Biomedical subjects

J D Mountz

Publications and source records attributed to J D Mountz.

At least 37 records · Page 2Linked to original sources

PubMed2001

Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity.

A novel anti-human DR5 monoclonal antibody, TRA-8, induces apoptosis of most tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive tumor cells both in vitro and in vivo. In contrast to both the membrane-bound form of human TRAIL, which induced severe hepatitis in mice, and the soluble form of human TRAIL, which induced apoptosis of normal human hepatocytes in vitro, TRA-8 did not induce significant cell death of normal human hepatocytes. However, both primary hepatocellular carcinoma cells and an established liver cancer cell line were highly susceptible to the killing mediated by TRA-8. We show here that elevated levels of cell-surface expression of DR5 and increased susceptibility to DR5-mediated apoptosis are characteristics of malignant tumor cells. In contrast, DR5 alone is not sufficient to trigger apoptosis of normal hepatocytes. Therefore, selective, specific targeting of DR5 with an agonistic antibody might be a safe and effective strategy for cancer therapy.

Animals↗

PubMed2001

Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus.

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap.

Adenoviridae↗

PubMed2001

Regulation of apoptosis of synovial fibroblasts.

In 1992, the first gene to cause systemic autoimmune disease in mice was identified as the Fas gene that is mutated in lymphoproliferative (lpr mice). These mice exhibited a defect in activation-induced cell death of T cells and B cells in vivo. This leads to the failure of proper clearance and removal of immune cells and defective downmodulation of an immune response. This then leads to the speculation that apoptosis defects, including defects in Fas, Fas ligand and Fas apoptosis signaling, may play a role in defective downmodulation of the hyperimmune response observed in human autoimmune diseases. Over the past 7 years, many scientists have analyzed different proapoptotic genes such as Fas, Fas ligand, Bcl-X, caspases as well as antiapoptosis pathways including defects in Fas and Fas ligand, Bcl-2 and caspase inhibitors. Potential genetic defects have been analyzed at the RNA, protein and functional level in humans with autoimmune disease. Somewhat surprisingly, most studies indicate that there is excessive apoptosis of PBMCs in autoimmune disease and human autoimmune disease suggesting that human autoimmune disease is not due to defective apoptosis of immune cells. Some studies indicate that there is decreased apoptosis of parenchymal cells such as RASF that undergo hyperplasia. Gene therapy and other modulators of apoptosis, such as wortmannin, can be used to faciliate apoptosis of RASF.

Animals↗

PubMed2000

Adeno-associated virus production of soluble tumor necrosis factor receptor neutralizes tumor necrosis factor alpha and reduces arthritis.

The major limitation of adenovirus is its association with induction of an inflammatory response and relatively short-term production of the gene therapy transgene product. Adeno-associated virus (AAV) is a 4.68-kb single-strand DNA virus that contains ITRs for viral replication and a packaging signal, and also has been engineered to contain therapeutic genes up to 5 kb in length. Transduction of recombinant AAV (rAAV) results in low inflammatory response and long-term expression. We have cloned a low-immunogenic form of human sTNFRI (sTNFRI2.6D) into AAV (rAAVsTNFRI). This vector was analyzed for its ability to transfect and neutralize the effect of TNF-alpha on primary rheumatoid arthritis synovial fibroblast (RASFs). The rAAVsTNFRI was transduced into the cells at 1.8 x 10(1), 1.8 x 10(2), and 1.8 x 10(3) viral particles per cell. There was greater than 90% neutralization of TNF-alpha at 1.8 x 10(3) viral particles/cell. There was a significant decrease in the synovial cell hyperplasia and cartilage and bone destruction in human TNF-alpha transgenic mice treated intraarticularly with rAAVsTNFRI. These results indicate that the low-immunogenic and long-term expressing vector, rAAVsTNFRI, can be used to deliver the soluble TNF-alpha in vitro and in vivo and effectively reduce the severity of arthritis.

Animals↗

PubMed2000

The prodomain of caspase-1 enhances Fas-mediated apoptosis through facilitation of caspase-8 activation.

