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• W2114203002 abstract "Lysosomal acid lipase (LAL) cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. In LAL gene-knockout (lal−/−) mice, blockage of cholesteryl ester and triglyceride metabolism led to abnormal organization of the thymus and spleen, as well as neutral lipid accumulation in these organs. LAL deficiency impaired T cell development in the thymus. Peripheral T cells were reduced dramatically in lal−/− mice, due largely to increased apoptosis and decreased proliferation of lal−/− T cells in the thymus and peripheral compartments. These lal−/− T cells lost the ability to respond to T cell receptor stimulation, including reduced expression of cell surface receptor CD69, abolishment of T cell proliferation, and decreased expression of T lymphokines after stimulation by either anti-CD3 plus anti-CD28 or phorbol-12-myristate-13-acetate and ionomycin. Differentiation of Th1 and Th2 CD4+ effector lymphocytes by T cell receptor stimulation was blocked in lal−/− mice. The ratio of CD4+CD25+FoxP3+ Tregs to CD4+ T cells was increased in lal−/− spleens. Bone marrow chimeras demonstrated retardation of T cell development and maturation in lal−/− mice due to defects in T cell precursors. Therefore, LAL, its downstream genes, and lipid mediators all play essential roles in development, homeostasis, and function of T cells. The altered development and function of lal−/− T cells contributes to disease formation in various organs during LAL deficiency. Lysosomal acid lipase (LAL) cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. In LAL gene-knockout (lal−/−) mice, blockage of cholesteryl ester and triglyceride metabolism led to abnormal organization of the thymus and spleen, as well as neutral lipid accumulation in these organs. LAL deficiency impaired T cell development in the thymus. Peripheral T cells were reduced dramatically in lal−/− mice, due largely to increased apoptosis and decreased proliferation of lal−/− T cells in the thymus and peripheral compartments. These lal−/− T cells lost the ability to respond to T cell receptor stimulation, including reduced expression of cell surface receptor CD69, abolishment of T cell proliferation, and decreased expression of T lymphokines after stimulation by either anti-CD3 plus anti-CD28 or phorbol-12-myristate-13-acetate and ionomycin. Differentiation of Th1 and Th2 CD4+ effector lymphocytes by T cell receptor stimulation was blocked in lal−/− mice. The ratio of CD4+CD25+FoxP3+ Tregs to CD4+ T cells was increased in lal−/− spleens. Bone marrow chimeras demonstrated retardation of T cell development and maturation in lal−/− mice due to defects in T cell precursors. Therefore, LAL, its downstream genes, and lipid mediators all play essential roles in development, homeostasis, and function of T cells. The altered development and function of lal−/− T cells contributes to disease formation in various organs during LAL deficiency. Lysosomal acid lipase (LAL) is a lysosomal hydrolase that is synthesized in rough endoplasmic reticulum and is cotranslationally glycosylated as it emerges in the endoplasmic reticulum lumen.1Du H Witte DP Grabowski GA Tissue and cellular specific expression of murine lysosomal acid lipase mRNA and protein.J Lipid Res. 1996; 37: 937-949Abstract Full Text PDF PubMed Google Scholar The low-density lipoprotein (LDL) receptor or other receptors on the plasma membranes of various cells can deliver low-density lipoprotein-bound cholesteryl esters and triglycerides to the lysosome. Cholesteryl esters and triglycerides are important components in neutral lipids that can be hydrolyzed by LAL in the lysosome of cells to generate free cholesterol and free fatty acids. Free