Apoptotic Thymocytes Research Articles

Of plaques and stones: the SR-B1 (scavenger receptor class B, type 1)

HepatologyVolume 26, Issue 4 p. 1072-1074 Hepatology ElsewhereFree Access Of plaques and stones: the SR-B1 (scavenger receptor class B, type 1) M A Arrese, M A ArreseSearch for more papers by this authorJ M Crawford, J M CrawfordSearch for more papers by this author M A Arrese, M A ArreseSearch for more papers by this authorJ M Crawford, J M CrawfordSearch for more papers by this author First published: 30 December 2003 https://doi.org/10.1002/hep.510260441Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Scheiber J, Fuchs M, Hermann E, Tauber G, Stange EF. Biliary cholesterol secretion and bile acid formation in the hamster: the role of newly synthesized cholesterol. J Lipid Res 1994; 35: 690– 697. 2 Cooper AD. Hepatic lipoprotein metabolism: recent molecular insights. Prog Liver Dis 1995; 13: 173– 200. 3 Lange Y, Steck TL. The role of intracellular cholesterol transport in cholesterol homeostasis. Trends Cell Biol 1996; 6: 205– 208. 4 Superko HR. Beyond LDL cholesterol reduction. Circulation 1996; 94: 2351– 2354. 5 Steinberg D. A docking receptor for HDL cholesterol esters. Science 1996; 271: 460– 461. 6 Rigotti A, Trigatti B, Babitt J, Penman M, Xu SZ, Krieger M. Scavenger receptor B-1–a cell surface receptor for HDL. Curr Opin Lipidol 1997; 8: 181– 188. 7 Acton S, Rigotti A, Landschulz DT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 1996; 271: 518– 520. 8 Babitt J, Trigatti B, Rigotti A, Smart EJ, Anderson RGW, Xu SZ, Krieger M. Murine SR-B1, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma caveolae. J Biol Chem 1997; 272: 13242– 13249. 9 Rigotti A, Edelman ER, Seifert P, Iqbal SN, DeMattos RB, Temel RE, Krieger M, et al. Regulation by adrenocorticotropic hormone of the in vivo expression of scavenger receptor class B type 1 (SR-B1), a high density lipoprotein receptor, in steroidogenic cells of the murine adrenal gland. J Biol Chem 1996; 271: 33545– 33549. 10 Landschulz KT, Pathak RK, Rigotti A, Krieger M, Hobbs HH. Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat. J Clin Invest 1996; 98: 984– 995. 11 Kozarsky KF, Donahee MH, Rigotti A, Iqbal SN, Edelman ER, Krieger M. Overexpression of the HDL receptor SR-B1 alters plasma HDL and bile cholesterol levels. Nature 1997; 387: 414– 417. 12 Thornton JR, Heaton KW, Macfarlane DG. A relation between high-density lipoprotein cholesterol and bile cholesterol saturation. Br Med J 1981; 283: 1352– 1354. 13 Petitti DB, Friedman GD, Klatsky AL. Association of a history of gallbladder disease with a reduced concentration of high-density-lipoprotein cholesterol. N Engl J Med 1981; 304: 1396– 1398. 14 Osono Y, Woollett LA, Marotti KR, Melchior GW, Dietschy JM. Centripetal cholesterol flux from extrahepatic organs to the liver is independent of the concentration of high density lipoprotein-cholesterol in plasma. Proc Natl Acad Sci U S A 1996; 93: 4114– 4119. 15 Robins SJ, Fasulo JM. High density lipoproteins, but not other lipoproteins, provide a vehicle for sterol transport to bile. J Clin Invest 1997; 99: 380– 384. 16 Crawford JM. Role of vesicle-mediated transport pathways in hepatocellular bile secretion. Semin Liver Dis 1996; 16: 169– 189. 17 Kadowaki H, Patton GM, Robins SJ. Metabolism of high density lipoprotein lipids by the rat liver: evidence for participation of hepatic lipase in the uptake of cholesteryl ester. J Lipid Res 1992; 33: 1689– 1698. 18 Elferink RPJ Oude, Tytgat GNJ, Groen AK. The role of mdr2 P-glycoprotein in hepatobiliary lipid transport. FASEB J 1997; 11: 19– 28. 19 Wang DQH, Paigen B, Carey MC. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice. Physical-chemistry of gallbladder bile. J Lipid Res 1997; 38: 1395– 1411. 20 Murao K, Terpstra V, Green SR, Kondratenko N, Steinberg D, Quehenberger O. Characterization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes. J Biol Chem 1997; 272: 17551– 17557. Citing Literature Volume26, Issue4October 1997Pages 1072-1074 ReferencesRelatedInformation

Purification and properties of DNase gamma from apoptotic rat thymocytes.