Caspase-1 (interleukin-1beta converting enzyme) is produced in the form of a latent precursor, which is cleaved to yield a prodomain in addition to the p20 and p10 subunits. It has been established that the (p20/p10)(2) heterotetramer processes the latent precursor of interleukin-1beta into an active form during apoptosis, but the function of the residual prodomain of caspase-1 (Pro-C1) has not been established. To evaluate the involvement of Pro-C1 in apoptosis, a Pro-C1 expression vector was transfected into the HeLa cell line, which is susceptible to Fas-mediated apoptosis. Expression of recombinant Pro-C1 in HeLa cells enhanced apoptosis mediated by Fas, but not etoposide-induced apoptosis. This enhancement of Fas-mediated apoptosis was abolished by inhibitors of caspase-8 (Ile-Glu-Thr-Asp-fluoromethyl ketone) and caspase-3 (Asp-Glu-Val-Asp-aldehyde) but was only slightly diminished by an inhibitor of caspase-1 (acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone). During apoptosis induced by an agonistic anti-Fas antibody, the activation of caspase-8 and caspase-3 was more pronounced and occurred more rapidly in HeLa/Pro-C1 cells than in the empty vector transfectant (HeLa/vec) cells; in contrast, caspase-1 was not activated in either HeLa/Pro-C1 or HeLa/vec cells. These results demonstrate an additional and novel function for caspase-1 in which Pro-C1 acts to enhance Fas-mediated apoptosis, most probably through facilitation of the activation of caspase-8.

Apoptosis↗

PubMed2000

Gene therapy that inhibits nuclear translocation of nuclear factor kappaB results in tumor necrosis factor alpha-induced apoptosis of human synovial fibroblasts.

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) increases the survival and proliferation of human rheumatoid arthritis (RA) cell lines. These experiments were designed to determine if inhibition of nuclear factor kappaB (NF-kappaB) nuclear translocation leads to increased apoptosis of TNFalpha-treated human RA cell lines. METHODS: We constructed an inhibitor of nuclear factor kappaB(IkappaB) dominant-negative adenovirus (AdCMVIkappaB-DN) and an X-linked inhibitor of apoptosis (XIAP) antisense adenovirus (AdCMVXIAP-AS). Primary RA synovial fibroblast (RASF) cell lines were transfected in vitro, and SV40-transformed RA synovial cell lines in SCID mice were transfected in vivo. Cells were treated with TNFalpha and analyzed for apoptosis. RESULTS: There was no apoptosis of primary RASF transfected in vitro with AdCMVIkappaB-DN alone. In contrast, there was apoptosis of >85% of cells treated with AdCMVIkappaB-DN plus TNFalpha. Primary RASF in SCID mice also exhibited high levels of apoptosis after in vivo transfection with AdCMVIkappaB-DN followed by treatment with TNFalpha. There was no apoptosis after treatment with AdCMVIkappaB-DN in the absence of TNFalpha. XIAP is an inhibitor of apoptosis which was up-regulated by TNFalpha, and this up-regulation was inhibited by AdCMVIkappaB-DN plus TNFalpha. Transfection of an AdCMVXIAP-AS gene therapy resulted in increased TNFa-induced apoptosis. CONCLUSION: AdCMVIkappaB-DN gene therapy greatly enhances apoptosis due to inhibition of an NF-kappaB-mediated antiapoptosis signaling pathway, and XIAP is a TNFalpha-inducible specific inhibitor of apoptosis in RA synovial cell lines. This and other modulators of TNF receptor or the Fas apoptosis pathway may be therapeutically beneficial in facilitating apoptosis of synovial tissue in patients with RA.

Animals↗

PubMed2000

Antigen presenting cells expressing Fas ligand down-modulate chronic inflammatory disease in Fas ligand-deficient mice.

We assessed the effect of modified antigen presenting cells (APCs) expressing high levels of Fas ligand (APC-FasL) on post-viral chronic inflammatory disease. FasL-deficient B6-gld/gld mice infected with murine cytomegalovirus (MCMV) cleared the virus from their lungs, kidneys, and livers within 2 weeks of infection. However, inflammation persisted in these organs for more than 8 weeks, with a chronically increased T-cell response to MCMV-infected APCs and production of autoantibodies. Administration of APC-AdFasL at 4 weeks suppressed this inflammation and diminished the T-cell response and autoantibody production. APC-AdFasL that had been transfected with ultraviolet-irradiated MCMV were more effective than uninfected APC-AdFasL in ameliorating the chronic inflammation. APC-AdFasL migrated preferentially to the spleen, where they triggered apoptosis of lymphocytes in the marginal zone of the spleen. These results confirm that Fas-mediated apoptosis is not required for clearance of virus, but is required for down-modulation of the virally induced chronic inflammatory response. This organwide effect of APC-AdFasL appears to be mediated by elimination of activated T lymphocytes in the spleen before their emigration to the target organs.

