Pyridinyl imidazoles inhibit IL-1 and TNF production at the protein level

The mechanism by which SK&F 86002 and other pyridinyl imidazoles inhibit the production of IL-1 and TNF from LPS-stimulated human monocytes was examined. Inhibition of IL-1 and TNF production was found to depend on the time of addition of SK&F 86002, with diminishing effect when added more than 2 h after LPS stimulation. Analysis of Western blots confirmed that both intracellular IL-1β and extracellular TNF were significantly reduced in response to SK&F 86002, but these reductions were not paralleled by changes in IL-1 and TNF mRNA.³⁵S methionine pulse and pulse-chase studies on IL-1 biosynthesis suggest that significant inhibition by SK&F 86002 and related compounds occurs at the translational level.

     

Pre-morbid human T-lymphotropic virus type I proviral load,rather than percentage of abnormal lymphocytes,is associated with an increased risk of aggressive adult T-cell leukemia/lymphoma

Out of 153 newly referred human T-lymphotropic virus type I infected patients, 42 (27%) had 5% or more abnormal lymphocytes, consistent with the diagnosis of smoldering adult T-cell leukemia/lymphoma. The abnormal lymphocyte percentage was higher in patients with human T-lymphotropic virus type I associated inflammatory disease compared with asymptomatic carriers (P=0.006). Over 4.5 years median follow up, 4 patients, all with 10 or more human T-lymphotropic virus type I DNA copies/100 peripheral blood mononuclear cells at presentation, but only one with 5% or more abnormal lymphocytes at presentation, developed adult T-cell leukemia/lymphoma. Thus, high pre-morbid human T-lymphotropic virus type I proviral load, rather than fulfilment of the classification criteria for smoldering adult T-cell leukemia/lymphoma, was associated with an increased risk of developing aggressive adult T-cell leukemia/lymphoma.     

Differential effects of the bicyclic imidazoles on cytokine biosynthesis in human monocytes and endothelial cells

The effects of bicyclic imidazoles on human monocyte and endothelial cell cytokine production were examined. These compounds constitute the CSAID^TM class of anti-inflammatories and are inhibitors of cytokine biosynthesis. The bicyclic imidazoles differ from glucocorticoids and phosphodiesterase inhibitors in their chemical structure as well as pharmacological profile. At optimal concentrations of LPS (50 ng/ml), SK&F 86002, a prototypic compound, inhibited IL-1 and TNF but not g-CSF or IRAP production in human monocytes. At suboptimal concentrations of LPS (50 pg/ml), IL-6 and IL-8 production were also inhibited. Inhibition of cytokine biosynthesis was stimulus independent. For example, induction of IL-1 or TNF expression by phosphatase 1 and 2A inhibitors (Okadaic acid or Calyculin A) and Vitamin D3-dependent induction of IL-1 or TNF was also inhibited. In addition, IL-8 production, but not ICAM/E-Selectin expression in IL-1-stimulated HUVEC, was inhibited at similar IC₅₀s. Taken together, the bicyclic imidazoles inhibit cytokine production selectively in a stimulus and cell type independent manner.     

Effects of pyridinyl imidazole compounds on murine TNF-α production

The effects of SK&F 86002 and other pyridinyl imidazole compounds on murine cytokine production were investigated.In vitro, SK&F 86002 inhibited LPS stimulated TNF- production by the RAW 264.7 cell line and by oil elicited peritoneal macrophages with an IC₅₀ of 5M. In general, the activity was reflective of previous results obtained with human monocytes as SK&F 86002 and its analogs demonstrated identical rank order potency for TNF- inhibition in both species. These compounds also inhibited TNF-in vivo in a murine model of endotoxin shock. Following oral administration, SK&F 86002 and its analogs reduced serum TNF- levels by >80% and afforded 100% protection from lethality. In contrast, tenidap, a novel anti-inflammatory drug, had minimal to no effect on murine TNF- production in the same assays. These data further extend the pharmacological profile of the pyridinyl imidazoles by demonstrating that these compounds potently inhibit murine TNF- production bothin vitro andin vivo.     

Effect of SK&F 86002 on cytokine production by human monocytes

Human monocytes respond to a variety of stimuliin vitro by producing a number of physiologically important macromolecules including the cytokines. SK&F 86002, a dual inhibitor of the arachidonate metabolism, has been shown to inhibit LPS induced IL-1 production in human monocytes. We examined its effect on the production of other cytokines which are coordinately expressed as a result of LPS stimulation such as tumor necrosis factor alpha (TNF), alpha interferon (IFN-A), interferon beta-2 (IL-6) and granulocyte colony stimulating factor (g-CSF). The IC₅₀ of SK&F 86002 for the TNF production was 5–8 M, and >20M for the other three cytokines. These IC₅₀s were significantly higher than that previously reported for IL-1 production (1–2M). Taken together these data indicate that the inhibitory effect of SK&F 86002 on IL-1 production is selective and the production of cytokines in drug treated monocytes can be differentially affected.     

