; American Type Culture Collection, Bethesda, MD) were incubated in vitro with cytokines. Cells were grown in complete Dulbecco's modified Eagle's medium and incubated at 37°C in a 5% CO2 atmosphere, resuspended at 2 × 106/ml in complete Dulbecco's modified Eagle's medium and plated overnight in 12-well culture dishes. The cells were cultured an additional 24 hours either without stimulation or with LPS (1 ng/ml, 10 ng/ml, 100 ng/ml, 1 μg/ml, or 10 μg/ml), poly(I:C) (50 μg/ml), with IFN-α (500 to 1000 U/ml) ± anti-IFN-α neutralizing antibody (1 to 2 μg/ml), or IFN-β (500 to 1000 U/ml) (all from PBL Biomedical Laboratories, Piscataway, NJ), or with interleukin (IL)-12 (10 to 20 ng/ml), tumor necrosis factor (TNF)-α (20 ng/ml), or IL-6 (5 ng/ml) (all from BD Biosciences, San Jose, CA). Cells were harvested at specified time points for RNA extraction using TRIzol. Total RNA was precipitated with isopropanol and the pellet washed with cold 75% (v/v) ethanol and resuspended in diethyl pyrocarbonate-treated water. One μg of RNA was treated with DNase I (Invitrogen) to remove genomic DNA and reverse transcribed to cDNA using Superscript first-strand synthesis system for RT-PCR (Invitrogen). One μl of cDNA was added to the PCR mixture containing 1× PCR buffer (2.5 mmol/L MgCl2, 400 μmol/L dNTPs), 0.025 U of TaqDNA polymerase (Invitrogen), 1 μmol/L each of forward and reverse primers, and diethyl pyrocarbonate-water in a 20-μl volume. Amplification was performed for 5 minutes at 94°C, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, extension at 72°C for 1 minute, and a final extension of 72°C for 10 minutes in a PTC-100 programmable thermal controller (MJ Research, Inc., Waltham, MA). PCR primers were synthesized by Invitrogen (Table 1).Table 1Primers Used for RT-PCRGeneForwardReversebpβ-Actin5′-TGGAATCCTGTGGCATCCTGAAAC-3′5′-TAAAACGCAGCTCAGTAACAGTCCG-3′348Mx1*IFN-I inducible gene.5′-GATCCGACTTCACTTCCAGATGG-3′5′-CATCTCAGTGGTAGTCAACCC-3′181IRF-7*IFN-I inducible gene.5′-TGCTGTTTGGAGACTGGCTAT-3′5′-TCCAAGCTCCCGGCTAAGT-3′359IP-10*IFN-I inducible gene.5′-ATCATCCCTGCGAGCCTAT-3′5′-ATTCTTGCTTCGGCAGTTAC-3′361ISG15*IFN-I inducible gene.5′-CAGAAGCAGACTCCTTAATTC-3′5′-AGACCTCATATATGTTGCTGTG-3′339CCL215′-ATGATGACTCTGAGCCTCC-3′5′-GAGCCCTTTCCTTTCTTTCC-3′346CCL195′-GCCTCAGATTATCTGCCAT-3′5′-AGACACAGGGCTCCTTCTGGT-3′340CCR75′-GAGAGACAAGAACCAAAAGCAC-3′5′-GGGAAGTAATTAGGAGGAAAAGG-3′394CXCL135′-ATGAGGCTCAGCACAGCAAC-3′5′-CCATTTGGCACGAGGATTCAC-3′245CXCR55′-ACTACCCACTAACCCTGGAC-3′5′-AGGTGATGTGGATGGAGAGGAG-3′409IL-12p405′-GAGTGGACTGGACTCCCGA-3′5′-CAAGTTCTTGGGCGGGTCTG-3′618IL-65′-CTGGTGACAACCACGGCCTTCCCT-3′5′-ATGCTTAGGCATAACGCACTAGGTT-3′600IL-45′-CGAAGAACACCACAGAGAGTGAGCT-3′5′-GACTCATTCATGGTGCAGCTTATCG-3′181IFN-γ5′-AGCGGCTGACTGAACTCAGATTGTA-3′5′-GTCACAGTTTTCAGCTGTATAGGG-3′24718S5′-CGGCTACCACATCCAAGGAA-3′5′-GCTGGAATTACCGCGGCT-3′198CCL225′-CCAGGACTACATCCGTCACC-3′5′-TGGCAGAAGAATAGGGCTTG-3′239CXCL125′-TGCTCTCTGCTTGCCTCCA-3′5′-GGTCCGTCAGGCTACAGAGGT-3′69* IFN-I inducible gene. Open table in a new tab Gene expression was quantified by real-time PCR. One μl of cDNA was added to a mixture containing 3.75 mmol/L MgCl2, 1.25 mmol/L dNTP mixture, 0.025 U of Amplitaq Gold, SYBR Green dye (Applied Biosystems, Foster City, CA), optimized concentrations of specific forward and reverse primers, and diethyl pyrocarbonate-treated water to a final volume of 20 μl. Amplification conditions were 95°C (10 minutes), followed by 45 cycles of 94°C (15 seconds), 60°C (25 