We previously identified three distinct DNA endonucleases, DNases alpha, beta and gamma, present in rat thymocyte nuclei. On the basis of their enzymic and biochemical properties, gamma-type DNase was regarded as a candidate for the apoptotic endonuclease. Here we purified DNase gamma to apparent homogeneity from apoptotic rat thymocyte nuclei induced by X-irradiation and characterized its properties in detail. The purified DNase gamma exhibited one predominant protein band on SDS/PAGE and an endonuclease activity in a zymography with an estimated molecular mass of 33 kDa. The molecular mass of the native form determined by G2000SW gel-filtration HPLC was 30 kDa. Amino acid analysis showed that the amino acid composition of DNase gamma was similar to that of rat DNase I (molecular mass 32 kDa) but different with regard to alanine and lysine residues. The N-terminal amino acid sequence of DNase gamma was revealed to be not identical with that of rat DNase I. In accordance with previous studies, homogeneously purified DNase gamma requires both Ca2+ and Mg2+ for activity. This requirement could be partially supplied by Mn2+. Of the bivalent metal ions tested, Co2+, Ni2+, Cu2+ and Zn2+ inhibited DNase gamma activity. These bivalent cations also suppressed apoptotic DNA fragmentation in rat thymocytes irradiated by X-rays. The same order of inhibitory ability was observed for these bivalent metal ions in vivo (in intact cells) and in vitro, suggesting that the suppression of apoptotic DNA fragmentation at the cellular level is due to the inhibition of DNase gamma. DNase gamma activity was found to exist at high levels in spleen, lymph node, thymus, liver and kidney, but little was present in brain, heart or pancreas. On the basis of these findings, together with previous data, we conclude that DNase gamma is a novel DNase I-like endonuclease responsible for internucleosomal cleavage of chromatin during thymic apoptosis.

Autoantibodies in systemic lupus erythematosus.

Research on pathogenic and nonpathogenic anti-DNA antibodies has shown that the charge and affinity for double-stranded DNA may not be adequate predictors of their pathogenicity. In turn, anti-DNA-induced renal damage could be caused by differences in fine specificities. Penetration of anti-double-stranded DNA monoclonal antibodies into live cells appears to require DNA or a DNA-like membrane receptor. Anti-U1 ribonucleoproteins penetrate live cells and induce apoptosis. HLA-DRB1*1501 or HLA-DRB1*1503, DQA1*0102, and DQB1*0602 haplotypes were increased in patients with systemic lupus erythematosus with anti-ribosomal protein P (anti-P) antibodies, particularly in whites, blacks, and Mexican-Americans. Anti-Ro/Sjögren's syndrome antigen A antibodies had a strong predictive diagnostic value for systemic lupus erythematosus among patients positive for antinuclear antibodies and negative for double-stranded DNA but had no utility among those positive for both antinuclear antibodies and anti-DNA antibodies. True antiphospholipid murine monoclonal antibodies display an unusual number of cationic residues in the H-chain CD3 region, whereas anti-beta 2-glycoprotein-I monoclonal antibodies lack them. The association of IgG anti-beta 2-glycoprotein-I with a history of thrombosis in systemic lupus erythematosus has been largely confirmed. Apoptotic thymocytes bind antiphospholipid antibodies in a beta 2-glycoprotein-I-dependent manner and may constitute a source of immunogen in autoimmune disease.

