Bhanu Sinha

Taming TNF: strategies to restrain this proinflammatory cytokine

Recent studies have demonstrated the essential role of tumor necrosis factor alpha (TNF-alpha) in rheumatoid arthritis and Crohns disease. This article discusses agents known to suppress the formation or activity of TNF-alpha, and summarizes clinical studies using anti-TNF-alpha antibodies.

Endogenous Adenosine Curtails Lipopolysaccharide‐Stimulated Tumour Necrosis Factor Synthesis

Recent studies have demonstrated the inhibitory effect of exogenous adenosine on TNF production. During inflammation endogenous adenosine levels are elevated and may be one of several anti‐inflammatory mediators that reduce TNF synthesis. In the present study the authors investigated this role of adenosine in freshly isolated human PBMC. The effect of endogenous adenosine on TNF formation was studied by four different approaches. First, adenosine deaminase was added to LPS‐stimulated mononuclear cells. This enzyme specifically deaminates extracellular adenosine to the inactive metabolite inosine. TNF production was augmented from baseline stimulation (LPS alone) of 3.5 ± 0.4 ng ml−1— 5.2 ± 0.9 ng ml−1 in the presence of 10 U ml−1 adenosine deaminase. Second, TNF production was determined after stimulation in the presence of dipyridamole, an inhibitor of cellular re‐uptake of adenosine which increases extracellular concentrations. TNF synthesis was reduced dose‐dependently from 3.1 ± 0.9 ng ml−1— 1.1 ± 0.2 ng ml−1 by 10 μm dipyridamole. Third, the adenosine A2 receptor antagonist 8‐(3‐chlorostyryl)caffeine (100 nm) enhanced TNF synthesis from a baseline of 3.7 ± 0.5 ng ml−1— 5.5 ± 0.9 ng ml−1. In contrast, no increase resulted from the addition of 100 nm of the specific A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine. Finally, the authors were able to show that suppression of TNF formation by the specific type IV phosphodiesterase inhibitor rolipram can be completely reversed by adenosine deaminase or by the application of the A2 receptor antagonist. The authors conclude that endogenous adenosine controls TNF production. This effect of adenosine may not only have a physiological role but also appears to contribute to the pharmacological inhibition of TNF synthesis by exogenous agents such as the specific type IV phosphodiesterase inhibitor rolipram.

Nitric oxide downregulates tumour necrosis factor in mRNA in RAW 264.7 cells

Nitric oxide (NO) and tumour necrosis factor (TNF) are essential mediators in a number of biological processes, including the immune response. TNF stimulates NO production via expression of inducible NO synthase (iNOS), with L-arginine being the only substrate. Previously, we demonstrated that, inversely, NO inhibits lipopolysaccharide (LPS)-induced TNF synthesis. We have now investigated whether this reduction of TNF bioactivity is also reflected at the level of TNF mRNA in the murine macrophage cell line RAW 264.7. TNF mRNA was quantified by Northern analysis using an alpha[33P]dCTP-labelled probe. Cells stimulated with 10 microg/ml LPS in the absence of L-arginine, in order to prevent endogenous NO formation, contained more TNF mRNA than cells supplied with 1 mM L-arginine at 14 h and 20 h after stimulation. By contrast, no difference was observed at 4 h. This time course is compatible with the involvement of iNOS. The half-life of TNF mRNA in the presence of NO was roughly half that observed under L-arginine-free conditions (41 min versus 77 min, respectively). L-citrulline (1 mM), which has been shown to be recycled in RAW 264.7 cells to L-arginine, completely restored attenuation of TNF bioactivity and TNF message to control levels obtained with 1 mM L-arginine. Together, these findings suggest that endogenous NO regulates TNF mRNA, mainly by reducing its half-life. In addition, a distinct additional band (approximately 1.4 kb) hybridizing with the TNF probe was consistently observed in non-stimulated cells. This may correspond to TNF mRNA specifically hydrolysed at the AU-rich region, possibly reflecting another control point for TNF expression.