cholesterol is released from lysosomes, which leads to activation of acyl-CoA:cholesterol acyltransferase and down-regulation of LDL receptor gene expression and 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity.2Assmann G Seedorf U Scriber CR Beaudet AL Sly WS Valle D Childs B Kinzle KW Metabolic and Molecular Bases of Inherited Diseases. McGraw-Hill, New York2001: 3551-3572Google Scholar LAL contributes to the homeostatic control of plasma lipoprotein levels and to the prevention of cellular lipid overload in various cells of the arterial wall. Therefore, LAL plays an essential role in modulation of cholesterol metabolism in all cells. In humans, LAL deficiency produces two human diseases, Wolman's disease and cholesteryl ester storage disease.2Assmann G Seedorf U Scriber CR Beaudet AL Sly WS Valle D Childs B Kinzle KW Metabolic and Molecular Bases of Inherited Diseases. McGraw-Hill, New York2001: 3551-3572Google Scholar, 3Aslanidis C Ries S Fehringer P Buchler C Klima H Schmitz G Genetic and biochemical evidence that CESD and Wolman disease are distinguished by residual lysosomal acid lipase activity.Genomics. 1996; 33: 85-93Crossref PubMed Scopus (128) Google Scholar In vivo, lal−/− mice appeared normal at birth, survived into adulthood, and were fertile. However, many organs including the liver, lung, spleen, adrenal glands, and small intestine developed severe pathogenic phenotypes due to elevated levels of cholesteryl ester and triglycerides in adult lal−/− mice.4Lian X Yan C Yang L Xu Y Du H Lysosomal acid lipase deficiency causes respiratory inflammation and destruction in the lung.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L801-L807Crossref PubMed Scopus (76) Google Scholar, 5Du H Heur M Duanmu M Grabowski GA Hui DY Witte DP Mishra J Lysosomal acid lipase-deficient mice: depletion of white and brown fat, severe hepatosplenomegaly, and shortened life span.J Lipid Res. 2001; 42: 489-500Abstract Full Text Full Text PDF PubMed Google Scholar, 6Du H Duanmu M Witte D Grabowski GA Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage.Hum Mol Genet. 1998; 7: 1347-1354Crossref PubMed Scopus (120) Google Scholar These pathogenic phenotypes resulted from malformation and malfunction of both residential cells and immune cells at the molecular and cellular levels. For example, in the lung, cytokines/chemokines, endopeptidases (eg, matrix metalloproteinases), apoptosis inhibitors, transcription factors, and oncogenes were expressed aberrantly.4Lian X Yan C Yang L Xu Y Du H Lysosomal acid lipase deficiency causes respiratory inflammation and destruction in the lung.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L801-L807Crossref PubMed Scopus (76) Google Scholar, 7Lian X Yan C Qin Y Knox L Li T Du H Neutral lipids and peroxisome proliferator-activated receptor-{gamma} control pulmonary gene expression and inflammation-triggered pathogenesis in lysosomal acid lipase knockout mice.Am J Pathol. 2005; 167: 813-821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar The Affymetrix GeneChip Microarray study demonstrated a correlation between gene profile changes and pathogenic progression in the lal−/− lung in an age-dependent manner. Some of these genes are downstream targets for lipid mediators (eg, peroxisome proliferator-activated receptor gamma), which can be activated by neutral lipid metabolic derivatives (hormonal ligands). Since lal−/− pathogenic phenotypes are highly associated with inflammation in multiple organs, immune cells, including myeloid lineage cells and lymphocyte lineage cells, must contribute significantly to disease formation. Altered differentiation and dysfunction of myeloid lineage cells (macrophages, neutrophils) have been reported previously to contribute to pathogenic progression in lal−/− mice.4Lian X Yan C Yang L Xu Y Du H Lysosomal acid lipase deficiency causes respiratory inflammation and destruction in the lung.