Still Immunodetectable Nuclear RNPs Are Extruded from the Cytoplasm of Spontaneously Apoptotic Thymocytes

The fate of different nuclear ribonucleoprotein (RNP) components was investigated during spontaneous apoptosis of thymocytes using specific monoclonal antibodies against snRNPs, hnRNPs, and ribosomal proteins at light and electron microscopy levels and by flow cytometry. It was found that, during apoptosis, nuclear RNP-containing structures (perichromatin granules, interchromatin granules, and perichromatin fibrils) segregate in the interchromatin space and cluster into heterogeneous aggregates of granules in which some of the structures may still be recognized morphologically. Along with the progress of apoptosis, the clusters are extruded from the nucleus into the cytoplasm, from which they are finally released via cytoplasmic extrusions. At all these stages, RNPs inside the clusters are always recognized by specific antibodies, even when they bleb out of the cell surface, thus suggesting that degradation of RNPs might be only partial during apoptosis. This could be potentially harmful in genetically susceptible subjects, as the appropriate MHC class II molecules may capture and present normally cryptic self-peptides. It is tempting to speculate that this event might have implications in the etiology of autoimmune diseases.

Macrophages lacking scavenger receptor A show a decrease in binding and uptake of acetylated low-density lipoprotein and of apoptotic thymocytes, but not of oxidatively damaged red blood cells.

Macrophage binding of oxidatively damaged red blood cells (OxRBC) and apoptotic thymocytes correlates in many instances with a loss of phospholipid bilayer asymmetry, i.e., with an increase in expression of phosphatidylserine on the outer leaflet of the plasma membrane. Oxidatively modified LDL (OxLDL) can compete for the binding of these ligands to macrophages. However, the receptor(s) responsible remains to be identified. The present studies show that mouse peritoneal macrophages totally lacking scavenger receptor A (SRA) bound OxRBC just as effectively as wild-type macrophages, whereas their binding and uptake of acetyl LDL was reduced by more than 80%. Binding of apoptotic thymocytes and binding of OxLDL were also reduced, but only by 20-30%. We conclude that SRA is not involved in the recognition of phosphatidylserine-rich membranes but contributes to the binding of OxLDL and apoptotic thymocytes. The binding of OxRBC was almost totally calcium-dependent, whereas the binding of apoptotic thymocytes was not, suggesting that the mechanisms involved in their uptake by macrophages under these conditions were different.

Characterization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes.

Recently, a murine scavenger receptor type B class I (SR-BI) was identified that binds high density lipoprotein (HDL) and mediates the selective uptake of cholesterol esters. The human CD36 and LIMPII analogous-1 (CLA-1) receptor shows high sequence homology with SR-BI, but their functional relationship has not been determined. Transfected cells expressing CLA-1 bound HDL with a Kd of about 35 microg/ml, similar to the Kd for HDL binding to rodent SR-BI. This binding resulted in an intracellular accumulation of HDL-derived [3H]cholesterol esters without internalization or degradation of 125I-apolipoprotein. CLA-1 was strongly expressed in the adrenal gland and was also abundant in liver and testis, suggesting that CLA-1, like SR-BI, could play a role in the metabolism of HDL. However, CLA-1 was also expressed in monocytes and, like SR-BI, recognized modified forms of low density lipoproteins as well as native LDL and anionic phospholipids. These findings suggest that CLA-1 might have additional physiological functions. We found that CLA-1 recognizes apoptotic thymocytes. Our results demonstrate that CLA-1, a close structural homologue of SR-BI, is also functionally related to SR-BI and may play an important role as a "docking receptor" for HDL in connection with selective uptake of cholesterol esters. An additional role in recognition of damaged cells is suggested by these studies.

Utilization of an in vitro assay to evaluate chromatin degradation by candidate apoptotic nucleases