Cicaprost and the type IV phosphodiesterase inhibitor, rolipram, synergize in suppression of tumor necrosis factor-alpha synthesis

Suppression of tumor necrosis factor-alpha (TNF) synthesis is one major target in pharmacological immunomodulation. We now showed the synergistic suppressive effect of the specific type IV phosphodiesterase inhibitor, rolipram, and of the stable prostacyclin analogue, cicaprost, on TNF synthesis. This effect was seen with lipopolysaccharide and Staphylococcus epidermidis as stimuli in human peripheral blood mononuclear cells and in whole blood. Lipopolysaccharide-induced TNF synthesis by mononuclear cells decreased from 3.4 ng/ml to 1.5 ng/ml in the presence of 100 nM rolipram and to 0.7 ng/ml in the presence of 10 nM cicaprost. The combination of both agents suppressed TNF synthesis more than 10-fold, to 0.3 ng/ml. Synergistic suppression was also demonstrated for TNF mRNA.

Amrinone suppresses the synthesis of tumor necrosis factor-alpha in human mononuclear cells

Tumor necrosis factor-alpha (TNF) exerts a wide spectrum of biological activities and contributes to the pathophysiology of septic shock. Elevated circulating levels of TNF have also been reported in patients with severe chronic heart failure. We studied the effect of amrinone, a class III cyclic nucleotide phosphodiesterase inhibitor used in the treatment of acute heart failure, on the synthesis of TNF in vitro. Peripheral blood mononuclear cells from healthy volunteers or cells of a permanent monoblast cell line were stimulated for 20 h with bacterial lipopolysaccharide and different doses of amrinone. TNF production is suppressed in a dose-dependent manner to a minimum of 9% of controls with 1000 microM of amrinone, reaching half-maximal inhibition at 80 microM amrinone. This effect appears to be mediated via cAMP, which accumulated nearly twofold in the presence of amrinone. Suppression of TNF synthesis by therapeutically administered phosphodiesterase inhibitors such as amrinone may contribute to their beneficial effect in the treatment of heart failure.

Radioimmunoassays for cyclic AMP cross-react with phosphodiesterase inhibitors and buffer components

We addressed the issue of cross-reactivity of several commonly used phosphodiesterase inhibitors with radioimmunoassays for cyclic AMP, after we had observed a considerably high cross-reactivity with a noncommercial antibody. Theophylline, pentoxifylline, penthydroxifylline (BL 194), albifylline (HWA 138), torbafylline (HWA 448), A 80 2715, isobutyl methylxanthine, and the nonmethylxanthines amrinone and rolipram were dissolved in supplemented and boiled cell culture medium (RPMI 1640). These samples were assayed for apparent cyclic AMP in two different, commercially available radioimmunoassay kits (based on polyclonal antibodies), applying the nonacetylated protocol. Cross-reactivity was dose-dependent and nonlinear. Samples containing theophylline and amrinone exhibited the strongest cross-reactivity in assay A (NEN/DuPont): 3.0 +/- 0.5(-nM) and 2.4 +/- 1.1 (-nM) apparent cyclic AMP +/- SD at 1-nM spike, respectively. With the more sensitive assay B (Amersham), higher concentrations of apparent cyclic AMP were detected: from 7.9 +/- 0.4 nM (for albifylline) to 3.5 +/- 0.1 nM (for rolipram). Values were calculated from standard curves set up in the respective assay buffer, where culture medium controls resulted in 1.8 +/- 0.3 nM and 3.1 +/- 0.1 nM for assay A and B, respectively. The culture medium interference increased with rising cyclic AMP concentrations. Although comparatively low, this degree of cross-reactivity is relevant for in vitro experiments. Phosphodiesterase inhibitors are commonly administered at millimolar concentrations, and resulting cyclic AMP levels are often in the nanomolar range. Neglecting these findings may lead to falsely high readouts of cyclic AMP concentrations.

Regulation of Cytokine Synthesis Through Cyclic Nucleotides

Recent studies have shown that the addition of phosphodiesterase inhibitors to mouse macrophages can suppress tumor necrosis factor (TNF) production [1, 2] putatively by raising the intracellular concentration of cyclic 3′, 5′-adenosine monophosphate cAMP [3]. Our current studies addressed three questions: First, is there a suppressive effect of phosphodiesterase inhibitors on TNF production by monocytes of human origin? Second, is this effect specific for TNF or does it also affect the synthesis of other cytokines such as interleukin-1β (IL-1β)? And third, is the effect of phosphodiesterase inhibitors on cytokine production mediated by accumulation of cAMP levels alone or also of cyclic 3′,5′-guanosine monophosphate (cGMP) levels?