seconds), 72°C (25 seconds), and a final extension at 72°C for 8 minutes using a DNA Engine Opticon 2 continuous fluorescence detector (MJ Research). Primers were synthesized by Invitrogen (Table 2). Transcripts were quantified using the comparative (2−ΔΔCt) method.Table 2Primers Used for Real-Time PCRGeneForwardReverseMx1*IFN-I inducible gene.5′-GATCCGACTTCACTTCCAGATGG-3′5′-CATCTCAGTGGTAGTCAACCC-3′ISG15*IFN-I inducible gene.5′-AGCGGAACAAGTCACGAAGAC-3′5′-TGGGGCTTTAGGCCATACTC-3′IP-10*IFN-I inducible gene.5′-CCTGCAGGATGATGGTCAAG-3′5′-GAATTCTTGCTTCGGCAGTT-3′IRF-7*IFN-I inducible gene.5′-TGCTGTTTGGAGACTGGCTAT-3′5′-TCCAAGCTCCCGGCTAAGT-3′TLR 35′-TCCGCCCTCTTCGTAACT TG-3′5′-TTGGCGGCTGGTAATCTTCT-3′TLR 45′-GAGGCAGCAGGTGGAATT GT-3′5′-TGCTCAGGATTCGAGGCTTT-3′TNFα5′-GGCAGGTCTACTTTGGAGTCATTGC-3′5′-ACATTCGAGGCTGCTCCAGTGAATTCGG-3′IL-45′-TCAACCCCCAGCTAGTTGTC-3′5′-TGTTCTTCGTTGCTGTGAGG-3′MyD885′-ACTGGCGGCCTGAGCAACTAGGA-3′5′-TGTCCCAAAGGAAACACACA-3′TRIF5′-CCTGTCAGCACGTTTTCTGT-3′5′-TCCGGACATGCTCTTTCTCT-3′TRAM5′-TGCAAACCATCAATGCCTTA-3′5′-TCAAATACAGACTCCCGGAAA-3′β-Actin5′-CCCACACTGTGCCCATCTAC-3′5′-CGCTCGGTCAGGATCTTCAT-3′* IFN-I inducible gene. Open table in a new tab The following labeled antibodies were used: anti-CD19-FITC or PE-Cy7; anti-CD8-PE; anti-CD4-PE-Cy5; anti-CD3-APC; anti-CD5-PE; anti-CD45R (B220)-PE-Cy5 or PerCP Cy5.5; anti-CD11b-APC; anti-I-Ad-FITC or PE; anti-CD11c-PE or APC; anti-CD86-biotin; and anti-CD40-biotin (all from BD Biosciences, San Jose, CA), anti-CD-11b-Pacific Blue (Caltag Laboratories, Burlingame, CA), and anti-PDCA-1-FITC (Miltenyi Biotec, Auburn, CA). For biotinylated reagents, avidin-PE-Cy5 was used (BD Biosciences). Biotinylated isotype controls were used to evaluate background fluorescence. Isolated peritoneal cells were washed in staining buffer (PBS supplemented with 0.1% NaN3 and 1% bovine serum albumin). After aliquotting 106 cells/well into 96-well round-bottom polystyrene plates (Costar, Corning, NY), the cells were preincubated for 10 minutes with 1 μg of anti-CD16 in 10 μl of staining buffer to block Fc binding. Primary antibodies were then added at pretitrated amounts and incubated for 20 minutes, followed by washing in staining buffer. After the addition of avidin-PE-Cy5 to appropriate wells, the cells were washed and resuspended in PBS supplemented with 1% paraformaldehyde. Data were acquired on a FACSCalibur (BD Biosciences) and analyzed with FCS Express Version 2 (DeNovo Software, Thornhill, ON, Canada). At least 20,000 events per sample were acquired and analyzed using size gating to exclude dead cells. Lipogranulomas were picked from the peritoneal wall and snap-frozen in OCT compound (Tissue-Tek; Sakura Fine-tek USA, Inc., Torrance, CA). Cryostat sections (5 μm) were collected on gelatin/chromium potassium sulfate-treated slides, fixed in cold acetone, and stored at −80°C. For staining, sections were air-dried at room temperature then quenched with 0.3% H2O2 for 10 minutes and blocked with 10% normal goat serum (Vector Laboratories, Burlingame, CA) for 30 minutes. Sections were then incubated with biotinylated anti-B220, -CD4, -CD11c, -CD11b (BD Biosciences), or peanut agglutinin (Vector) for 1 hour followed by incubation with horseradish peroxidase-streptavidin (Vector) for 30 minutes and aminoethylcarbazole. For peripheral lymph node addressin staining, sections were incubated with purified rat anti-mouse PNAd (MECA-79, BD Biosciences), and then with biotinylated mouse anti-rat IgM (BD