Adenoviridae↗

PubMed2000

Serum levels of soluble Fas correlate with indices of organ damage in systemic lupus erythematosus.

OBJECTIVE: To evaluate whether the levels of soluble form of the Fas apoptosis antigen (sCD95/sFas) varied from those of healthy control subjects in a group of patients with systemic lupus erythematosus (SLE). This was done to determine whether sFas has a role in either the disease activity or the organ damage in SLE. METHODS: Serum levels of sFas were measured over a period of 4 y (277 determinations) in 39 Arab patients with SLE and 22 age-, gender-, and race-matched healthy controls using double antibody ELISA. SLEDAI scores for disease activity and SLICC/ACR scores for cumulative organ damage were determined. Serum levels of acute phase reactants, complement, inflammatory cell counts, levels of autoantibodies, and kidney and liver function test results were obtained retrospectively from clinical records. RESULTS: sFas levels were significantly higher in patients with SLE (n = 39, 277 determinations) (0.60 ng/ml +/- 0.38) than in healthy controls (n = 22) (0.26 ng/ml +/- 0.11) (P < 0.00001). The levels of sFas correlated with SLICC/ACR (r = 0.36; P < 0.02), but not with SLEDAI. sFas correlated with renal and liver function tests measured by s-creatinine (r = 0.38; P < 0.0001), creatinine clearance (r = -0.30, P < 0.001), s-albumin (r = -0.28, P < 0.0001), and ALT (r = 0.35; P < 0.00001), but did not correlate with the levels of acute phase reactants. CONCLUSION: sFas is elevated in sera of SLE patient. Since sFas correlates with indices of organ damage but not with disease activity, it may be a marker of organ damage in SLE and may act to protect certain organs from further damage by inhibiting Fas-mediated apoptosis.

Adolescent↗

PubMed1999

Induction of specific T cell tolerance by Fas ligand-expressing antigen-presenting cells.

Autocrine interaction of Fas and Fas ligand leads to apoptosis of activated T cells, a process that is critical for the maintenance of peripheral T cell tolerance. Paracrine interactions of Fas ligand with T cells also may play an important role in the maintenance of tolerance, as Fas ligand can create immune-privileged sites and prevent graft rejection by inducing apoptosis in T cells. We surmised that APCs that express Fas ligand might directly induce apoptosis of T cells during presentation of Ag to the T cells, thus inducing Ag-specific, systemic T cell tolerance. Here, we show that profound, specific T cell unresponsiveness to alloantigen was induced by treatment of H-2k mice with H-2b APCs that expressed Fas ligand and that profound T cell unresponsiveness specific for the H-Y Ag was induced by treatment of H-2Db/H-Y TCR transgenic female mice with H-2Db/H-Y APCs that expressed Fas ligand. The induction of this systemic T cell tolerance required the expression of Fas ligand on the APCs as well as the expression of Fas on the T cells. The tolerance was restricted to the Ag presented by the APCs. The rapid and profound clonal deletion of the Ag-specific, peripheral T cells mediated by the Fas ligand-expressing APCs contributed to the induction of tolerance. These findings demonstrate that Ag-specific T cell tolerance can be induced by APCs that express Fas ligand and suggest a novel function for APCs in the induction of T cell apoptosis. Furthermore, they indicate a novel immunointervention strategy for treatment of graft rejection and autoantigen-specific autoimmune diseases.

Animals↗

PubMed1999

Specific targeting of activated endothelium in rat adjuvant arthritis with a 99mTc-radiolabeled E-selectin-binding peptide.

