Monoclonal antibody to a structure expressed on a subpopulation of rat CD8 T cell subsets.

We have developed a monoclonal antibody, RTS-1, which can divide a rat CD8 (+) peripheral T cell population into two functionally distinct subsets. The cell-surface structure defined by this antibody is a glycoprotein with a molecular weight of 220 kDa found to be a high molecular isoform of rat CD45 antigen. CD4 (+) T cells were not stained by RTS-1 antibody. The cytotoxic T cell-enriched population did not express RTS-1 epitope on the cell surface. CD8 (+) spleen cells as well as RTS-1(+)CD8(+)T cells exhibited strong inhibition on mitogen-induced immunoglobulin G production by rat B cells. Furthermore, RTS-1 antibody, but not the control antibody, abolished CD8(+)T cell-mediated inhibition of immunoglobulin G production by rat B cells. These data suggest that RTS-1 antibody recognizes a unique determinant of rat CD45 antigen that is expressed on a fraction of CD8(+) cells.     

Biosynthesis of inter-alpha-trypsin inhibitor and alpha 1-microglobulin in a human hepatoma cell line.

1. Biosynthesis of alpha 1-microglobulin and inter-alpha-trypsin inhibitor was investigated in a human hepatoma cell line HepG-2. 2. alpha 1-Microglobulin was translated as a precursor common with the light chain of inter-alpha-trypsin inhibitor. 3. alpha 1-Microglobulin was synthesized and secreted into the growth medium within 30 min. 4. Processing of inter-alpha-trypsin-inhibitor-related proteins appeared slow and incomplete. The light chain was connected via a chondroitinsulphate to a heavy chain to form a 125,000-Mr protein and secreted within 1-4 hr.     

bFGF suppresses serum-deprivation-induced apoptosis in a human lens epithelial cell line.

There is increasing evidence that basic fibroblast growth factor (bFGF) plays an important role in cell proliferation, differentiation, and survival in various systems. In the eye, although a truncated, dominant negative bFGF receptor in transgenic mice induced defective lens development and caused lens fiber cells to display characteristics of apoptosis, there is little direct evidence of the effect of bFGF on lens epithelial cell apoptosis. Our study examines the effects of bFGF on programmed cell death induced by serum deprivation using a human lens epithelial cell line. Cells supplemented with 20% fetal bovine serum were used as normal controls. Over a period of 7 days, the addition of 100 ng/ml bFGF effectively suppressed serum-deprived apoptosis. The expression of gamma-crystallin and major intrinsic protein, which are markers of lens cell differentiation, was not detected. Also there was no significant difference in cell proliferation between serum-deprived cells with or without bFGF. ICE (caspase-1) was expressed under both the conditions, but the level of expression between the two groups was not substantially different. bcl-2 and c-myc were upregulated only in bFGF-treated cells. Thus we speculate that the inhibitory effect of bFGF on apoptosis is through the upregulation of the inhibitor of apoptosis, instead of downregulation of the initiator. This effect appears to be independent of lens cell differentiation and proliferation.     

Biosynthesis of calcitonin by a rat medullary thyroid carcinoma cell line

The 32-amino acid form of the peptide hormone calcitonin is the product of a series of post-translational processing steps of a 13,400-dalton precursor, procalcitonin. We have now identified the steps involved in proteolytic paring of the precursor to the mature secretory form. Cultures of the CA-77 cell line were radiolabeled and the various forms of calcitonin were isolated by specific immunoprecipitation followed by fractionation on gel filtration and reversed-phase high performance liquid chromatography. Pulse-chase kinetics showed that procalcitonin was cleaved to a 6,500-dalton biosynthetic intermediate which was subsequently processed to the size of mature calcitonin (3,400 daltons). Partial microsequencing of the [35S] methionine-labeled intermediate indicated that the sequence consisted of the COOH-terminal 52 residues of procalcitonin. Partial microsequencing of the [35S]methionine- or [3H]proline-labeled 3,400-dalton species revealed that it was indistinguishable from naturally occurring, amidated calcitonin. These data define the major pathway for calcitonin biosynthesis in this neoplastic cell line and presumably in normal cells.     

A second subunit of CD8 is expressed in human T cells.

