Mary L. Spengler

Tumor necrosis factor-alpha (TNF) is a macrophage-derived peptide that is known to be an important mediator in various physiologic and immunologic events. Although the effector function of TNF has received recent attention, there is relatively little information regarding factors that regulate TNF expression. Highly Ia-positive murine peritoneal macrophages obtained via complete Freund's adjuvant elicitation were challenged with lipopolysaccharide (LPS) and assessed for the production and regulation of TNF at the cellular and molecular levels. In response to 1 microgram/ml LPS, the kinetics of functionally active TNF reached a maximum at approximately 3-4 h. The plateau in TNF levels was concomitant with an accelerated increase in prostaglandin E2 production. The addition of exogenous PGE2 demonstrated a dose-dependent reduction in LPS-induced TNF activity at the cellular level, as well as a significant reduction in TNF mRNA accumulation as assessed by Northern blot and in situ hybridization analysis. The reduction in LPS-stimulated mRNA accumulation by PGE2 was shown to occur at least at the level of transcription, since nuclear run-off analysis showed a specific reduction in TNF transcripts. These studies demonstrate that PGE2 can regulate macrophage-derived TNF gene expression.

The circadian clock controls many physiological parameters including immune response to infectious agents, which is mediated by activation of the transcription factor NF-κB. It is widely accepted that circadian regulation is based on periodic changes in gene expression that are triggered by transcriptional activity of the CLOCK/BMAL1 complex. Through the use of a mouse model system we show that daily variations in the intensity of the NF-κB response to a variety of immunomodulators are mediated by core circadian protein CLOCK, which can up-regulate NF-κB-mediated transcription in the absence of BMAL1; moreover, BMAL1 counteracts the CLOCK-dependent increase in the activation of NF-κB-responsive genes. Consistent with its regulatory function, CLOCK is found in protein complexes with the p65 subunit of NF-κB, and its overexpression correlates with an increase in specific phosphorylated and acetylated transcriptionally active forms of p65. In addition, activation of NF-κB in response to immunostimuli in mouse embryonic fibroblasts and primary hepatocytes isolated from Clock-deficient mice is significantly reduced compared with WT cells, whereas Clock-Δ19 mutation, which reduces the transactivation capacity of CLOCK on E-box-containing circadian promoters, has no effect on the ability of CLOCK to up-regulate NF-κB-responsive promoters. These findings establish a molecular link between two essential determinants of the circadian and immune mechanisms, the transcription factors CLOCK and NF-κB, respectively.

Tumor necrosis factor (TNF) is an oncolytic peptide that may also exert many other biologic effects. Experimentally, immunologically activated mononuclear phagocytes stimulated with endotoxin (LPS) produce TNF, while resting mononuclear phagocytes stimulated with LPS produce little TNF. To date, the ability of human alveolar macrophages (AMs) to produce TNF has not been clearly delineated. As pulmonary sarcoidosis is a granulomatous inflammatory disorder characterized by immunologically activated AMs, we investigated the production of TNF by AMs obtained by bronchoalveolar lavage from 7 normal volunteers and 13 patients with pulmonary sarcoidosis. The AMs were cultured with and without LPS, and TNF production was assessed by an in vitro cytotoxicity assay. Unstimulated sarcoid and normal AMs produced little TNF, but LPS stimulation enhanced TNF production by both normal and sarcoid AMs. Furthermore, LPS-stimulated sarcoid AMs produced more TNF than normal AMs (84.9 +/- 16.7 versus 32.5 +/- 10.2 units/million cells, P less than 0.05). It is concluded that human AMs can produce TNF and that sarcoid AMs are primed and can produce significantly more TNF, compared with normal AMs.

The circadian clock regulates biological processes from gene expression to organism behavior in a precise, sustained rhythm that is generated at the unicellular level by coordinated function of interlocked transcriptional feedback loops and post-translational modifications of core clock proteins. CLOCK phosphorylation regulates transcriptional activity, cellular localization and stability; however little is known about the specific residues and enzymes involved. We have identified a conserved cluster of serines that include, Ser431, which is a prerequisite phosphorylation site for the generation of BMAL dependent phospho-primed CLOCK and for the potential GSK-3 phosphorylation at Ser427. Mutational analysis and protein stability assays indicate that this serine cluster functions as a phospho-degron. Through the use of GSK-3 activators/inhibitors and kinase assays, we demonstrate that GSK-3beta regulates the degron site by increasing CLOCK phosphorylation/degradation, which correlates with an increase in the expression of CLOCK responsive promoters. Stabilization of phospho-deficient CLOCK delays the phase of oscillation in synchronized fibroblasts. This investigation begins the characterization of a complex phospho-regulatory site that controls the activity and degradation of CLOCK, a core transcription factor that is essential for circadian behavior.

The regulation of lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF) production by prostaglandin E2 (PGE2), forskolin, and dibutyryl cyclic AMP (cAMP) was examined at the cellular and molecular levels. The above three agents could suppress LPS (100 ng/ml)-stimulated TNF production by immunologically activated murine macrophages (M phi s) in a dose-dependent manner. The concomitant addition of PGE2, dibutyryl cAMP, or forskolin to LPS-challenged M phi s resulted in 50% inhibition of TNF production at 10(-7), 3 X 10(-6), and 3 X 10(-5) M, respectively. Interestingly, delaying the addition of PGE2 or dibutyryl cAMP by 1.5 h post-LPS stimulation was also effective in suppressing the production of TNF bioactivity, but only dibutyryl cAMP was effective when its addition was delayed by 3 h. Northern (RNA) blot analysis of mRNA isolated from LPS-challenged M phi s treated with PGE2 or dibutyryl cAMP corroborated the bioactivity data. The delayed addition of PGE2 or dibutyryl cAMP by 1.5 h post-LPS stimulation resulted in a suppression of TNF mRNA accumulation by 50 to 70%. These data support the concept that LPS is a potent stimulus for M phi-derived TNF production and that this mediator is a very proximal signal in LPS-mediated disease states. Thus, therapeutic approaches that target the suppression of TNF in LPS-dependent disease states may be limited by the rapid expression of this mediator.