Apoptosis is commonly associated with the catabolism of the genome in the dying cell. The chromatin degradation occurs in essentially two forms: (1) internucleosomal DNA cleavage to generate oligonucleosomal-length fragments (180-200 bp and multiples thereof), and (2) cleavage of higher order chromatin structures to generate approximately 30-50 Kb fragments. To investigate this component of apoptosis and identify the nuclease(s) responsible, we have developed and utilized an in vitro assay that recapitulates the genomic destruction seen during apoptosis in vivo and allows the simultaneous analysis of both forms of DNA degradation from the same sample. Using this assay we evaluated the digestion patterns of several candidate apoptotic nucleases: DNase I, DNase II, and cyclophilin (NUC18) as well as the bacterial enzyme micrococcal nuclease (not thought to be involved in apoptosis). Chromatin degraded by DNase I formed a smear of DNA on conventional static-field agarose gels and approximately amp;30 - 50 Kb DNA fragments on pulsed field gels. In contrast, DNase II, at a physiologically relevant pH, had no effect on the integrity of HeLa chromatin in either analysis. Similar to DNase I, cyclophilin C produced only approximately 30-50 Kb DNA fragments but did not generate internucleosomal fragments. In contrast, micrococcal nuclease generated both oligonucleosomal and approximately 30-50 Kb DNA fragments. Nuclear extracts from glucocorticoid-treated apoptotic thymocytes generated oligonucleosomal DNA fragments and the larger approximately 30-50 Kb DNA fragments, fully recapitulating both types of apoptotic DNA degradation. Previously, differential sensitivity of nucleases to inhibition by Zn2+ was used to argue that two distinct enzymes mediate approximately 30-50 Kb DNA cleavage and internucleosomal DNA degradation. While, the nuclease activity present in thymocyte nuclear extracts was differentially sensitive to inhibition by Zn2+ during short term incubations it was not during prolonged digestions, suggesting that differences in DNA detection are likely to account for previous results. Together our studies show that none of the nucleases commonly associated with apoptosis could fully recapitulate the DNA degradation seen in vivo.

The Condensation of Chromatin in Apoptotic Thymocytes Shows a Specific Structural Change

Chromatin condensation and DNA cleavage at internucleosomal sites have been recognized early as hallmarks of apoptosis, and it has been suggested that extensive DNA chain scission could directly result in the formation of dense chromatin bodies. Here we have shown that no causal relationship exists between DNA degradation and chromatin condensation in glucocorticoid-induced thymocyte apoptosis. The chromatin rearrangement occurred independent of as well as prior to DNA cleavage and involved a specific conformational change at the nucleosome level. In the early stages of the process, the core particles appeared to be tightly packed face-to-face in smooth 11-nm filaments that progressively folded to generate a closely woven network. The network finally collapsed, producing dense apoptotic bodies. Since trypsin digestion relaxed condensed chromatin and histone H4 underwent appreciable deacetylation in the apoptotic cell, we suggest that changes in the DNA-histone interactions represented a major modulating factor of condensation.

The semiconductor elements arsenic and indium induce apoptosis in rat thymocytes

Indium arsenide and gallium arsenide are important new materials in the semiconductor industry due to their superior electronic properties in comparison with the older silicon-based materials. Animal experiments have shown that exposure to these compounds induces marked alterations in gene expression and immune response. Toxicity to the immune system has frequently been related to T and B cell apoptosis. In the present study we show that the semiconductor elements indium (In) and arsenic (As) are able to induce apoptosis in rat thymocytes in vitro. The results show that exposure to InCl 3 (1, 10, or 100 μM) or Na AsO 2 (0.01, 0.1, or 1 μM) induced DNA laddering after 6 h of incubation without compromising cell viability. These results were corroborated by flow cytometry analysis of propidium iodide-loaded cells, showing a typical high hypodiploid DNA peak in apoptotic thymocytes. Higher doses of In (1 mM) or As (10–100 μM) induced cell death by necrosis. These data indicate that In and As can induce apoptosis and necrosis in T lymphocytes in a dose-dependent manner, which may be of relevance for their immunotoxicity.

Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro.

Numerous immature thymocytes undergo apoptosis and are rapidly engulfed by phagocytic thymic macrophages. The macrophage surface receptors involved in apoptotic thymocyte recognition are unknown. We have examined the role of the class A macrophage scavenger receptor (SR-A) in the engulfment of apoptotic thymocytes. Uptake of steroid-treated apoptotic thymocytes by thymic and inflammatory-elicited SR-A positive macrophages is partially inhibited by an anti-SR-A mAb and more completely by a range of scavenger receptor ligands. Thymic macrophages from mice with targeted disruption of the SR-A gene show a 50% reduction in phagocytosis of apoptotic thymocytes in vitro. These data suggest that SR-A may play a role in the clearance of dying cells in the thymus.

