Hybridoma fusions with hamster hosts were undertaken to generate mAbs to mouse spleen dendritic cells. Two mAb were obtained and used to uncover the distinct integrins of these APC. One, 2E6, bound a determinant common to all members of the CD11/CD18 family, most likely the shared 90 kD CD18 beta chain. 2E6 immunoprecipitated the characteristic beta 2 integrin heterodimers from lymphocytes (p180, 90; CD11a) and macrophages (p170,90; CD11b), but from dendritic cells, a p150,90 (presumably CD11c) integrin was the predominant species. 2E6 inhibited the binding function of the CD11a and CD11b integrins on B cells and macrophages in appropriate assays, but 2E6 exerted little or no inhibition on the clustering of dendritic cells to T cells early in primary MLR, suggesting a CD11/CD18-independent mechanism for this binding. The second mAb, N418, precipitated a 150, 90 kD heterodimer that shared the 2E6 CD18 epitope. This N418 epitope may be the murine homologue of the previously characterized human CD11c molecule, but the epitope was only detected on dendritic cells. N418 did not react with peritoneal macrophages, anti-Ig-induced spleen B blasts, or bulk lymph node cells. When used to stain sections of spleen, N418 stained dendritic cells in the T-dependent areas, much like anti-class II mAbs that were also generated in these fusions. In addition, N418 revealed nests of dendritic cells that punctuated the rim of marginal zone macrophages between red and white pulp. This localization positioned most dendritic cells at regions where arterial vessels and T cells enter the white pulp. We conclude that the p150, 90 heterodimer is the major beta 2 integrin of spleen dendritic cells, and we speculate that it may function to localize these APC at sites that permit access to the recirculating pool of resting T cells.

Transcription of the antiatherogenic protein apolipoprotein AI is regulated by the thyroid hormone, L-triiodothyronine. Transient transfection and electrophoretic mobility shift assays were used to identify the cis-acting elements involved. In transient transfection assays, hormone bound to either thyroid hormone receptor alpha or beta exerts a positive effect through a thyroid hormone response element, site A (-208 to -193). In the absence of site A, liganded receptor alpha or beta have a negative effect on promoter activity. This negative effect is mediated by a 40 bp fragment spanning nucleotides -46 to -7. Closer examination of this region of the gene shows there to be a negative thyroid hormone response element at position -25 to -20 which is fused to the 3' end of the TATA element. Electrophoretic mobility shift assays show that bacterially expressed chicken or rat thyroid hormone receptor alpha 1 binds to site A, either as a homodimer or as a heterodimer with the human 9-cis-retinoic acid receptor alpha. In contrast, the negative thyroid hormone responsive element binds chicken thyroid hormone receptor alpha exclusively as a monomer. Site-directed mutagenesis of the negative thyroid hormone response element abolished the inhibitory effects of the hormone and increased basal promoter activity by up to 40-fold. These data suggest that functional positive and negative thyroid hormone response elements coexist within the rat apolipoprotein AI promoter and both elements contribute to the control of apolipoprotein AI gene expression.

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We studied the effects of transfection of the normal c-Ha-ras gene, ras" '12, and its oncogenic mutant, rasVal2, on expression of the a-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasval-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasval2 suppressed the activity of enhancer and promoter regions containing A+T-rich sequences (AT motif). In contrast, rasval-12 did not affect the promoter activity of the albumin and human hepatitis B virus pre-SI genes even though these promoters contain homologous A+T-rich elements. ras transfection appeared to induce phosphorylation of nuclear proteins that interact with the AFP AT motif, since gel mobility analysis revealed the formation of slow-moving complexes which was reversed by phosphatase treatment. However, similar changes in complex formation were observed with the albumin and hepatitis B surface antigen pre-Sl promoters. Therefore, this effect alone cannot explain the specific down regulation of the AFP promoter and enhancer activity. ras-mediated suppression of the AFP gene may reflect the process of developmental gene regulation in which AFP gene transcription is controlled by a G-protein-linked signal transduction cascade triggered by external growth stimuli.

