Restoration of abated T cell stimulation activity of mature dendritic cells.

Freshly isolated and cultured mature dendritic cells (DC) rapidly lose their high capacity to activate T cells. This diminished T cell activation activity is due, in part, to dramatically reduced expression of the costimulation ligands B7-1 (CD80) and B7-2 (CD86). Here we show that cultivation of mature DC with the lymphokines TNFalpha, GM-CSF, or TNFalpha plus GM-CSF, fully restored and enhanced their T-cell stimulation activity by two to threefold, four to fivefold, or six to sevenfold, respectively. The restored T-cell stimulation activity was directly correlated with induced and increased levels of CD80 (16-fold) and CD86 (10-fold) expression by the lymphokine-cultured DC. These results should facilitate many current DC-vaccination efforts towards generating more potent T cell responses.

Melatonin inhibition of cancer growth in vivo involves suppression of tumor fatty acid metabolism via melatonin receptor-mediated signal transduction events.

The growth of rat hepatoma 7288CTC in vivo is stimulated by the uptake of linoleic acid (LA) and its metabolism to 13-hydroxyoctadecadienoic acid (13-HODE), an important mitogenic signaling molecule within this tumor. Conversely, the growth of a variety of experimental cancers in vivo is inhibited by either physiological or pharmacological levels of the pineal gland hormone melatonin, although the mechanism(s) are unknown. We tested the hypothesis that the mechanism of melatonin's anticancer action in vivo involves the inhibition of tumor LA uptake and metabolism to 13-HODE in hepatoma 7288CTC. Tumor uptake of LA and release of 13-HODE, measured in tissue-isolated rat hepatoma 7288CTC at 4-h intervals over a 24-h period, were highest during the light phase and lowest during the mid-dark phase, when plasma melatonin levels were lowest and highest, respectively. Pinealectomy eliminated this rhythm of tumor LA uptake and 13-HODE production, indicating that it was driven by the circadian melatonin rhythm. Perfusion of tissue-isolated tumors in situ with melatonin (1 nM) rapidly and reversibly inhibited the uptake of plasma fatty acids (FAs), including LA, and its metabolism to 13-HODE. These inhibitory effects of melatonin on tumor FA uptake and 13-HODE release were completely reversed by perfusion of tumors in situ with melatonin receptor antagonist S-20928, pertussis toxin, forskolin, or 8-bromo-cAMP. Perfusion of tumors in situ with melatonin also decreased tumor [3H]thymidine incorporation and DNA content; these effects on DNA synthesis were also prevented by the coperfusion of tumors with melatonin and S-20928, pertussis toxin, forskolin, 8-Br-cAMP, or 13-HODE. Pinealectomy stimulated tumor growth, LA uptake and metabolism to 13-HODE, and FA storage in hepatoma 7288CTC, whereas melatonin administration (200 microg/day) was inhibitory in vivo. Northern blot analysis revealed that, compared with normal liver tissue, hepatoma 7288CTC overexpressed mRNA transcripts for a plasma membrane-associated FA transport protein (FATP). FATP mRNA expression was unaffected by the treatment of tumor-bearing rats with daily afternoon melatonin injections or exposure to constant light. These results support a novel mechanism of tumor growth inhibition by melatonin involving a melatonin receptor-mediated suppression of cAMP levels, resulting in diminished tumor FA transport, possibly via decreased FATP function. The inhibition of these signal transduction events by melatonin culminates in the suppression of LA uptake, LA metabolism to the mitogenic signaling molecule 13-HODE, and cancer growth.

New actions of melatonin on tumor metabolism and growth.

Melatonin is an important inhibitor of cancer growth promotion while the essential polyunsaturated fatty acid, linoleic acid is an important promoter of cancer progression. Following its rapid uptake by tumor tissue, linoleic acid is oxidized via a lipoxygenase to the growth-signaling molecule, 13-hydroxyoctadecadienoic acid (13-HODE) which stimulates epidermal growth factor (EGF)-dependent mitogenesis. The uptake of plasma linoleic acid and its metabolism to 13-HODE by rat hepatoma 7288CTC, which expresses both fatty acid transport protein and melatonin receptors, is inhibited by melatonin in a circadian-dependent manner. This inhibitory effect of melatonin is reversible with either pertussis toxin, forskolin or cAMP. While melatonin inhibits tumor linoleic acid uptake, metabolism and growth, pinealectomy or constant light exposure stimulates these processes. Thus, melatonin and linoleic acid represent two important environmental signals that interact in a unique manner to regulate tumor progression and ultimately the host-cancer balance.

Biologically active recombinant rat granulocyte macrophage colony-stimulating factor produced in Escherichia coli.

