Discovery of novel ansamycins possessing potent inhibitory activity in a cell-based oncostatin M signalling assay.

We describe the isolation and characterisation of novel non-benzoquinone ansamycin metabolites related to geldanamycin from a culture of Streptomyces sp. S6699. The compounds possess potent inhibitory activity in a cell-based assay measuring inhibition of oncostatin M signalling in a reporter cell line utilising a secreted placental alkaline phosphatase (sPAP) readout. In this paper we report the isolation and structure elucidation of the compounds and describe some of their biological properties.

A novel (6S)-4,6-dimethyldodeca-2E,4E-dienoyl ester of phomalactone and related alpha-pyrone esters from a Phomopsis sp. with cytokine production inhibitory activity.

A series of novel 6-substituted 5,6-dihydro-5-hydroxy-alpha-pyrone esters, 1 approximately 3, isolated from fermentations of a Phomopsis sp. (Xenova culture collection no. X22502) have been identified as inhibitors of lipopolysaccharide (LPS)-induced cytokine production. These include the (6S)-4,6-dimethyldodecadien-2E,4E-dienoyl ester of phomalactone, 1, and two analogues bearing a prop-2E-enoic acid moiety at the 6-position of the alpha-pyrone ring. (6S)-4,6-Dimethyl-2E,4E-dienoic acid, 4, and a hydroxylated analogue, 5, were also isolated and characterised. The most potent cytokine production inhibitor was 1, which inhibited LPS-induced tumour necrosis factor alpha (TNFalpha) production by U937 cells and LPS-induced interleukin 1beta (IL-1beta) production by peripheral blood mononuclear cells (PBMC) with IC50 values of 80 nM and 190 nM respectively. The effect of 1 in PBMC was selective for IL-1beta relative to TNFalpha. The inhibition of IL-1beta production by 1 involved a post-translational mechanism of action at the level of IL-1beta secretion as demonstrated by the lack of an effect on cell-associated IL-1beta production. 1 showed no effect on the activity of caspase 1 in cytosolic extracts from the THP1 monocytic cell line.

Inhibition of endotoxin-induced TNF-alpha production in macrophages by 5Z-7-oxo-zeaenol and other fungal resorcylic acid lactones.

Resorcylic acid lactones are fungal metabolites that exhibit a wide range of biological properties which includes oestrogenic, antifungal, phytotoxic and anti-inflammatory activity. The capacity of 5Z-7-oxo-zeaenol, a resorcylic lactone of fungal origin and six naturally occurring analogues to inhibit lipopolysaccharide (LPS)-induced cytokine production in phorbol 12-myristate-13-acetate (PMA)-treated cultured myelomonocytic cells (U937) was compared. The activity of the natural analogues in the U937 assay varied over 10(4)-fold, with 5Z-7-oxo-zeaenol the most potent of those tested inhibiting tumour necrosis factor-alpha (TNF alpha) production in these cells with IC50 of 6 nM. The isomeric 7-oxo-zeaenol and structurally more distant monorden (radicicol) were the next most active compounds with IC50 approximately 500 nM, and zearalenone, the least active with IC50 > 400 microM. 5Z-7-oxo-zeaenol retained activity in LPS-stimulated peripheral blood mononuclear cells with an IC50 of 10-25 nM. This compound also inhibited LPS-induced TNF alpha production in whole blood experiments (IC50 100-1000 nM) and lowered serum levels of TNF alpha in mice when administered prior to LPS. 5Z-7-oxo-zeaenol was shown to inhibit the phosphorylation and activation of mitogen-activated protein kinase (MAPK) induced by LPS. These data are consistent with a mechanism of action at or upstream of MAPK with resultant downstream effects. This series of naturally occurring analogues represents an interesting group of compounds with diverse biological properties. Of this series, 5Z-7-oxo-zeanenol has exceptionally potent anti-inflammatory properties exhibited by its strong inhibition of cytokine production.

Differential effects on TNF alpha production by pharmacological agents with varying molecular sites of action.