We report here the isolation of an IgG Fc receptor from normal human T lymphocytes. The purified receptor has a nonreduced and a reduced component of molecular weights 120,000 and 60,000, respectively, and it was functionally active in the in vitro blocking of rosette formation between T lymphocytes and IgG-coated ox erythrocytes. An antiserum raised to the Fc receptor, and an isolated F(ab')2 fragment of this antiserum, also blocked rosette formation between T sensitive to irradiation and high-dose corticosteroid treatment (9), have diminished RNA content (10), and have reduced surface charge (11) when compared with the majority of T lymphocytes. In addition, at least some Ty cells can suppress immunoglobulin production and can participate in natural killer-cell activity and antibody-dependent, cell-mediated cytotoxicity (12)(13)(14)(15). Because the isolated IgG Fc receptor described in this report has retained functional activity and the antiserum raised against it has selective affinity for the receptor, the methods reported here provide a means for the chemical characterization of the receptor and a practical approach for the analysis of the biologic role of Fc receptor-positive T cells. METHODS Fc Receptor Isolation. Mononuclear cells from the peripheral blood of normal controls were isolated on FicollHypaque (16) and were depleted of phagocytic cells by allowing them to ingest carbonyl iron (13). T cells were rosetted with neuraminidase-treated sheep erythrocytes, separated on Ficoll-Hypaque, and the attached sheep erythrocytes were lysed by 0.1 M Tris-HCl, pH 7.2/0.83% NH4CI (13). Judged to be 96% pure by rerosetting with sheep erythrocytes and by the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3645 lack of surface Ig and nonspecific esterase-positive cells, 108 T cells were suspended in 0.13 M NaCI/0.5 mM MgCl2/0.02 M Hepes (pH 6.0; 1 vol)/0.5 M sucrose (1 vol) at a concentration of 5 X 107 cells/ml and were subjected to nitrogen cavitation at a pressure of 800 psi for 20 min (Artisan Bomb, Artisan Industries, Waltham, MA). The 2-ml supernatant material obtained after centrifugation (100,000 X g for 1 hr at 00C) was subjected to a 3-hr electrodialysis against distilled water using a current of 40 mA to remove salts and to fragment the T-cell membranes. Neutralization was achieved by adding small amounts of triethylamine. (The electrodialysis procedure, a new method for cell membrane disruption, will be described more fully elsewhere by C. Galanos.) The dissolved T-cell membranes were then immediately passed over a 1 X 15 cm column of Sepharose 4B to which immunoelectrophoretically pure, pooled heat-aggregated (17) human IgG had been covalently bonded (18). After washing the column with 5-column volumes of phosphate-buffered saline, the adherent fraction was removed by a passage of 0.1 M glycine-HCI, pH 2.7. The eluted material w...

The eye is continually subjected to ambient radiation. The benign function of this light is to direct vision and circadian rhythm. However, under intense light from artificial sources such as lasers and operating room microscopes the natural protection of eye against light damage may be undermined. The damage can be a result of a photochemical, thermal or mechanical injury which can all disrupt ocular tissues . The mechani sm for the above damage involves the production free radical and reactive oxygen intermediates either as a primary effect of the absorbance of the light or as a secondary effect due to an inflammatory response to heat or mechanical injury. This can induce a cytokine cascade as well as induce the increased production of nitric oxide.Glutathione mimics can block light induced free radical and oxygen radical intermediates in the eye. For instance, singlet oxygen is scavenged at a rate of 106 M1 s1 hydroxyl radical is scavenged at a rate of l0 W1 S-and superoxide is scavenged at a rate of 10 M1 s 1 In addition thiols block nitric oxide (NO) and other intermediates involved in an inflammatory response. Glutathione mimics which have offered in vivo protection against light damage to the lens and retina, have the potential to protect against laser-induced damage to the human eye.

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Oestrogens protect against ischaemic heart disease in the post-menopausal female by increasing serum concentrations of apolipoprotein (apo) AI and the abundance of high-density lipoprotein particles. In men and experimental male animals, the administration of oestrogen has variable effects on apo AI expression. As the major mode of oestrogen action on target genes involves regulating promoter activity and hence transcription, oestrogen is expected to alter transcription of the apo AI gene. To test this hypothesis, the effect of 17 -oestradiol (E 2 ), on rat apo AI promoter activity in male hepatoma HuH-7 cells, was tested by cotransfecting a reporter template, pAI.474.CAT containing 474 to 7 of the rat apo AI promoter and an oestrogen receptor (ER) expression vector, pCMV-ER. Transfected cells exposed to E 2 showed a dose-dependent decrease in chloramphenicol acetyltransferase (CAT)-activity, with a maximum 91 1·5% reduction at 1 µM E 2 . Deletional analysis of the promoter localized the inhibitory effect of ER and E 2 to site B ( 170 to 144) with an adjacent 5 contiguous motif, site S ( 186 to 171) acting as an amplifier. HuH-7 cell nuclear extracts showed binding activities with both sites S and B, but recombinant human ER did not. Furthermore, nuclear extracts from E 2 -treated HuH-7 cells showed weaker binding activity to site B, but not to site S. In summary, the inhibitory effect of ER and E 2 on rat apo AI gene activity is mediated by a promoter element, site B. This inhibitory effect arises from a mechanism that does not involve direct ER binding to the B-element. The conclusion that E 2 inhibits apo AI transcription was confirmed in vivo. Treatment of male adult Sprague-Dawley rats with up to 200 µg E 2 for 7 days decreased apo AI protein and hepatic mRNA by 72 21% and 68 1·4% respectively. Results of 'run-on' transcription of the apo AI gene in isolated hepatic nuclei showed a 55% decrease in hormone-treated male rats. These findings suggest that E 2 exerts primarily an inhibitory effect within male hepatic nuclei.