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L801-L807Crossref PubMed Scopus (76) Google Scholar, 7Lian X Yan C Qin Y Knox L Li T Du H Neutral lipids and peroxisome proliferator-activated receptor-{gamma} control pulmonary gene expression and inflammation-triggered pathogenesis in lysosomal acid lipase knockout mice.Am J Pathol. 2005; 167: 813-821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 8Yan C Lian X Li Y Dai Y White A Qin Y Li H Hume DA Du H Macrophage-specific expression of human lysosomal acid lipase corrects inflammation and pathogenic phenotypes in lal-/- mice.Am J Pathol. 2006; 169: 916-926Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar In these mice, Kupffer cells (liver macrophages), bronchoalveolar macrophages, and intestinal macrophages appeared abnormal (foamy) with neutral lipid accumulation and aberrant cytokine/chemokine secretion.4Lian X Yan C Yang L Xu Y Du H Lysosomal acid lipase deficiency causes respiratory inflammation and destruction in the lung.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L801-L807Crossref PubMed Scopus (76) Google Scholar, 5Du H Heur M Duanmu M Grabowski GA Hui DY Witte DP Mishra J Lysosomal acid lipase-deficient mice: depletion of white and brown fat, severe hepatosplenomegaly, and shortened life span.J Lipid Res. 2001; 42: 489-500Abstract Full Text Full Text PDF PubMed Google Scholar, 8Yan C Lian X Li Y Dai Y White A Qin Y Li H Hume DA Du H Macrophage-specific expression of human lysosomal acid lipase corrects inflammation and pathogenic phenotypes in lal-/- mice.Am J Pathol. 2006; 169: 916-926Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar In a cell type-specific rescue experiment, a myeloid-specific doxycycline-inducible transgenic mouse system was generated to specifically express wild-type hLAL in lal−/− myeloid lineage cells under the control of the c-fms promoter. Expression of LAL in myeloid lineage cells corrected myeloid cell abnormalities. As a result, the pathogenic phenotypes in the lung, liver and small intestine of lal−/− mice were partially corrected.8Yan C Lian X Li Y Dai Y White A Qin Y Li H Hume DA Du H Macrophage-specific expression of human lysosomal acid lipase corrects inflammation and pathogenic phenotypes in lal-/- mice.Am J Pathol. 2006; 169: 916-926Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar On the other hand, the function of neutral lipids in lymphocytes, especially in T cells, has not been investigated in lal−/− mice. This report will test the hypothesis that LAL deficiency influences T cell development, homeostasis and function. Limited reports have shown that triacylglycerol accumulation in T cell was associated with T cell apoptosis.9Al-Saffar NM Titley JC Robertson D Clarke PA Jackson LE Leach MO Ronen SM Apoptosis is associated with triacylglycerol accumulation in Jurkat T-cells.Br J Cancer. 2002; 86: 963-970Crossref PubMed Scopus (107) Google Scholar In this report, development, maturation and function of T cells were systematically examined in lal−/− mice. The blockage of the cholesteryl ester and triglyceride metabolic pathway in lal−/− mice caused disorganization of immune tissues (eg, thymus, spleen), retardation of T cell development in the thymus, reduced T cell numbers and loss of T cell function in responding to T cell receptor (TCR) stimulation. These results support the concept that neutral lipids, their downstream genes and lipid mediators play essential roles in development and function of T cells, which contribute to abnormal inflammatory responses and disease formation in various organs