OBJECTIVE: To determine the potential of an E-selectin-binding peptide (ESbp) to specifically bind activated endothelium in rheumatoid arthritis (RA) animal models. METHODS: ESbp (KYDGDITWDQLWDLMK; 2,027 daltons) was labeled with biotin and 99mTc. The affinity of ESbp derivatives for E-selectin was measured by enzyme-linked immunosorbent assay. The binding of biotin-ESbp was compared with that of an anti-E-selectin antibody, by immunohistochemical analyses of human synovial sections and sections from the Mycoplasma pulmonis MRL-lpr/lpr mouse arthritis model. 99mTc-ESbp was sequentially imaged in vivo with a gamma camera in the rat adjuvant-induced arthritis model. RESULTS: E-selectin expression was detected in human RA synovium and mouse arthritic synovium using biotin-ESbp. Both biotin-ESbp and 99mTc-labeled ESbp had high affinity for E-selectin (dissociation constant 2-5 nM). In vivo imaging showed specific binding of 99mTc-ESbp to the rat ankle joint prior to clinical manifestations of inflammation. CONCLUSION: These results demonstrate that activated endothelium can be targeted with 99mTc-ESbp. The specificity of targeting can be used to evaluate up-regulation of E-selectin in RA models, and to follow changes in this up-regulation during treatment trials.

Amino Acid Sequence↗

PubMed1999

Herpes simplex virus type 2 infection induced apoptosis in peritoneal macrophages independent of Fas and tumor necrosis factor-receptor signaling.

Freshly isolated macrophages from mature mice are poorly or nonpermissive for infections with HSV. However, despite lack of significant viral replication, HSV infection has been demonstrated to induce substantial cell death among macrophages. To determine if HSV-induced cytotoxicity of macrophages is due to apoptosis, peritoneal macrophages were obtained from C57BL/6 (B6) mice, and apoptosis was analyzed following HSV-2 infection in vitro. Macrophages underwent apoptosis upon HSV-2 infection indicated by annexin V staining, labeling of DNA strand breaks and electronmicroscopy. Apoptosis was associated with macrophage activation demonstrated by upregulation of MHC class II and Mac-1 surface expression. Though there was also an upregulation of Fas (Apo-1/CD95) and tumor necrosis factor (TNF)-receptor 1 (TNF-R1) pathways, inhibition of Fas by soluble Fas and blocking of TNF-alpha using a TNF-binding protein did not prevent HSV-induced apoptosis. Moreover, apoptosis was not impaired in HSV-2 infected macrophages from Fas-deficient B6-lpr/lpr mice suggesting involvement of other apoptosis pathways, or activation of Fas or TNF-R pathways downstream of the receptor level. The present results demonstrate that HSV-2 infection leads to activation and subsequent apoptosis in peritoneal macrophages independent of Fas or TNF-R1 signaling.

Animals↗

PubMed1999

Regulation of Fas-mediated apoptosis in CD2-fas transgenic mice.

Fas-mediated apoptosis is an essential mechanism for maintenance of immune homeostasis. The expression of Fas is regulated at transcriptional and protein levels. Furthermore, several death domain molecules and caspases are crucial downstream mediators and executioners of Fas-mediated apoptosis. A tightly regulated interaction of these molecules ensures normal immune functions, including the execution of activation-induced cell death, T-cell mediated cytotoxicity, and surveillance of immune privileged tissues. In contrast, abnormally increased or decreased Fas-mediated apoptosis is a major pathogenic mechanism of several diseases, including systemic or tissue-specific autoimmune diseases and immune deficiency. Two CD2-fas transgenic mouse lines are described here to demonstrate the importance of controlling Fas-mediated apoptosis. Correction of Fas in Fas-mutant mice restored apoptosis function and ameliorated autoimmune symptoms, whereas a long-term enhancement of Fas expression in Fas-normal mice resulted in an increased acute-phase response and renal amyloidosis in aged transgenic mice.

Acute-Phase Reaction↗

PubMed1999

Apoptosis and cell death in the endocrine system.