DNA fragmentation during thymic apoptosis is catalyzed by DNase γ

The physiological and pathological importance of cell death by apoptosis has recently been recognized. One of the hallmarks of apoptosis is the enzymatic cleavage of genomic DNA into nucleosomal oligomers. The identification of an endonuclease responsible for apoptosis might help to explain how this cell suicide is regulated and why DNA is cleaved. Here, we found that γ type of DNase was retained in apoptotic rat thymocyte nuclei. Homogeneously purified DNase γ (Mr = 33 kDa) from the apoptotic nuclei was revealed to be a Ca2+/Mg2+-dependent endonuclease and inhibited by Zn2+. This enzyme cleaved chromosomal DNA with 3′-hydroxyl (OH) and 5′-phosphoryl (P) ends. The cleavage ends and its divalent cation dependencies match those observed in apoptotic thymocytes induced by X-irradiation or glucocorticoid treatment, indicating that this endonuclease is a central component of the thymic apoptosis machinery.

Cellular catabolism in apoptosis: DNA degradation and endonuclease activation.

Recent research has focused on identifying the biochemical events associated with the apoptotic process. These include specific degradation of the chromatin which was described by Wyllie in 1980 [1], with the report of the appearance of discretely sized DNA fragments from apoptotic rat thymocytes. The fragments corresponded in size to strands of DNA that were cleaved at internucleosomal regions and create a 'ladder pattern' when electrophoresed on an agarose gel. Because of its near universality, internucleosomal DNA degradation is considered a diagnostic hallmark of cells undergoing apoptosis. It is of great interest to identify the enzymes involved, and some of the candidates will be discussed.

Anti-phospholipid autoantibodies bind to apoptotic, but not viable, thymocytes in a beta 2-glycoprotein I-dependent manner.

Anti-phospholipid autoantibodies (aPL) are associated with a clinical syndrome of hypercoagulability, thrombocytopenia, and fetal loss. Several groups have shown that the in vitro target of many aPL is not a pure phospholipid Ag, but is either a complex between anionic phospholipid and the plasma protein beta2-glycoprotein I (beta 2GPI) or the protein beta 2GPI alone. Anionic phospholipids are normally absent from the extracellular surface of cell membranes but redistribute from the inner to the outer leaflet during apoptosis. We show that aPL bind specifically to apoptotic, but not viable, thymocytes, and that binding is dependent upon the presence of beta 2GPI. Moreover, we show that beta 2GPI binds selectively to the surface of apoptotic thymocytes to generate an epitope for antiphospholipid autoantibodies. These findings suggest that apoptotic cells may be the natural immunogen and/or target for aPL. Moreover, we propose that the interaction of circulating beta 2GPI with redistributed anionic phospholipid may itself generate a novel ligand by which apoptotic cells are recognized directly for phagocytic clearance.

Activation of the Na(+)/H(+) antiporter, Na+/HCO3(-)/CO3(2-) cotransporter, or Cl(-)/HCO3(-) exchanger in spontaneous thymocyte apoptosis.

This study was undertaken to define the role of ion transporters on the apoptosis of thymocytes. Culture conditions, such as the ionic strength of NaCl, Ca2+, the buffer system (HCO3-/CO2), and the pH, could influence the spontaneous apoptosis of thymocytes. Depletion of NaCl in the culture medium either delayed or completely inhibited the apoptotic process of thymocytes, while its restoration led to a dose-dependent apoptosis. A high concentration (100 microM) of Ca2+ induced thymocyte apoptosis in the nominal absence of NaCl, whereas a low concentration (10 microM) enhanced apoptosis in the presence of 138 mM NaCl. Thymocytes had a higher spontaneous apoptotic rate in cultures without HCO3-/CO2 than in those with HCO3-/CO2. The thymocyte apoptosis completely ceased in medium at pH 6.0 and was considerably enhanced at pH 7.6. Intracellular pH, determined with the pH-sensitive bis-carboxyethyl carboxyfluorescein probe, was higher in apoptotic thymocytes than in nonapoptotic cells. Spontaneous apoptosis occurred in cells with alkaline intracellular condition, whereas it was considerably retarded in cells under acidified conditions. Amiloride analogue, including 5-(N,N-dimethyl)-amiloride (Na+/H+ antiporter), 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (Cl-/HCO3- exchanger), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (Na+/HCO3-/CO3(2-) cotransporter), inhibited the spontaneous thymocyte apoptosis. In contrast, neither cycloheximide nor actinomycin D had the same effect. In addition, thymocyte apoptosis was enhanced by PMA, but inhibited by forskolin. Taken together, thymocytes cultured in vitro underwent apoptosis with increased intracellular pH via activation of Na+/H+ antiporter, Na+/HCO3-/CO3(2-) cotransporter, or Cl-/HCO3- exchanger. This process does not require de novo protein synthesis.