in lal−/− mice. All scientific protocols involving the use of animals have been approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine and followed guidelines established by the Panel on Euthanasia of the American Veterinary Medical Association. Protocols involving the use of recombinant DNA or biohazardous materials have been reviewed by the Biosafety Committee of Indiana University School of Medicine and followed guidelines established by the National Institutes of Health. Animals were housed under Institutional Animal Care and Use Committee-approved conditions in a secured animal facility at Indiana University School of Medicine. The single-cell suspensions from the thymus and spleen of lal+/+ and lal−/− mice were obtained by grinding and filtration through nylon mesh into fluorescence-activated cell sorting (FACS) buffer (PBS, 2% fetal bovine serum, 0.01% sodium azide). Bone marrow cells were flushed from femurs and resuspended in FACS buffer as previously described.10Yan C Lian X Dai Y Wang X Qu P White A Qin Y Du H Gene delivery by the hSP-B promoter to lung alveolar type II epithelial cells in LAL-knockout mice through bone marrow mesenchymal stem cells.Gene Ther. 2007; 14: 1461-1470Crossref PubMed Scopus (18) Google Scholar Peripheral blood mononuclear cells were obtained after red blood cell lysis. Lung single cell suspension was prepared as previously described.11Li Y Du H Qin Y Roberts J Cummings OW Yan C Activation of the signal transducers and activators of the transcription 3 pathway in alveolar epithelial cells induces inflammation and adenocarcinomas in mouse lung.Cancer Res. 2007; 67: 8494-8503Crossref PubMed Scopus (121) Google Scholar Briefly, the mouse chest cavity was opened using surgical dissection, and the inferior vena cava and abdominal aorta were clamped. The left atrium was opened by incision, and the right ventricle was infused with 1× PBS to remove any residual blood in the pulmonary vasculature. The lung was cut into small pieces and placed in RPMI 1640 containing 5% fetal bovine serum, collagenase (Sigma-Aldrich, St. Louis, MO), and DNase. After 40 minutes of collagenase digestion at 37°C, lungs were further disrupted by aspiration through an 18-gauge needle. Approximately 1 to 2 × 106 cells from various organs in FACS buffer were stained with isotype control or primary antibodies. The primary antibodies CD4 (GK1.5), CD8 (53–6.7), CD69 (H1.2F3), CD25 (PC61.5), CD44 (IM7), TCRβ (H57–597), FoxP3 (150D/E4), CD90.2 (thy1.2, 53–2.1), anti-B220 (RA3–6B2), and CD11b (M1/70) were purchased from eBiosciences (San Diego, CA). FoxP3 intracellular staining was performed according to the protocol from eBiosciences. Cells were analyzed on a LSRII machine (BD Biosciences, San Jose, CA). Data were analyzed using the BD FACStation Software (BD Biosciences). The total number of positive cells was calculated as gated viable cells. Quadrants were assigned using isotype control mAb. Fluorescein isothiocyanate (FITC)-, phycoerythrin-, phycoerythrin cytochrome- and allophycocyanin-conjugated mAbs with irrelevant specificities were used as isotype control. Oil red O staining followed a previously published procedure.4Lian X Yan C Yang L Xu Y Du H Lysosomal acid lipase deficiency causes respiratory inflammation and destruction in the lung.