Inflammatory diseases of the endocrine system--such as thyroiditis, diabetes, and Graves' disease--are considered to be autoimmune in origin. More recently, these and other autoimmune diseases have been associated with defects in Fas apoptosis. The mutation of the Fas or Fas ligand (FasL) has been observed in a minority of patients with autoimmune disease. However, dysfunction of the Fas apoptosis signaling pathway or production of soluble factors, including sFas and sFasL, may be more prevalent. Certain endocrine tissues, such as the testes, are immune privilege sites. Defects in Fas and FasL expression in immune privilege sites can trigger an inflammatory response. Other factors that trigger inflammatory diseases of the thyroid or islets may be loss of self tolerance, leading to an autoimmune response. An infectious trigger or other environmental agent can initiate organ damage, leading to release of new antigens that initiate the autoreactive process. We have developed a murine cytomegalovirus model of Sjögren's syndrome in which defects in the Fas/FasL pathway are necessary to enable chronic inflammation, even after the initial virus has been cleared. Another interaction between the endocrine system and apoptosis is by direct hormone interaction. This is exemplified by the orphan steroid receptor Nur77. Nur77 is important for T cell apoptosis after signaling through CD3. We have demonstrated that a dominant-negative Nur77 transgenic mouse exhibits a defect in thymic selection of T cells. Therefore, there are many potential mechanisms by which endocrine glands or hormones can affect the Fas apoptosis pathway, resulting in either cell death or a chronic inflammatory disease in the endocrine system, leading to hypothyroidism and diabetes. This inflammatory dysfunction can be reversed by a dominant-negative I kappa B that prevents nuclear translocation of NF-kappa B. We have developed antigen-specific, antigen-presenting cells that express high levels of FasL that can prevent tissue-specific inflammatory disease. Treatment with these cells prevents development of diabetes in NOD mice. Further understanding of the role and regulation of apoptosis in diseases of the endocrine system (e.g., diabetes, thyroiditis) should lead to better methods of treatment and prevention of these diseases.

Animals↗

PubMed1998

Apoptosis mediated by Fas but not tumor necrosis factor receptor 1 prevents chronic disease in mice infected with murine cytomegalovirus.

The role of Fas- and TNF-receptor 1 (TNF-R1)-mediated apoptosis in the clearance of virally infected cells and in the regulation of the immune response was analyzed after murine cytomegalovirus (MCMV) infection of C57BL/6 (B6)-+/+ mice, Fas-mutant B6-lpr/lpr mice, TNF-R1 knockout B6-tnfr0/0 mice, and double-deficient B6-tnfr0/0 lpr/lpr mice. There was approximately equivalent clearance of MCMV in B6-+/+, B6-tnfr0/0, and B6-lpr/lpr mice, and by day 28 no infectious virus could be detected in the liver, kidney, lung, or peritoneal exudate. However, delayed virus clearance was observed in B6-tnfr0/0 lpr/lpr mice. An acute inflammatory response occurred in the liver, lung, and kidney of all mice, which was most severe 7 d after MCMV infection, but resolved by day 28 in B6-+/+ and B6-tnfr0/0 mice, but not in B6-lpr/lpr or B6-tnfr0/0 lpr/lpr mice. These results indicate that apoptosis mediated by either Fas or TNF-R1 is sufficient for rapid clearance of the virus. However, apoptosis induced by Fas, but not TNF-R1, is required for the downmodulation of the immune response to the virus and prevention of a chronic inflammatory reaction.

Animals↗

PubMed1998

Inhibition of tumor necrosis factor alpha decreases inflammation and prolongs adenovirus gene expression in lung and liver.

The clinical application of adenoviral gene therapy currently is impeded by the potent host immune response to the virus, which limits the duration of its effects. In these studies, we investigated the role of TNF-alpha and of a soluble TNF receptor (TNF-bp) in the inflammatory response and expression of a lacZ-expressing adenovirus (AdCMVlacZ) in the liver and lung of mice. The expression of the recombinant adenovirus was studied in mouse liver and lung by determining the activity of the lacZ gene product of the adenovirus. The mononuclear cell inflammatory response was determined histologically at different times after intravenous or intranasal administration of AdCMVlacZ. The cytotoxic T cell and antibody response to the adenovirus was determined. Treatment with TNF-bp reduced circulating levels of TNF-alpha, greatly reduced the inflammatory response, and resulted in prolonged expression of lacZ for up to 30 days in the liver and lung after either intravenous or intranasal administration of adenovirus. Treatment with TNF-bp had no effect on anti-adenovirus antibodies and induction of cytotoxic T cells 30 days after administration of AdCMVlacZ. These results indicate that TNF-alpha is the primary factor driving the early inflammatory response leading to elimination of adenovirus-infected cells in the liver and lung and that TNF-bp is capable of inhibiting these effects.

Adenoviridae↗

PubMed1998

Effect of age and apoptosis on the mouse homologue of the huWRN gene.