Negative selection by endogenous antigen and superantigen occurs at multiple thymic sites.

The site of negative selection in the thymus has been inferred from a range of different experiments. Analysis of thymic deletion of V beta 5+, V beta 11+ or V beta 17a+ cells in H-2E transgenic mice led to the theory that negative selection occurs predominantly in the medulla (specifically, through presentation by medullary dendritic cells). Other experiments investigated whether transgenic TCR are deleted at the double-positive (DP) or single-positive stage following encounter with peptide ligand: by flow cytometric analysis deletion is generally found to occur at the DP thymocyte stage and as these cells are found predominantly in the cortex, it has been inferred that this is the key site of negative selection. The visualization of apoptotic thymocytes in situ has recently been reported for specific examples of negative selection. Using a panel of TCR transgenic lines in which negative selection occurs at different stages of thymocyte development, we have used TUNEL staining to analyse the anatomical sites of thymocyte apoptosis. For the first time we have been able to compare directly the sites of deletion induced by the endogenous cognate peptides or by endogenous superantigen. We show that generalization from the medullary deletion of V beta 5+, V beta 11+ or V beta 17a+ cells by the endogenous superantigens Mtv 8 and 9 from limited examples of cortical deletion by exogenous peptide administered to TCR transgenic mice is over-simplified. Apoptotic thymocytes in mice lacking Mtv superantigens are indeed localized in the cortex. However, when deletion is induced by cognate self peptide, apoptosis can occur in the cortex, the medulla or at the junction between the two.

Decreased production of reactive oxygen intermediates is an early event during in vitro apoptosis of rat thymocytes

Thymocyte apoptosis is one of the best characterized experimental models of apoptosis that can be induced by a variety of stimuli such as glucocorticoids, ionizing radiation, antibodies, and toxins. Recently, it has been suggested that oxidative stress is a common mediator of apoptosis. However, little is known about the production and possible function of reactive oxygen intermediates (ROI) in thymocytes. We used a highly sensitive flow cytometric assay with the hydrogen peroxide-sensitive dye, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), to measure intracellular ROI production in rat thymocytes, to study its primary sources, and to compare ROI levels in normal and apoptotic thymocytes. Apoptosis was induced by incubating the cells in the presence or absence of dexamethasone (Dex) at 37°C in vitro. Normal thymocytes spontaneously produced significant amounts of ROI. Catalase or superoxide dismutase did not affect this intracellular fluorescence, presumably due to their failure to penetrate into the cells. However, N-acetyl- l-cysteine significantly attenuated the fluorescence in a dose-dependent manner. Significant inhibition of the intracellular fluorescence was also observed by addition of N-nitro- l-arginine methyl ester (L-NAME), that could not be reversed by L-arginine. The addition of N-nitro-D-arginine methyl ester (D-NAME) also caused considerable inhibition. This indicates that the inhibition by L-NAME or D-NAME is due to a direct scavenging effect, and nitric oxide production is not likely to be involved. In contrast to neutrophils and macrophages whose superoxide anions are released from membrane-bound NADPH oxidase, the production of ROI in thymocytes is likely to originate mainly from mitochondria, as indicated by the inhibitory effect of the addition of rotenone or antimycin A. The addition of lymphocyte simulators phytohemagglutinin (PHA), concanavalin A (Con A), or phorbol 12-myristate 13-acetate (PMA) enhanced intracellular fluorescence of thymocytes. This increase was abrogated by addition of rotenone or antimycin A. The ROI production was decreased with time after incubation of the thymocytes for 1, 3, and 6 h in vitro. The appearance of apoptosis of thymocytes in vitro, as indicated by DNA content of cells by flow cytometry and DNA ladder formation in agarose gel electrophoresis, was delayed, as compared to the time course of the decreased ROI production. The addition of Dex to the culture medium accelerated both of these processes. The results suggest that a decreased spontaneous production of ROI in thymocytes precedes the spontaneous in vitro apoptosis and Dex exaggerates these changes

Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes.