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L801-L807Crossref PubMed Scopus (76) Google Scholar, 5Du H Heur M Duanmu M Grabowski GA Hui DY Witte DP Mishra J Lysosomal acid lipase-deficient mice: depletion of white and brown fat, severe hepatosplenomegaly, and shortened life span.J Lipid Res. 2001; 42: 489-500Abstract Full Text Full Text PDF PubMed Google Scholar Briefly, frozen tissue sections were prepared from lal+/+ or lal−/− thymus and spleen after a standard cryostat procedure. Tissue section slides were stained with oil red O solution (0.5% in propylene glycol) in a 60°C oven for 10 minutes and placed in 85% propylene glycol for 1 minute. Slides were counterstained in hematoxylin. The spleen and thymus from lal+/+ and lal−/− mice were washed with PBS and dehydrated by a series of increasing ethanol concentrations, followed by paraffin embedding. Five-μm tissue sections were incubated at 4°C overnight with rat anti-mouse CD3 (145–2C11, 1:500; BD Biosciences Clontech, Mountain View, CA), LAMP-2 (M3/84, 1:400; Santa Cruz Biotechnology, CA) as the primary antibody. Tissue sections were washed and treated with biotinylated secondary antibodies. The signals were detected with a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) to visualize the signals following a procedure recommended by the manufacturer. CD4+ and CD8+ T cells from lal+/+ and lal−/− spleen were purified with an antibody-linked magnet-activated cell sorter (Miltenyi Biotec Inc.) according to the manufacturer's instructions. For TCR stimulation studies, the sorted T cells were cultured in 96-wells coated with anti-CD3 mAb (145–2C11, 2 μg/ml) alone or in combination with anti-CD28 mAb (37.51.1, 5 μg/ml). In other experiments, T cells were cultured in the presence of phorbol-12-myristate-13-acetate (PMA) (10 nmol/L) and ionomycin (1 μg/ml). For in vitro proliferation, CD4+ T cells were incubated in PBS containing 1 μmol/L Carboxyfluorescein diacetate succinimidyl diester (CFSE, Molecular Probes, Eugene, Oregon) at 37°C for 15 minutes and then pelleted and resuspended in complete medium for 30 minutes. CD4+ T cells were washed twice in PBS and then cultured in 96-well round-bottom plates in complete medium alone, or with the addition of anti-CD3 plus anti-CD28 mAb. After 3 days, cells were harvested, and stained with allophycocyanin-labeled anti-CD4 mAb (eBiosciences), and analyzed using flow cytometry (BD Biosciences). Analyses were performed on 1 × 105 viable cells gated according to their forward/side scatter profile and CD4 expression. Data were analyzed using CellQuest software (BD Biosciences). After T cells were cultured in wells coated with anti-CD3 and/or anti-CD28 for 24 to 72 hours, supernatants were harvested. In certain experiment, T cells were cultured in the presence of PMA and ionomycin. Levels of interleukin (IL)-2, IL-4, and interferon (IFN)-γ in the supernatants were measured using DueSet ELISA kits for mouse IL-2, IL-4, and IFN-γ according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). CD4+ and CD8+ T cells were sorted from the spleen in lal+/+ and lal−/− mice, and stimulated with plate-bound anti-CD3 mAb (2 μg/ml, clone 145–2C11) plus anti-CD28 mAb (5 μg/ml, clone 37.51.1) for 16 or 40 hours. Total RNA was isolated using Qiagen RNeasy Midi Kit according to the manufacturer's instruction (Qiagen Inc., Valencia, CA). Total mRNA (750 ng) was reversely transcribed using TaqMan Reverse Transcription Reagent as recommended by the manufacturer (Applied Biosystems, Branchburg, NJ) on GeneAmp PCR system 9700 (Applied Biosystems) with a suggested cycling protocol of 25°C for 10 minutes, 48°C for 30 minutes, and 95°C for 5 minutes. For real-time PCR, a total of 5 μl of cDNA was amplified by a pair of sequence-specific DNA oligonucleotide primers (Table 1) for apoptosis molecules or cytokines in a 50 μl reaction solution containing SYBR Green PCR Master Mix (Applied Biosystems). Reactions were analyzed using the Relative Quantification Assay of 7500 System Sequence Detection Software (Applied Biosystems). The default cycling protocol for this software was 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Dissociation curves were measured