A rapid assay for measuring the metabolism of [3H]-retinoic acid in cell cultures.

A simple method was developed to detect the metabolism of [3H]-retinoic acid to polar products using intact tumor cells in culture. Unaltered [3H]-retinoic acid was separated from more polar metabolites using C18-bonded solid phase extraction cartridges. Separation of unaltered retinoic acid and polar metabolites was confirmed by HPLC. The murine mammary carcinoma cell line TA3 Ha used in these studies converted 40% to 50% of added radioactive retinoic acid to polar metabolites released into the culture medium during a 4-hr incubation period. Metabolism of [3H]-retinoic acid by TA3 Ha cells was inhibited by the cytochrome P-450 inhibitors ketoconazole, clotrimazole, and liarozole. The simplicity and rapidity of this assay should make it useful for evaluating compounds as inhibitors of retinoic acid metabolism.

Characterization of a high affinity interleukin-1 (IL-1) specific binding site in a human synovial sarcoma (Hs431) cell line.

A subpopulation of cells was derived from the Hs431 connective tissue sarcoma cell line which possessed high affinity (estimated Kd = 0.38-0.55 nM) binding sites for human recombinant [125I]-IL-1 alpha. Binding at 4 degrees C was slow approaching equilibrium by 4 hrs. Dissociation of [125I]-IL-1 alpha was also slow and unaffected by high concentrations of cold ligand. The binding site also underwent ligand-induced internalization at 37 degrees C. An Mr = 83,000 protein was identified in affinity crosslinking studies. Despite these similarities to previously reported IL-1 receptors, Hs431 cells did not exhibit biological responses to IL-1 which have been observed in other cell lines. IL-1 did not induce PGE2 or collagenase synthesis. IL-1 also failed to induce ornithine decarboxylase activity (ODC) or stimulate [3H]-thymidine incorporation. In contrast, the Hs431 cells did contain a functional epidermal growth factor (EGF) receptor as determined from binding studies, protein kinase activity, induction of ODC, and stimulation of [3H]-thymidine incorporation. Thus, the refractoriness of Hs431 cells to IL-1 was fairly specific and did not result from a generalized defect associated with cell transformation.

Natural killing of cytomegalovirus-infected fibroblasts by human mononuclear leucocytes.

The ability of human peripheral blood mononuclear (MN) cells to lyse uninfected and cytomegalovirus (CMV) infected human fibroblasts was determined in a 51Cr-release assay. Maximal release was obtained with 6-day infected fibroblasts incubated with MN cells for 24 hr. A linear relationship existed between E/T ratios of 12.5:1 to 100:1 and lysis of CMV-infected targets. Donor immune status had no effect on the magnitude of killing of infected or uninfected targets. Killing was mediated by non-B, predominantly non-T, Fc receptor-bearing cells. Preincubation of effector cells with interferon enhanced killing of both CMV-infected and uninfected fibroblasts, but infected targets were more effectively killed. These results indicated a possible role for natural killer cells in recovery from CMV infection.

Lymphocyte blastogenesis and interferon production in adult human leukocyte cultures stimulated with cytomegalovirus antigens.

Lymphocyte blastogenesis and interferon production were measured in adult human leukocyte cultures stimulated with purified or crude cytomegalovirus antigens. Leukocytes from seropositive adults underwent blastogenesis when stimulated with purified or crude Towne strain antigen, whereas neither antigen stimulated blastogenesis in cultures from seronegative donors. The concentrations of antigens yielding maximal blastogenesis varied among the individuals tested. When cultures from seropositive individuals were stimulated with antigens prepared from three different CMV strains--AD-169, Towne, and Davis--comparable levels of blastogenesis were detected. Type 1 interferon was detected in supernatants of cultures stimulated with crude antigens regardless of the immune status of the donor. In contrast, when purified antigen was used as the stimulant, only cultures obtained from seropositive individuals produced detectable levels of interferon, which appeared to be predominantly type 2 or immune interferon.

Identification of some interleukin-1 (IL-1) sensitive organs in vivo using the induction of ornithine decarboxylase (ODC) following injection of recombinant IL-1 beta in mice.

Intraperitoneal (i.p.) and intravenous (i.v.) injection of human recombinant interleukin-1 beta (rIL-1 beta) in mice produced a 2-4 fold induction of ornithine decarboxylase (ODC) in liver and heart six hours after administration. Lymphoid organs (thymus and spleen) and brain did not respond to rIL-1 beta administration with significant increases in ODC. IL-1-induced responses in heart seemed not to be secondary to stress induced catecholamine release since the beta-adrenergic antagonist propranolol did not inhibit the induction of ODC produced by injection rIL-1 beta. Injection of 10 micrograms bacterial lipopolysaccharide (LPS), an inducer of IL-1 synthesis, exhibited a pattern of tissue responsiveness which was distinct from the responses elicited by rIL-1 beta, most notably a marked 5-fold induction of ODC in spleen. The differences in the responses of various organs to rIL-1 beta vs. LPS suggested that the in vivo effects of LPS may involve more than stimulation of the release of IL-1. The identification of heart as an IL-1 sensitive tissue merits further study to define the contribution of IL-1 to cardiac physiology and pathophysiology.