Biosciences), followed by horseradish peroxidase-streptavidin (Vector). Sections were counterstained with Mayer's hematoxylin and dilute ammonia. Unfractionated peritoneal exudate cells, peritoneal cells depleted of CD11c+ cells, and the CD11c+ fraction were plated in 96-well plates (105 cells/well) and treated with LPS (10 μg/ml), CpG ODN no. 1826 (10 μg/ml), or poly (I:C) (50 μg/ml). Culture supernatants were collected 24 hours later and TNF-α and IL-12 ELISAs were performed using hamster monoclonal and rabbit polyclonal antibodies (TNF-α) or rat monoclonal antibody pairs (IL-12) from BD Biosciences. After incubation with biotinylated cytokine-specific antibodies, streptavidin-alkaline phosphatase (1:1000 dilution; Southern Biotechnology Associates, Birmingham, AL) was added for 30 minutes at 22°C, and the reaction was developed. Peritoneal washings obtained by lavage were assayed for IFN-β (antibodies from PBL Biomedical Laboratories, Piscataway, NJ), IFN-γ, IL-6, and IL-12 (antibodies from BD Biosciences) in the same manner. Optical density was converted to concentration based on standard curves produced using recombinant cytokines using a four-parameter logistic equation (Softmax Pro 3.1 software; Molecular Devices Corporation, Sunnyvale, CA). Both TMPD and mineral oil induce chronic inflammation when injected into the peritoneal cavity. Because the onset of IgG autoantibody production in TMPD-treated mice is between 12 and 20 weeks after intraperitoneal injection of the oil, we compared the peritoneal inflammatory responses to TMPD and mineral oil (a mixture of hydrocarbons that does not induce lupus in mice) at these time points (Figure 1, A–C). At 12 weeks, the inflammatory response to TMPD (and to a lesser extent mineral oil) was predominated by a striking influx of CD11b+ cells that did not bear B-cell (CD19, B220) or dendritic cell (CD11c) markers, and thus were probably monocytes or monocytes/macrophages (Figure 1, A–C; Table 3). At the same time, the number of B cells decreased in comparison with resident peritoneal cells from untreated mice (Figure 1, B and C). At 12 weeks, there were marked differences in CD19+/B220+ B cells (2.2% in TMPD-treated versus 27.6% in mineral oil-treated mice) and the number of I-Ad+ cells (6.6% TMPD-treated versus 32.4% mineral oil-treated mice). Representative flow cytometry data are shown in Figure 1D. Although the percentage of B1/macrophages by scatter was significantly higher in TMPD-treated mice, very few of these were B1 cells, which would be reflected in the I-Ad+ and CD11bint population (Figure 1D). Additional staining with CD19, CD5, and B220 confirmed that B1 cells were markedly decreased in TMPD-treated mice (data not shown). Dendritic cells (CD11c+) represented ∼5% of the peritoneal exudate cells both in TMPD- and in mineral oil-treated mice. Because of the higher peritoneal cell counts, the absolute numbers of peritoneal dendritic cells were greater in TMPD- versus mineral oil-treated mice at 12 weeks (Figure 1C). Normal resident peritoneal cells contained very few dendritic cells. The expression of CD86 on peritoneal DCs from TMPD- versus mineral oil-treated mice was comparable (Figure 1D).Table 3Absolute Cell Counts in Lipogranulomas and Peritoneal Exudate at 20 WeeksLipogranulomasPeritoneal exudateAbsolute cell count (×10−6)TMPDMineral oilTMPDMineral oilTotal cells7.5 ± 1.51.6 ± 0.215 ± 4.68.5 ± 0.1B cells CD19+, CD5−0.5 ± 0.1 (6.4%)0.08 ± 0.03 (4.5%)1.8 ± 0.8 (11.8%)1.2 ± 0.3 (13.2%) CD19+, CD5+0.01 ± 0.003 (0.1%)0.001 ± 0.0001 (0.07%)0.1 ± 0.006 (0.06%)0.01 ± 0.002 (0.1%)T cells CD3+1.4 ± 0.2 (19%)0.2 ± 0.1 (16%)3.9 ± 1.3 (26%)1.5 ± 0.8 (17%) CD3+, CD4+0.6 ± 0.08 (8%)0.1 ± 0.04 (8%)2.6 ± 0.8 (17%)1.0 ± 0.6 (11%) CD3+, CD4−*Because the CD8 antigen is partially sensitive to collagenase/dispase treatment, the CD4−, CD8+ and double-negative (CD4−, CD8−) subsets are combined and reported as CD3+, CD4−.0.8 (11%)0.1 (8%)1.3 (8%)0.5 (6%)Antigen-presenting cells CD11c+, CD11b−0.04 ± 0.005 (0.6%)0.02 ± 0.006 (1%)0.03 ± 0.01 (0.3%)0.04 ± 0.006 (0.6%) CD11c+, CD11b+0.4 ± 0.09 (5%)0.2 ± 0.06 (9%)0.09 ± 0.02 (0.6%)0.1 ± 0.01 (1%) CD11c−, CD11b+1.9 ± 0.5 (24%)0.6 ± 0.3 (20%)3.8 ± 1.8 (23%)2.9 ± 0.3 (34%)* Because the CD8 antigen is partially sensitive to collagenase/dispase treatment, the CD4−, CD8+ and double-negative (CD4−, CD8−) subsets are combined and reported as CD3+, CD4−. Open table in a new tab Between 12 and 20 weeks after injection of TMPD or mineral oil, the numbers of total B cells increased somewhat in both TMPD- and mineral oil-treated mice (Figure 1C, Table 3). T cell counts were greatly increased in TMPD- versus mineral oil-treated mice at 12 weeks, but did not increase further at 20 weeks (Figure 1C, Table 3). The absolute number of T cells in the peritoneal exudate of TMPD-treated mice was approximately twice that in mineral oil-treated mice; in both cases the ratio of CD4+/CD8+ cells was ∼2:1. Between 12 and 20 weeks, both the percentage and the absolute number of CD11c+ dendritic cells in the peritoneal exudate decreased in both TMPD- and mineral oil-treated mice, concomitant with the appearance of relatively large numbers of dendritic cells in the lipogranulomas (Figure 1, B and C; Table 3). These data suggest that intraperitoneal administration of TMPD stimulated an early influx of monocytes, dendritic cells, and T cells followed by an influx of B lymphocytes. TMPD was relatively more potent than mineral oil in attracting T lymphocytes into the peritoneal exudate and promoting the formation of lipogranulomas with large numbers of monocytes, dendritic cells, and T and B lymphocytes at 20 weeks (Table 3). Chronic inflammation induced by either TMPD or mineral oil causes the formation of lipogranulomas adherent to the mesothelial surfaces of the peritoneal lining.19Potter M MacCardle RC Histology of developing plasma cell neoplasia induced by mineral oil in BALB/c mice.J Natl Cancer Inst. 1964; 33: 497-515PubMed Google Scholar, 20Leak LV Potter M Mayfield WJ Response of the peritoneal mesothelium to the mineral oil, pristane.Curr Top Microbiol Immunol. 1985; 122: 221-233PubMed Google Scholar, 21Cruickshank B Thomas MJ Mineral oil (follicular) lipidosis: II. histologic studies of spleen, liver, lymph nodes, and bone marrow.Hum Pathol. 1984; 15: 731-737Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 22Dincsoy HP Weesner RE MacGee J Lipogranulomas in non-fatty human livers: a mineral oil induced environmental disease.Am J Clin Pathol. 