to determine the quality for each pair of DNA oligonucleotide primers. All samples were measured in triplicate. Real-time PCR was normalized by glyceraldehyde-3-phosphate dehydrogenase mRNA expression. Fold changes were determined by 2(ddCt), in which ddCT = dCt (lal+/+ sample) − dCt (lal−/− sample). The expression level of lal+/+ samples was set as 1.Table 1Primers for Real-Time PCRGeneForward primerReverse primermApaf-15′-CAGTAATGGCGTCTTGTCAGTGA-3′5′-CGTTGATATTGAGTGGCCTGACT-3′mBak5′-CAACCCCGAGATGGACAACT-3′5′-GACCCACCTGACCCAAGATG-3′mBax5′-GGGCCCACCAGCTCTGA-3′5′-TGGATGAAACCCTGTAGCAAAA-3′mBid5′-GGCGTCTGCGTGGTGATT-3′5′-ACAAGCTGTGTAGCTCCAAGCA-3′mCasp35′-TGCTTACTCTACAGCACCTGGTTACT-3′5′-TGAACCACGACCCGTCCTT-3′mCasp65′-CGGGCAGGTGAAAGTAAAACA-3′5′-GGATCGAACACTTCCCTACTTTTG-3′mCasp75′-CCGTCCACAATGACTGCTCTT-3′5′-GGTCCTCCTCAGAGGCTTTTC-3′mCasp85′-CGCCAAGAGAACAAGACAGTGA-3′5′-CGAGGTTTGTTCTTCATTTGGTAA-3′mCasp95′-AACGACCTGACTGCCAAGAAA-3′5′-GGTTCCGGTGTGCCATCTC-3′mCideA5′-CAGCAGCCTGCAGGAACTTAT-3′5′-ACCAGGCCAGTTGTGATGACT-3′mFasL5′-CGAGGAGTGTGGCCCATT T-3′5′-TCCAGAGGGATGGACCTTGA-3′mGranzyme B5′-AAAGCTGAAGAGTAAGGCCAAGAG-3′5′-CGCCTGGGCAGGTTGAG-3′mIL-1β5′-TTGACGGACCCCAAAAGATG- 3′5′-CAGGACAGCCCAGGTCAAA-3′mIL-25′-AAACTAAAGGGCTCTGACAACACA-3′5′-CACCACAGTTGCTGACTCATCA-3′mIL-45′-TTGAACGAGGTCACAGGAGAAG-3′5′-AGGACGTTTGGCACATCCA-3′mIL-65′-GAGGCTTAATTACACATGTTC-3′5′-TGCCATTGCACAACTCTTTTCT-3′mIL-105′-GCTGCGGACTGCCTTCA-3′5′-TCCAGCTGGTCCTTTGTTTGA-3′mIL-135′-AGCAACATCACACAAGACCAGACT-3′5′-CCAGGTCCACACTCCATACCA-3′mIL-165′-GCTCCCTGCATGGTGACAA-3′5′-CACCCTGTTCTGTCCCTTTGA-3′mIL-175′-TCTGTGTCTCTGATGCTGTTGCT-3′5′-CGCTGCTGCCTTCACTGTAG-3′mIFNγ5′-CAAGGCGAAAAAGGATGCAT-3′5′-CTGGACCTGTGGGTTGTTGAC-3′mTNFα5′-CCCCAAAGGGATGAGAAGTTC-3′5′-TGAGGGTCTGGGCCATAGAA-3′GAPDH5′-GGCCATCAAGCCAGAGCTT-3′5′-CCAAACCATCACTGACACTCAGA-3′ Open table in a new tab Dual staining with FITC-annexinV and propidium iodide (PI) was performed to detect cells undergoing apoptosis using an Annexin V-FITC kit (BD Biosciences, Bedford, MA). T cells from different tissues of lal+/+ and lal−/− mice were stained with surface markers and washed twice with PBS. After resuspension of labeled cells in annexin V-binding buffer containing FITC-conjugated annexin V, PI was added into samples and incubated on ice for 10 minutes. Cells were analyzed on LSRII within 1 hour. Viable cells were defined by FITC negative (FITC−) and PI negative (PI−) population. Early apoptotic cells were defined by FITC positive (FITC+) and PI− population. Nonspecific binding was blocked by pre-incubating the cells with rat IgG (10 μg/ml) and anti-FcII/III. CD4+CD25+ regulatory T cells (Treg) from lal+/+ and lal−/− mice (CD45.2 mice) were sorted using negative selection with the anti-CD4 MACS system, followed by positive selection with an anti-CD25 MACS column (Miltenyi Biotech, Anburn, CA). CD4+CD25− responder cells from CD45.1+ mice were purified from the spleen and labeled with CFSE (2.5 μmol/L). To assess Treg suppressor activity, freshly isolated Tregs were co-cultured with CD4+CD25− T cells at different ratios in the presence of 5 × 105 irradiated (3000 rad) T cell-depleted autologous splenocytes. Cells were incubated at 37°C with 5% CO2 for 4 days with stimulation of anti-CD3 antibody (2 μg/ml) plus anti-CD28 antibody (2 μg/ml) or PBS (control). Responder cells, gated with CD45.1 antibody staining, were plotted for proliferation analysis. Cell proliferation was measured by CFSE dilution. The acquired FACS data were analyzed with the CellQuest program. Bone marrow was flushed from femurs and tibias of 8 to 10-week-old donor lal−/− or lal+/+ mice. Mature lymphocytes were depleted from the bone marrow cell preparation by using CD4 and CD8 antibody-linked magnet-activated cell sorting (Miltenyi Biotech, Auburn, CA). These