The CD8 glycoprotein plays important functions in T cell development and in T cell activation. In rodents, CD8 is a heterodimer, consisting of an alpha-chain (Lyt2) and a beta-chain (Lyt3). In humans, only the alpha-chain has been detected, and it has been thought that CD8 consists of homodimers of this protein. We have isolated functional cDNA clones encoding human CD8 beta, and show that the CD8 beta protein is expressed on the surface of CD8+ human T cells. cDNA clones encoding multiple forms of the human CD8 beta-chain have been isolated and characterized. These structural variants, which are likely to arise by alternative splicing, differ in the sequences encoding the cytoplasmic domain, which can consist of 19, 30, or 52 amino acids. One of the cDNAs lacks nucleotide sequences corresponding to a hydrophobic transmembrane domain, and may encode a secreted CD8 beta protein. The protein product of the human CD8 beta gene can be detected by a recently described anti-CD8 monoclonal antibody, 597. Expression of the epitope recognized by this antibody requires co-expression of the CD8 alpha and CD8 beta gene products. About 90% of human CD8 alpha positive thymocytes and peripheral blood lymphocytes express CD8 beta at the cell surface. Expression of the CD8 beta chain is thus conserved between human and rodents, and the variant CD8 beta polypeptides may have distinct roles in T cell function and development.     

Retinoic acid induces a specific membrane glycoprotein in human epithelial cell lines

Retinoic acid (RA) inhibits growth, increases the cytokeratin content, and alters the cytoskeleton of the human cervical cell line NHIK 3025. Using RA-treated NHIK 3025 cells as immunogen we prepared murine monoclonal antibodies (IgG1) which recognized an RA-induced cell-surface antigen which could not be detected in untreated NHIK 3025 cells. Analysis of the Triton soluble proteins by SDS-gel electrophoresis and immunoblotting revealed that the cell-surface antigen is a 140-kDa glycoprotein (gp140). gp140 was also shown to be induced by RA in HeLa S3 cells and constitutively expressed in the human trophoblast cell line BeWo. gp140 was also detected in other human epithelial cell lines, but not in human hematopoietic cells. Expression of gp140 was induced in HeLa S3 cells by nanomolar concentrations of RA, and in NHIK 3025 cells by micromolar amounts (1-10 microM). The glycoprotein was detectable 3-6 h following exposure to RA and its expression was reversible upon removal of RA from the medium. Our results indicate that gp140 is a newly identified RA-inducible epithelial membrane glycoprotein which may represent a phenotypic differentiation marker for epithelial cells.     

Biosynthesis of sterols and bile acids in rat liver epithelial cell lines

A rat liver epithelial cell line growing in a serum-supplemented medium expressed biosynthetic pathways of bile sterols and of free and conjugated chenodeoxycholic and cholic acids, the main primary bile acids of the liver. They were identified and measured by gas chromatography-mass spectrometry. The bile steroid secretion in the serum-supplemented cell line was established upon incubation in a serum-free medium which was demonstrated to sustain cell growth, allowing elimination of the interference of exogenous bile steroids and effectors. The free bile acid secretion was also expressed in a subline adapted to proliferate in this serum-free medium, i.e., a basal medium supplemented with 4 g/l albumin carrying 7.6 muequiv./l of a mixture of six long-chain free fatty acids but without any addition of hormones and growth factors. In addition, the rat liver epithelial cell line growing in the serum-supplemented medium maintained, with time, a steady-state of bile acid secretion over a lifespan of 500 days. In the two types of liver epithelial cell lines, dexamethasone and chenodeoxycholic acid supplementation exerted, individually, either a stimulating or an inhibiting effect on the bile acid secretion concurrently with the hydroxylation of chenodeoxycholic acid into alpha-muricholic acid.     

Biosynthesis of thyrotropin-releasing hormone by a rat medullary thyroid carcinoma cell line

Prepro-thyrotropin-releasing hormone (TRH) messenger RNA was detected in the rat medullary thyroid carcinoma cell line CA77. The RNA of 1.6 kilobases comigrated with that found in rat hypothalamus. Using three radioimmunoassays specific for pro-TRH-derived peptides, we demonstrated that CA77 cells synthesize high levels of immunoreactive TRH and all of the other pro-TRH-derived peptides identified in hypothalamic tissue. The relative levels of the pro-TRH-derived peptides also indicate that CA77 cells process the TRH precursor in a manner similar to hypothalamic tissue. CA77 cells provide a promising model system for further studies of prepro-TRH gene regulation and post-translational maturation.     

Biosynthesis of platelet glycoprotein V expressed as a single subunit or in association with GPIb-IX.