1982; 78: 35-41PubMed Google Scholar However, relatively little is known about the organization of these structures. Lipogranulomas induced by TMPD (Figure 2A) were more compact and nodular than those induced by mineral oil (Figure 2B). Hematoxylin and eosin (H&E) staining of TMPD-induced granulomas revealed a predominantly mononuclear cell infiltrate surrounding numerous oil droplets (Figure 2C). Neovascularization of TMPD-induced lipogranulomas was apparent by H&E staining (Figure 2D). The blood vessels were strongly positive for peripheral lymph node addressin (MECA-79 staining), a marker for high endothelial venules (Figure 2E). Serial sections through individual TMPD-induced lipogranulomas revealed the presence of B220+ B cells, CD4+ T cells, and CD11c+ dendritic cells (Figure 2, F–H). In some cases, there was organization into discrete areas consisting of B cells, or T cells plus dendritic cells (Figure 2, F–H). However, the B cells did not stain with a germinal center marker, peanut agglutinin (Figure 2I). The lipogranulomas also contained large numbers of CD11b+ cells, which were probably mainly monocytes, macrophages, and dendritic cells (not shown). Thus, the overall structure of TMPD-induced peritoneal lipogranulomas resembled that of secondary lymphoid tissue, although with some differences, such as absence of peanut agglutinin staining and FDC-M1+ follicular dendritic cells (not shown). Immunohistochemistry of mineral oil-induced lipogranulomas also revealed the presence of MECA-79+ high endothelial venules (Figure 2J), B220+ B cells, and CD4+ T cells (Figure 2, K and L), but there was no evidence of organization into discrete T-cell/dendritic cell and B-cell zones as seen in some TMPD lipogranulomas. When the lipogranulomas were dissociated with collagenase/dispase and analyzed by flow cytometry, it was apparent that lipogranulomas from TMPD-treated mice were considerably more cellular than those from mineral oil-treated mice (sevenfold higher total cells) (Table 3). This was reflected in the absolute numbers of B cells (sixfold increased), T cells (sixfold increased), monocytes (threefold increased), and both CD11c+/CD11b+ and CD11c+/CD11b− dendritic cells (twofold increased). As a percentage of total cells, dendritic cells were more abundant in lipogranulomas than in peritoneal exudate (6 to 10% in lipogranulomas from TMPD- or mineral oil-treated mice versus 1 to 2% in peritoneal exudate), suggesting that they might home specifically to the lipogranulomas. As high endothelial venules are a source of the T-cell/dendritic cell attractant chemokines SLC (CCL21) and ELC (CCL19),23Gunn MD Tangemann K Tam C Cyster JG Rosen SD Williams LT A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes.Proc Natl Acad Sci USA. 1998; 95: 258-263Crossref PubMed Scopus (854) Google Scholar, 24Baekkevold ES Yamanaka T Palframan RT Carlsen HS Reinholt FP von Andrian UH Brandtzaeg P Haraldsen G The CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment.J Exp Med. 2001; 193: 1105-1112Crossref PubMed Scopus (319) Google Scholar we looked for expression of CCL21/CCL19 as well as the B-cell attractant chemokine BLC (CXCL13) in TMPD and mineral oil lipogranulomas (Figure 3). Both TMPD and mineral oil-induced lipogranulomas expressed mRNA for CCL21 and CCL19, ligands for the CC-chemokine receptor CCR7, but levels appeared higher in TMPD versus mineral oil lipogranulomas. Expression of CCR7, the receptor on T cells and DCs for CCL21/CCL19, also was detected. In addition, TMPD and mineral oil lipogranulomas expressed the CXC-chemokine receptor CXCR5 and its ligand CXCL13 at high levels. Two other chemokines implicated in the organization of secondary lymphoid tissue, CXCL12 (SDF-1) and CCL22 (MDC), also were detected in both TMPD and mineral oil lipogranulomas (Figure 3, bottom). The data suggest that
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