cells were referred to as T cell-depleted bone marrow cells. Three-month old lal−/− or lal+/+ recipient mice were lethally irradiated with 1000 rad of γ-irradiation and rested 1 day before receiving cells. Recipient mice were injected with 2.5 to 5 × 106 T cell-depleted bone marrow cells in 500 μl 1× PBS via tail vein. Reconstituted mice were analyzed 10 weeks later. Three-month-old lal+/+ and lal−/− mice were injected i.p. with 20 μg of either control hamster IgG or anti-CD3ε mAb (2C11, BD Bioscience). After 3 hours, spleen cells were isolated from animals as described above and stained with FITC- or phycoerythrin-conjugated anti-CD4 (GK1.5), anti-CD8 (53–6.7), and anti-CD69 (H1.2F3) for flow cytometry analysis. Blood samples were collected from lal+/+ and lal−/− mice after 3 hours i.p. injection of anti-CD3ε mAb or control hamster IgG. Plasma was isolated by centrifugation at 5000 rpm for 10 minutes at 4°C. LDL-cholesterol was measured using N-GENEOUS LDL-cholesterol reagent (Genzyme, Cambridge, Massachusetts) follow by manufactured protocol. Briefly, 150 μl of Reagent 1 was added to 3 μl of plasma and incubated in room temperature for 10 minutes. Detergent in Reagent 1 solubilized non-LDL lipoprotein particles. Cholesteryl esters released free cholesterol that reacted with cholesterol oxidase to generate hydrogen peroxide. The latter was consumed by horseradish peroxidase in the presence of 4-aminoantipyrine to generate a colorless solution. The addition of Reagent 2 (50 μl) solubilized LDL particles and produced a color proportional to the amount of cholesterol present in the sample. The color was measured at 660 nm. LDL-cholesterol calibrator (Genzyme) (concentration ranged from 175 to 886 mg/dL) was used as standard. Lipoprotein (d <1.21) was isolated from lal+/+ and lal−/− mice 3 hours post-injection of anti-CD3 antibody or control IgG. Lipoprotein (50 μg) in final volume of 0.33 ml of 1 × PBS was incubated with TBARS reagent (0.67 ml; 15% trichloroacetic acid, 0.375% thiobarbituric acid, 0.25 N hydrochloric acid) at 95°C for 30 minutes. Samples were centrifuged to remove precipitate (10,000 × g for 15 minutes at room temperature), and read absorbance at 535 nm. Diluted maloaldehyde (0 to 10 nmol) was used as standard. A paired Student's t-test or analysis of variance was used to evaluate the significance of the differences. Statistical analysis was performed with SPSS version 11.5, with a value of P < 0.05 regarded as statistically significant. The thymus is divided into two main compartments, the cortex and the medulla. H&E staining revealed that the lal−/− mouse thymic architecture appeared to be disturbed histologically, lacking the distinct cortical and medullary structures (Figure 1A). Oil red O staining identified massive neutral lipid accumulation in lal−/− thymi with age progression. Biochemical measurement showed increased levels of triglycerides and cholesterol in lal−/− thymi at various age groups (Figure 1B). As we reported previously, macrophages are the major cells for lipid storage due to LAL deficiency.4Lian X Yan C Yang L Xu Y Du H Lysosomal acid lipase deficiency causes respiratory inflammation and destruction in the lung.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L801-L807Crossref PubMed Scopus (76) Google Scholar, 8Yan C Lian X Li Y Dai Y White A Qin Y Li H Hume DA Du H Macrophage-specific expression of human lysosomal acid lipase corrects inflammation and pathogenic phenotypes in lal-/- mice.Am J Pathol. 