This study describes the activation conditions for tumor necrosis factor-alpha (TNF alpha) production in myelomonocytic U937 cells and human primary peripheral blood monocytes in response to lipopolysaccharide (LPS) and/or phorbol 12-myristate 13-acetate (PMA). PMA itself induced only low levels of TNF alpha production with delayed kinetics (e.g. 0.758 +/- 0.128 ng/ml from U937 cells after 48 h) while LPS induced greater levels of TNF alpha production in less time (e.g. 2.083 +/- 0.96 ng/ml from monocytes in 24 h). Pharmacological agents with various molecular sites of action were used to validate the two systems, with the protein serine-threonine kinase inhibitors staurosporine and Ro-31-8220, the protein tyrosine kinase inhibitor herbimycin A (HBA) and dexamethasone exhibiting the greatest potency (IC50S 5-350 nM). In contrast to the effect on TNF alpha production, PMA induced strong phosphorylation/activation of p42/p44mapk in monocytes by 10 min determined in a mobility shift assay, while LPS was a weaker inducer. Additionally, staurosporine (to LPS and PMA) and HBA (to LPS only) inhibited the activation of these mitogen-activated protein kinase (MAPK) isoforms at doses 10-100 fold higher than those required to inhibit maximal TNF alpha production. These data indicate the involvement of the p42/p44mapk signalling pathway in LPS-induced pro-inflammatory cytokine production but suggest that other signalling pathways are also implicated in this phenomenon.

Development of microglia and macrophages in the postnatal rat pituitary.

Microglia and macrophages, immunolabelled with F4/80 (which binds a 160-kDa plasmalemmal glycoprotein) and OX-42 (which labels the complement type 3 receptor, CR3), were identified in the neuro- and adenohypophyses, respectively, of postnatal rats from day 1 to adulthood. In the neurohypophysis, the numerical density (cells/mm2) of microglia increased from postnatal day 1 to day 7 but was then unchanged from the adult density. In the adenohypophysis, the numerical density of macrophages increased from postnatal day 1 to day 21. The increasing size of the pituitary meant that the total number of such cells increased rapidly in the neurohypophysis up to day 14, but was then essentially unchanged; in the adenohypophysis macrophages increased in proportion to the increasing size of the gland up to day 21. Proliferation of the mononuclear cells was analysed by the immunodetection of bromodeoxyuridine incorporation into the nuclei of microglia and macrophages. F4/80-immunoreactive cells incorporating bromodeoxyuridine were found on all the postnatal days studied. The proportion of such cells in the neurohypophysis was high from postnatal day 1 to day 14 and in the adenohypophysis was maximal on day 14, decreasing in both parts of the pituitary by day 21. The estimated total number of proliferating cells was maximal in both parts of the pituitary on day 14. In both parts, OX-42-immunoreactive cells were less numerous than F4/80-immunoreactive cells up to postnatal day 14; CR3 expression may therefore be associated with maturation of these cells. Neurohypophysial microglia increased in size to postnatal day 7, consistent with the assumption of a 'compact' microglial morphology; adenohypophysial macrophages did not change in size over the postnatal period. Throughout the period studied, neurohypophysial microglia were significantly more densely distributed and larger in size than adenohypophysial macrophages. Neurohypophysial microglia phagocytose terminals of neurosecretory neurons from day 7, concurrent with the development of a distinct perivascular space. In the adenohypophysis, the perivascular space was present from birth and macrophages were not phagocytic. There are, therefore, considerable differences in the density, morphology and activity between the populations of myelomonocytic cells in the postnatal rat neuro- and adenohypophyses.

Interleukin-10 stimulation of phosphatidylinositol 3-kinase and p70 S6 kinase is required for the proliferative but not the antiinflammatory effects of the cytokine.

Interleukin-10 (IL-10) is a powerful suppressor of the proinflammatory monokine production by lipopolysaccharide-stimulated monocytes as well as a T- and B-cell growth cofactor. The signal transduction cascades initiated by IL-10 ligation to its cognate receptor remain to be elucidated. Here, we demonstrate that in both primary monocytes and the D36 cell line, IL-10 rapidly and transiently stimulated phosphatidylinositol 3-kinase activity associated with the p85 subunit of the enzyme. IL-10 also activated p70 S6 kinase in both cell types. The activation of both of these kinases was sensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase. The activation of p70 S6 kinase was also inhibited by the immunosuppressive drug rapamycin. Both rapamycin and wortmannin inhibited the IL-10-induced proliferation of D36 cells but in contrast had no effect on the antiinflammatory effects of the cytokine on lipopolysaccharide-stimulated monocytes. Similar results on D36 proliferation and lipopolysaccharide-stimulated monocyte inhibition by IL-10 were obtained with another phosphatidylinositol 3-kinase inhibitor, LY294002. This suggests that the activation of phosphatidylinositol 3-kinase and p70 S6 kinase is involved in the proliferative functions of IL-10 and that other as yet uncharacterized pathways affect the suppressive effects on monocytes, indicating that multiple and distinct signaling pathways mediate the various pleiotropic activities of IL-10. Furthermore, these findings suggest that it may be possible in the future to modulate the antiinflammatory effects of IL-10 for therapeutic benefit without disrupting other functions of the cytokine.