Rat granulocyte macrophage colony-stimulating factor (rGM-CSF) cDNA was amplified and cloned, and recombinant-rGM-CSF (R-rGM-CSF) was expressed and isolated from Escherichia coli. The synthesis of R-rGM-CSF was directed by a modified, inducible maltose binding protein (MBP) gene fusion expression vector, pMTR-23, and secreted to the periplasm. The vector pMTR-23 contains a new multiple cloning site encoding a unique thrombin-sensitive cleavage site and short spacer arm which facilitates separation of the MBP from the foreign protein domain of hybrid proteins. Biologically active R-rGM-CSF was rapidly purified by a combination of affinity and ion exchange chromatography, with a yield of 1.5 mg of R-rGM-CSF per liter of cultured cells. The purified R-rGM-CSF, like the native molecule, exhibits potent biological activity in two rGM-CSF-specific assays, considerably enhancing the accessory activity of rat dendritic cells and stimulating the differentiation of dendritic cells from fresh cultures of rat bone marrow cells. Although dendritic cells are difficult to isolate from tissues, the availability of R-rGM-CSF should now facilitate the development of large numbers of dendritic cells and the understanding of their regulatory role in the immune response.

Conditioned medium from activated rat macrophages and the recombinant factors, IL-1 beta and GM-CSF, enhance the accessory activity of dendritic cells.

Low density lymph node cells (LD-LNC; 5% of total unfractionated LNC) contain 95% of the accessory activity required for responses of T lymphocytes to mitogens. Significantly greater responses to mitogens occur when T lymphocytes are added to LD-LNC that have been exposed overnight to silica, in comparison to responses occurring with LD-LNC incubated without silica. Conditioned medium (CM) from silica-treated LD-LNC is itself able to mediate enhanced responses; i.e., when LD-LNC are exposed overnight to CM alone and mitogen-treated T lymphocytes added the next day. The enhancing activity found in CM from LD-LNC exposed to silica is produced by macrophages; however, their low accessory activity is not enhanced by CM. In contrast, dendritic cells isolated from LD-LNC exposed to silica or to CM show significantly increased accessory activity, but dendritic cells do not produce the enhancing activity found in CM. CM lacks IL-2 activity and does not have any effect on the responses of untreated or mitogen-treated T lymphocytes alone. Thus, macrophages produce the enhancing activity and dendritic cells respond to it. Maximum enhancement of dendritic cell accessory activity requires overnight exposure to CM; once induced, accessory activity is not further modulated after continued incubation in the presence or absence of CM. LD-LNC, adherent peritoneal exudate cells, and adherent thioglycollate-induced peritoneal exudate cells produce enhancing activity after exposure to silica, LPS, and silica plus LPS. After gel filtration of a CM produced by silica plus LPS, enhancing activity shows a broad molecular weight distribution between 20 and 55 kD. IL-1 is present in CM and shows a more narrow molecular weight distribution that falls within the lower molecular weight range for enhancing activity. Silica treatment by itself produces CM containing little IL-1, but abundant enhancing activity; gel filtration of this CM shows that the distribution of enhancing activity is confined more narrowly to the higher molecular weight range, suggesting that IL-1 is one of several factors that enhances the accessory activity of dendritic cells. Recombinant human IL-1 beta does have enhancing activity, but of the other recombinant factors tested only mouse GM-CSF also has enhancing activity. Human IL-1 alpha, tumor necrosis factor alpha, IL-4, rat IL-3 and rat IFN-gamma, as well as L cell-conditioned medium containing M-CSF, lack enhancing activity.

The effect of silica treatment on accessory cell-dependent rat T lymphocyte proliferation.

Previous work has shown that purified rat macrophages lack both accessory activity for T lymphocyte responses to mitogens and stimulatory activity in a mixed leukocyte reaction, in marked contrast to the potent activity of dendritic cells. This study was designed to re-evaluate macrophages as accessory cells by treating various cell preparations with either silica or L-leucine methyl ester, which have been reported to be toxic to macrophages, and then determining the effect of the treated cells on responses to the mitogens, sodium periodate or concanavalin A. These studies indicated that treatment with L-leucine methyl ester failed to kill rat macrophages or dendritic cells, whereas silica was specifically toxic for rat macrophages. The studies therefore focused on silica. Co-culturing mitogen-treated lymph node cells with silica over a wide range of concentrations had no effect on responses. The same results were obtained if mitogen-treated lymphocytes were enriched with lymph node macrophages and dendritic cells and then co-cultured with silica. Preparations containing both macrophages and dendritic cells were incubated with silica for 24 h to ensure the death of virtually all macrophages; upon the addition of mitogen-treated lymphocytes, the macrophage-depleted accessory cells induced vigorous proliferative responses. Peritoneal exudate cells showed variable, but low accessory activity that increased after incubation with silica. Elimination of more than 90% of the macrophages from peritoneal exudate cells, as determined by staining for non-specific esterase, failed to eliminate this accessory activity. Taken together, these findings confirm and extend the conclusion that rat macrophages lack or have exceedingly low accessory activity.