2006; 169: 916-926Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Indeed, Mac3 antibody (a macrophage marker) staining showed massive macrophage infiltration in the lal−/− thymus, which was colocalized in the same area of lipid accumulation (Figure 1C). In general, the size of thymus and mean thymocyte weight were significantly reduced in lal−/− mice compared with age-matched lal+/+ control mice (Table 2). This thymic hypoplasia became more evident with age progression. The ratio between thymus weight versus body weight was severely reduced in lal−/− mice as well. These pathogenic results suggest a correlation between LAL deficiency and T cell development in the thymus of lal−/− mice.Table 2Thymus Weight (TW) and TW/BW from lal+/+ and lal−/− Mice*TW, thymus weight; BW, body weight; 3 mol/L, 5 mol/L, 7 mol/L: 3, 5, 7-month-old; Shown are combined data from four independent experiments (3 mice per group per experiment).Thymus weight (mg)TW/BW (%)Mice3 mo5 mo7 mo3 mo5 mo7 moLal+/+66.17 ± 6.3876.67 ± 19.6153.52 ± 12.400.20 ± 0.020.26 ± 0.070.14 ± 0.03lal−/ −39.87 ± 7.00†Significant difference between lal+/+ and lal−/− mice. The P values were determined by student's t-test. Changes were significant with‡P < 0.05 as compared with lal+/+ mice.41.63 ± 10.64†Significant difference between lal+/+ and lal−/− mice. The P values were determined by student's t-test. Changes were significant with‡P < 0.05 as compared with lal+/+ mice.28.67 ± 3.57†Significant difference between lal+/+ and lal−/− mice. The P values were determined by student's t-test. Changes were significant with‡P < 0.05 as compared with lal+/+ mice.0.16 ± 0.030.14 ± 0.04†Significant difference between lal+/+ and lal−/− mice. The P values were determined by student's t-test. Changes were significant with‡P < 0.05 as compared with lal+/+ mice.0.09 ± 0.01†Significant difference between lal+/+ and lal−/− mice. The P values were determined by student's t-test. Changes were significant with‡P < 0.05 as compared with lal+/+ mice.* TW, thymus weight; BW, body weight; 3 mol/L, 5 mol/L, 7 mol/L: 3, 5, 7-month-old; Shown are combined data from four independent experiments (3 mice per group per experiment).† Significant difference between lal+/+ and lal−/− mice. The P values were determined by student's t-test. Changes were significant with‡ P < 0.05 as compared with lal+/+ mice. Open table in a new tab The thymus is the most important organ for T cell development. The total cellular numbers in the lal−/− thymus were significantly reduced compared with those in age-matched lal+/+ mice (Figure 2A). Next, T cells at different developmental stages were evaluated in lal+/+ and lal−/− mice. T cell development in the thymus can be divided into various stages, including CD4−CD8− double-negative (DN), CD4+CD8+ double-positive (DP), and CD4+CD8−/CD4−CD8+ single-positive (SP) stages. T cells in almost all these stages were significantly reduced in lal−/− mice compared with those in lal+/+ mice, except that decrease of CD4 SP cells was only observed at 9 months of age (Figure 2A). At 9 months old, the DP thymocytes were almost absent. Therefore, the blockage of T cell development occurred before the DP stage. Importantly, the CD4:CD8 ratio was significantly increased in the lal−/− thymus (Figure 2B). To determine possible alterations at the earliest stages of intrathymic T cell development, the earliest stages of thymocyte subsets were analyzed as defined by CD44 and CD25 markers, including DN1 (CD44+CD25−), DN2 (CD44+CD25+), DN3" @default.
• W2114203002 created "2016-06-24" @default.
• W2114203002 creator A5007071486 @default.
• W2114203002 creator A5027881339 @default.
• W2114203002 creator A5046799481 @default.
• W2114203002 creator A5080575998 @default.
• W2114203002 date "2009-03-01" @default.
• W2114203002 modified "2023-10-16" @default.
• W2114203002 title "Critical Roles of Lysosomal Acid Lipase in T Cell Development and Function" @default.
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