In dexamethasone-treated thymocyte cultures an increase in nuclear proteolytic activity paralleled chromatin fragmentation and the appearance of small apoptotic cells. The elevation of nuclear proteolytic activity was accompanied by site-specific degradation of nuclear mitotic apparatus protein and lamin B, two essential components of the nuclear matrix. Nuclear mitotic apparatus protein phosphorylation and cleavage into 200 and 48 kDa fragments occurred within 30 minutes of dexamethasone treatment. Cleavage of lamin B, which generated a fragment of 46 kDa consistent with the central rod domain of the protein, was also detected after 30 minutes of exposure to the steroid hormone. The level of lamin B phosphorylation did not change as a result of the dexamethasone treatment and the lamina did not solubilize until the later stages of apoptosis. Initial DNA breaks, detected by the terminal transferase-mediated dUTP-biotin nick end labeling assay, occurred throughout the nuclei and solubilization of lamina was not required for this process to commence. The data presented in this paper support a model of apoptotic nuclear destruction brought about by the site-specific proteolysis of key structural proteins. Both the nuclear mitotic apparatus protein and lamin B were specifically targeted by protease(s) at early stages of the cell death pathway, which possibly initiate the cascade of degradative events in apoptosis.

Characterization of the spontaneous apoptosis of rat thymocytes in vitro.

Unlike splenic or blood lymphocytes, rat thymocytes spontaneously undergo continuously increasing apoptosis during culture. In this study we characterized apoptotic thymus cells of rats according to cell size, nuclear dye binding and surface marker expression. Furthermore, the effects of cell density in culture, the age of the donor animals, glucocorticoids, and inhibition of protein synthesis were studied. It was found that: (1) apoptotic rat thymocytes are recognized in flow cytometry as small, acridine orange low, CD4low, CD8high cells; (2) the rate of apoptosis is dependent on the cell density in the culture in a biphasic manner; (3) thymic apoptosis increases with age of the donor animal in fresh, as well as in 24-hour cultivated cell suspensions; (4) neither adrenalectomy nor in vivo or in vitro treatment with the glucocorticoid antagonist RU 38486 influenced spontaneous apoptosis of thymocytes, and (5) inhibition of protein synthesis, which decreases apoptosis induced by corticosterone, had no effect on spontaneous apoptosis of thymocytes.

Structural Evidence for Thymocyte Apoptosis and Clearance Mechanism of Apoptotic Thymocytes in the Thymus

Structural Evidence for Thymocyte Apoptosis and Clearance Mechanism of Apoptotic Thymocytes in the Thymus

スカベンジャー受容体遺伝子欠損マウスのマクロファージ系細胞の解析

Macrophage scavenger receptors (MSR) have been implicated in atherogenesis via receptor mediated uptake of modified lipoproteins and are reportedly cation independent adhesion molecules on macrophages, but their major physiological functions remain obscure. Thus to elucidate their in vivo role, mice lacking both type I and type II MSR were created by gene targeting. Both the heterozygotes and homozygotes in the MSR mutation were normal in appearance and growth, and were fertile. The uptake of oxdized low density lipoprotein (LDL), acetyl LDL and advanced glycosylation end product (AGE) by peritoneal macrophages derived from homozygotes declined to between 25 and 40% as compared with those from the wild type. Numbers of adhesive MSR defected macrophages on a plastic surfaces were <50% compared with those of the wild type. Hepatic granulomas induced by zymosan injection in homozygotes were larger, diffuse and less circumscribed compared with those of the wild type. With regard to phagocytic clearance for apoptotic thymocytes, it has been clarified that MSR accounts for approximetely half of the phagocytic activity required for the uptake of apoptotic thymocytes.These results indicate that the MRS not only functions to uptake modified LDL but also participates widely in the removal of foreign bodies and waste materials. Moreover, MSR appears to exert multifunctional roles of endocytosis, adhesion and phagocytosis in macrophages.