Immunophenotypic evidence for distinct populations of microglia in the rat hypothalamo-neurohypophysial system.

The morphology, distribution and immunophenotype of microglia throughout the adult rat hypothalamo-neurohypophysial system was examined. Four macrophage-associated antibodies (OX-42, F4/80, ED1 and ED2) were used; the expression of major histocompatibility complex antigens was investigated by use of antibodies against OX-6, OX-17 (MHC class II) and OX-18 (MHC class I). Three distinct types of microglia were identified. The first was located in the magnocellular nuclei; these 'radially branched' ('ramified') microglia had round cell bodies and long branched processes, and were strongly immunoreactive only for OX-42. The second was located outside the blood-brain barrier in the median eminence, pituitary stalk and neurohypophysis often close to blood vessels; these 'compact' microglia had irregular cell bodies and shorter processes, and were strongly labelled by OX-42 and F4/80, weakly labelled by OX-18, and generally unlabelled by ED1, ED2, OX-6 and OX-17. The third type was found in small numbers throughout the system at the surface of the nervous tissue or around blood vessels; these 'perivascular' microglia were elongated cells with no branching processes, and were strongly labelled by ED1, ED2, OX-18, OX-6, OX-17 and F4/80 antibodies but showed variable OX-42 immunoreactivity. Cells in a perivascular location were heterogeneous with respect to their immunophenotype. The presence in the normal adult rat hypothalamo-neurohypophysial system of MHC class-II molecules (OX-6 and OX-17) on a sub-set of perivascular microglia suggests that these cells are capable of presenting antigen to T lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Perivascular microglia in the rat neural lobe engulf magnocellular secretory terminals during osmotic stimulation.

The response of microglia in the rat neural lobe to osmotic stimulation has been studied. Microglia were identified by immunoreactivity for the macrophage markers OX-42 and F4/80. The numerical density of microglia did not change significantly with osmotic stimulation but microglia in the perivascular space partially or completely enclosed significantly greater numbers of neurosecretory terminals in osmotically stimulated animals.

Specific radioimmunoassays for IL 1 alpha and IL 1 beta in plasma at physiological and acidic pH: determination of immunoreactive forms by gel filtration and radioligand binding studies.

We have developed specific radioimmunoassays for interleukin 1 alpha (IL 1 alpha) and interleukin 1 beta (IL 1 beta) and applied these successfully to the measurement of interleukin 1 (IL 1) in neat plasma. Further characterization of the plasma immunoreactive forms of IL 1 was done using Sephadex G-75 chromatography and TSKG2000 high performance gel permeation chromatography. This revealed the immunoreactivity to be associated with a high molecular weight fraction for both IL 1 alpha and IL 1 beta. Incubation of plasma with iodinated IL 1 alpha and beta showed that there was a time-dependent association of tracer with the high molecular weight fraction and that this was predominantly with IL 1 beta. The activity was displaceable with unlabeled IL 1 beta, which together with the chromatography results, suggested that IL 1 beta is protein-bound in plasma. Furthermore, we have shown that under acid conditions both tracer and endogenous IL 1 beta immunoreactivity migrate as a low (17 kD) molecular weight fraction. This suggests that dissociation from a high molecular weight binder has occurred. Acid treatment of plasma raised the immunoreactive IL 1 beta level, but had no effect on IL 1 alpha levels, confirming the specificity of a binder to IL 1 beta, as shown by the tracer experiments. These results suggest that plasma contains high molecular weight binders of IL 1, particularly IL 1 beta, and that these may play a role in regulating the distribution, clearance and bioactivity of circulating IL 1.