Werner's syndrome (WS) is an inherited disease with clinical symptoms which resemble premature aging. The Werner's syndrome gene (WRN), which is located on human chromosome 8p12, encodes a predicted protein of 1432 amino acids and shows significant similarity to DNA helicases. We have cloned the full-length mouse cDNA homologue of the human WRN gene encoding a predicted protein of 1320 amino acids and have obtained a full-length 70 kb genomic clone containing the moWRN gene. This gene has been mapped to chromosome 8A3 in mice. The expression of the moWRN gene was increased during apoptosis after IL-2 deprivation, and decreased in the spleen of aged mice. Lymphoid cells isolated from a patient with WS exhibited increased apoptosis after incubation with anti-Fas but not after incubation with the topoisomerase inhibitor VP16. RNase protection reviled dysregulation of the ICE family of apoptosis molecules in the WS cell line. These results indicate that the WS helicase is involved in certain pathways of apoptosis, and defective WS gene expression leads to accumulation of cells that are highly susceptibility to Fas-induced apoptosis.

Aging↗

PubMed1998

Autocrine and paracrine apoptosis are mediated by differential regulation of Fas ligand activity in two distinct Jurkat T cell populations.

Fas ligand (FasL) produced by activated T cells mediates autocrine-induced apoptosis to limit T cell expansion. To investigate the regulation of FasL activity, Jurkat cells were stably transfected with a 2.3-kb fragment of human FasL promoter that controlled the expression of a GFP reporter gene. Two populations of Jurkat cells with different levels of GFP expression were obtained. One population constitutively expressed high levels of GFP (GFP+), while the other population expressed low levels of GFP (GFP-). The level of GFP expression in the two populations correlated with their levels of FasL transcription and its functional activity. Autocrine regulation of apoptosis was demonstrated by increased FasL activity after stimulation of GFP- cells with anti-CD3, phorbyl myristyl acetate plus ionomycin, or Con A. Paracrine regulation of apoptosis was suggested by the induction of apoptosis of GFP- cells after coculture with unstimulated GFP+ cells. GFP+ cells exhibited a decreased sensitivity to FasL-mediated apoptosis compared with GFP- cells. Furthermore, the cell surface expression of Fas and CD4 was lower on GFP+ cells than GFP- cells, whereas the expression of CD45RO was higher. A decreased level of IL-2 was produced by GFP+ cells after phorbyl myristyl acetate and ionomycin stimulation. Our results indicate that a subpopulation of T cells that express low levels of FasL and IL-2, which are responsive to up-regulation of these molecules after activation, can undergo apoptosis either by suicide after activation or by a paracrine pathway mediated by T cells that constitutively express higher levels of FasL.

Apoptosis↗

PubMed1998

Fas/Fas ligand signaling during gestational T cell development.

Most thymocytes express high levels of Fas Ag (Apo-1/CD95); however, the role of Fas/Fas ligand-mediated apoptosis in thymocyte development remains unclear. During gestational development of thymocytes in C57BL/6(B6) +/+ mice, the highest levels of Fas ligand mRNA and Fas ligand protein expression were detected at gestational day (GD) 15, and there was a ninefold decrease in Fas ligand mRNA expression between GD 15 and 17 accompanied by a sixfold increase in Fas mRNA. Apoptotic thymocytes were first detected in the medulla at GD 15, and increasing numbers of cortical clusters and scattered, single apoptotic cells were present on GD 16 and 17. Thus, early apoptosis correlated with high expression of Fas ligand. High levels of Fas ligand mRNA were maintained throughout gestational development in thymocytes of Fas-deficient B6-lpr/lpr mice, but cortical clusters and scattered apoptotic cells were decreased relative to B6 +/+ mice before GD 17. Kinetic analysis of fetal thymic organ cultures treated with anti-Fas Ab demonstrated that thymocytes become sensitive to Fas-mediated apoptosis during the transition from the CD4-CD8- to the CD4+CD8+ phenotype. More mature CD4+CD8+ thymocytes and CD4+ and CD8+ thymocytes became resistant to Fas-mediated apoptosis after GD 17, despite high expression of Fas. However, low avidity engagement of the TCR on Fas-sensitive CD4+CD8+ thymocytes before GD 17 induced resistance to Fas-mediated apoptosis. The present results indicate that Fas plays a critical role in mediating apoptosis during early gestational thymocyte development and that thymocytes that receive a survival signal through TCR/CD3 become resistant to Fas-mediated apoptosis.

Animals↗