Glycoprotein (GP) V is noncovalently linked to GPIbalpha, GPIbbeta and GPIX within the platelet GPIb-V-IX complex, a receptor for von Willebrand factor and thrombin. Two functions have been ascribed to GPV, namely, the modulation of thrombin- and collagen-dependent platelet responses. The biosynthesis of this molecule was investigated in pulse-chase metabolic labelling experiments performed in CHO cell lines transfected with GPV, alone or in the presence of GPIb-IX. GPV could not be detected at the surface of cells expressing the single subunit but was found instead as a soluble form in the culture medium. In pulse-chase studies, an immature 70 kDa protein was detected in cell lysates, whereas a fully processed 80-82 kDa form was only observed in the culture supernatants at later chase times. Immature GPV was N-glycosylated and retained before the medial Golgi while the secreted molecule contained complex sialylated sugars. The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected by inhibitors of proteasome, calpain or metalloproteinases. When GPV was cotransfected with GPIb-IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence-activated cell sorting and confocal microscopy analyses. Surface expressed GPV was processed from an immature 70 kDa form to produce a mature 80 kDa protein, processing similar to the intracellular trafficking of GPIbalpha. These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb-V-IX complex.     

Thiazolidinedione inhibits production of RANTES in a cytokine-treated human lung epithelial cell line.

The chemokine RANTES is a potent chemoattractant for eosinophils. RANTES is produced by lung epithelial cells during eosinophil-rich inflammatory diseases such as asthma. In this study, we examined the effects of thiazolidinediones (TZD) on RANTES expression in a human lung epithelial cell line, A549. In A549 cells, interleukin-1beta and tumor necrosis factor-alpha induced endogenous RANTES protein secretion, mRNA expression, and promoter activity. The TZD inhibited these effects. Our data indicate that the suppression of the expression of RANTES can be accomplished by TZD treatment, raising the possibility that TZD might be of therapeutic value in diseases such as asthma.     

Effects of PUVA on a human skin epithelial cell line

An established human epithelial cell line was exposed to photoactivated 8-methoxy psoralen (PUVA) during exponential growth. Effects of PUVA treatment on cell growth were measured by cell kinetic methods (counting of cell numbers, flow cytometric measurements (FCM) of DNA and calculations of labelling indices (LI)). Doses of 8-methoxy psoralen and UVA were comparable to those used in patients. The cell number in PUVA treated cultures remained almost constant, and very few mitoses were seen for 144 h. About 9 h after PUVA, both FCM and LI showed an increase in the fraction of cells in S-phase, reaching a maximum of 85-90% after 24 h. DNA synthesis took place at a low rate in these cells. FCM showed an increasing fraction of polyploid cells after PUVA treatment. The possibility that inhibition of cell proliferation is one of the main effects of PUVA, is discussed.     

Modulation of human fetal hepatocyte survival and differentiation by interactions with a rat liver epithelial cell line

Our recent studies have shown that chick embryo epiphyseal cartilage synthesizes three distinct species of proteoglycan (PG-H, PG-Lb, and PG-Lt) which are analogous in having glycosaminoglycan side chains of the chondroitin (dermatan) sulfate type but different from one another in regard to the structure of core protein. In the present report, the expression of PG-H and PG-Lb has been studied in developing chick hind limbs (stages 19-33), using antibodies specific for these substances in indirect immunofluorescence. At the onset of cartilage morphogenesis (stage 24), PG-H became recognizable in the cartilage primordia, whereas a parallel section stained for PG-Lb showed no reaction. The first evidence of PG-Lb appearance was seen in a stage 28 cartilage (e.g., tibia) in which the cells in the middiaphysis became elongated in a direction perpendicular to the long axis of the cartilage. The PG-Lb fluorescence was confined to the zone of these flattened, disc-like cells, whereas the fluorescence for PG-H was uniformly distributed throughout the cartilage. With further development of cartilage (stage 29 approximately), the zone of flattened cells spread proximally and distally, and simultaneously large hypertrophied cells appeared at the diaphyseal region. During these zonal changes of cell morphology, the PG-Lb fluorescence remained restricted to the zone of flattened cells. Parallel sections stained for PG-H, in contrast, showed an evenly distributed pattern of the PG-H fluorescence throughout the cartilage. The results indicate that the appearance of PG-Lb is closely associated with the zonal changes of cell shape and orientation along the proximal-distal axis of the developing limb cartilage, and further suggest that the flattened chondrocytes in this particular zone have undergone additional changes in gene expression to form an extracellular matrix of still another chemical property.