Lung fibroblasts inhibit activation-induced death of T cells through PGE(2)-dependent mechanisms.

Activation-induced cell death (AICD) is a regulatory mechanism eliminating excess activated T cells, mainly through a Fas/Fas ligand-dependent mechanism. The goal of this study was to determine whether mouse primary lung fibroblasts are capable of modulating AICD. Using T cell hybridoma DO11.10, we found that fibroblasts in coculture rescue T cells from AICD. Fibroblast-conditioned medium (FCM) also inhibited apoptosis of T cells activated with immobilized anti-CD3 antibody. The effects of lung fibroblasts are mediated, in part, by secreted prostaglandin E(2) (PGE(2)) because treatment of fibroblasts with indomethacin decreased antiapoptotic activity of FCM. Addition of exogenous PGE(2) to FCM from fibroblast cultures treated with indomethacin restored the inhibitory activity of FCM. Expression of Fas receptor and Fas ligand by anti-CD3-activated DO11.10 cells was not affected by PGE(2). However, the same concentrations of PGE(2) significantly downregulated activation of caspase-8 and caspase-3. These results demonstrate that lung fibroblasts inhibit the AICD of T cells by secreting PGE(2), which downregulates caspase activation and apoptosis.

Application of DNA topoisomerase-activated adapters to riboprobe synthesis.

Topoisomerase-activated adapters for rapid incorporation of the T7 promoter into PCR products were made by hybridizing synthetic oligonucleotides and activating vaccinia DNA topoisomerase I. The adapters were used to incorporate the T7 promoter sequence into PCR products amplified from cDNA and genomic DNA. Modified PCR products were used as templates to synthesize digoxigenin-labeled sense and cRNA probes by in vitro transcription with phage T7 RNA polymerase. The red/green cones were labeled by the antisense probe, but no specific signal was produced by the sense probe. These results demonstrate that topoisomerase-activated adapters provide a powerful and convenient tool for the rapid modification of PCR products.

Expression of the protein tyrosine phosphatase, phosphatase of regenerating liver 1, in the outer segments of primate cone photoreceptors.

Foveal cone photoreceptors are morphologically distinct and, presumably, express unique transcripts. We have identified a cDNA clone encoding the protein tyrosine phosphatase (PTP), phosphatase of regenerating liver 1 (PRL-1) in a screen for genes that are enriched in monkey fovea. PRL-1 was originally isolated as an immediate early gene in regenerating liver [R.H. Diamond, D.E. Cressman, T.M. Laz, C.S. Abrams, R. Taub, PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth, Mol. Cell Biol. 14 (1994) 3752-3762]. On cDNA Southern blots of human and monkey retina, radiolabeled PRL-1 cDNA hybridized to a single mRNA species of about 2.5 kb that was most intense in fovea-enriched samples. The monkey PRL-1 deduced amino acid sequence is identical to human, rat and mouse PRL-1. Affinity-purified antibodies directed against PRL-1 preferentially labeled cone photoreceptor cells and a subpopulation of bipolar cells in monkey retina. Immunoreactivity in cones was confined to red and green, but not to blue, cones and was restricted to the outer segments. Immunolocalization also revealed that PRL-1 protein expression was non-nuclear, suggesting that its function in the retina may be unrelated to its role in other tissues where it is expressed primarily in nuclei. Although both foveal and extrafoveal cones were PRL-1 reactive, the high abundance of PRL-1 mRNAs detected in monkey fovea correlates with the high concentration of cones in the fovea. The PRL-1 gene is located on chromosome 6q within an interval that also contains the genes that cause two hereditary retinal dystrophies. These studies demonstrate novel expression of the PRL-1 gene in the neural retina and suggest the phosphatase activity of PRL-1 may modulate normal cone photoreceptor cell function.

Contact Effects of Mononuclear Cells on L929 Fibroblast Morphological Phenotype.

We studied the contact interactions of human peripheral blood mononuclear cells (MNC) and transformed mouse fibroblast cell line L929 (L-cells), namely their effects on morphological phenotype of L-cells. The morphological characteristics of the fibroblast, (cell area, nucleus-cytoplasmic ratio, cell spreading, cell shape) were estimated with the aid of fight scanning microscopy, followed by computer image analysis. Contact interaction between fibroblasts and MNC caused normalization of morphological phenotype of the fibroblasts (increase of cell area, shape factor, spreading and decrease of nucleus-cytoplasmic ratio). This phenomenon was revealed by analysis of both average morphological characteristics and population contents. Only a particular subpopulation of L-cells, but not the whole population, was shown to be normalized by the effects of MNC. For elucidation of responsible MNC population, which was capable of influencing on fibroblast morphological phenotype, we separated MNC in several cell types: adherent cells, non-adherent cells, which were separated in E-rosetting cells, and non-E-rosetting cells. Only non-adherent and E-rosetting cells could normalize the morphological phenotype. E-rosetting population consisted of 85% of CD3(+) (T lymphocytes) and 15percnt; of CD56(+)/CD16(+) (natural killers). Supernatants of MNC, and MNC cocultured L-cells, obtained after 24 h incubation in the cell culture medium, were not able to normalize the morphological phenotype of the fibroblasts. They had small denormalizing effect on the fibroblasts (decrease of cell area, shape factor, spreading and increase of nucleus-cytoplasmic ratio). The results of this study indicate that the contact interaction between MNC and fibroblasts may normalize transformed fibroblast morphological phenotype. The dependence of fibroblast functional state on their morphological phenotype imply the presence of regulatory mechanism of contact interaction between fibroblasts and MNC, which determines many live processes in fibroblast.