Effect of a human immunodeficiency virus protease inhibitor on human monocyte function.

Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). Encoded by the HIV genome are several precursor proteins that undergo proteolytic cleavage to yield functional proteins. The env precursor protein is cleaved by a cellular protease. The gag precursor protein of HIV (p55), however, is cleaved by a virally encoded aspartate protease (HIV Protease). Cleavage of p55 is required for viral maturation and infectivity. There are also several host cell aspartate proteases that serve important homeostatic functions. Cathepsins D and E are lysosomal aspartate proteases which are believed to play an important role in macrophage function, and it has been suggested that inhibition of these enzymes by an HIV protease inhibitor may exacerbate immunosuppression in AIDS patients. We have studied the effect of SK&F 107461 (a hydroxyethylene dipeptide isostere inhibitor of HIV protease), on various host defense functions of human monocytes. Pepstatin A (an inhibitor of most aspartate proteases) and leupeptin (an inhibitor of serine and cysteine proteases) were included as controls. Although less potent than the prototypic aspartate protease inhibitor pepstatin, SK&F 107461 inhibited partially purified cathepsin D in vitro. However, in cell-based assays, SK&F 107461 had no effect on the degradation of hemoglobin, antigen processing of the protein antigen streptokinase, or secretion of 17-kD IL-1 beta by monocytes at concentrations which inhibit maturation of intracellular virus in HIV infected monocytes. Furthermore, SK&F 107461 had no effect on constitutive candidacidal activity. In contrast, leupeptin and pepstatin A partially inhibited accessory cell function of monocytes in the proliferative response to the recall antigen streptokinase. In addition, leupeptin partially inhibited degradation of hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pyridinyl imidazole compounds on murine TNF-α production

The effects of SK&F 86002 and other pyridinyl imidazole compounds on murine cytokine production were investigated.In vitro, SK&F 86002 inhibited LPS stimulated TNF-α production by the RAW 264.7 cell line and by oil elicited peritoneal macrophages with an IC₅₀ of 5μM. In general, the activity was reflective of previous results obtained with human monocytes as SK&F 86002 and its analogs demonstrated identical rank order potency for TNF-α inhibition in both species. These compounds also inhibited TNF-αin vivo in a murine model of endotoxin shock. Following oral administration, SK&F 86002 and its analogs reduced serum TNF-α levels by >80% and afforded 100% protection from lethality. In contrast, tenidap, a novel anti-inflammatory drug, had minimal to no effect on murine TNF-α production in the same assays. These data further extend the pharmacological profile of the pyridinyl imidazoles by demonstrating that these compounds potently inhibit murine TNF-α production bothin vitro andin vivo.     

Pyridinyl imidazoles inhibit IL-1 and TNF production at the protein level

The mechanism by which SK&F 86002 and other pyridinyl imidazoles inhibit the production of IL-1 and TNF from LPS-stimulated human monocytes was examined. Inhibition of IL-1 and TNF production was found to depend on the time of addition of SK&F 86002, with diminishing effect when added more than 2 h after LPS stimulation. Analysis of Western blots confirmed that both intracellular IL-1 and extracellular TNF were significantly reduced in response to SK&F 86002, but these reductions were not paralleled by changes in IL-1 and TNF mRNA.³⁵S methionine pulse and pulse-chase studies on IL-1 biosynthesis suggest that significant inhibition by SK&F 86002 and related compounds occurs at the translational level.     

A modified assay for interleukin-1 (IL-1)

A simple and reliable biological assay for interleukin-1 (IL-1) was developed, based on the production of interleukin-2 (IL-2) from the EL-4 murine T-cell lymphoma cell line, in the presence of 2-5 X 10(-7) M calcium ionophore A23187. The assay was generally performed in 2 stages ((a) IL-1-dependent IL-2 production, and (b) IL-2 assay) and took 36-48 h to complete. This assay was found to be 10-25 times more sensitive than the mouse thymus cell assay, was not sensitive to the presence of bacterial endotoxin, and had the advantage of not requiring the use of animal tissue as a source of cells. The assay was used in our laboratory to detect human, mouse, rat, and rabbit IL-1 of all isoelectric-point types.