Elasticity and adhesion of resting and lipopolysaccharide-stimulated macrophages.

Colloidal Force Microscopy was employed to study the viscoelastic and adhesive properties of macrophages upon stimulation with lipopolysaccharide (LPS). Force vs. distance measurements were performed. The adhesion of LPS-stimulated cells (separation force=37+/-3 nN) was almost twice as high as that of resting macrophages (16+/-1 nN). Upon retraction pulling of membrane tethers was observed. Tether lengths and forces at which rupture take place did not depend on stimulation. The reduced Young's modulus K, a measure of cytoskeleton elasticity, was three times lower than that of the control. The data show that LPS has profound effects on cytomechanical and adhesion properties of macrophages.

Intracellular LPS inhibits the activity of potassium channels and fails to activate NFkappaB in human macrophages.

Although much has been learned about signal transduction mechanisms and binding proteins involved in lipopolysaccharides (LPS)-induced activation of monocytes/macrophages, little is known about the ability of internalized LPS to activate cells. To approach this question we either exposed macrophages to LPS or microinjected the cells with LPS before studying early cellular events associated with LPS-mediated macrophage activation. We measured membrane currents and translocation of NFkappaB to the nucleus. Using the whole-cell patch clamp technique ion channels were analyzed and characterized as K+ sensitive inward and outward currents. Exogenous LPS was shown to increase the voltage-dependent outward current whereas the voltage-dependent inward current was unaffected. However when cells were microinjected with LPS both inward and outward current were completely abolished. The presence of LPS within the cells did not prevent them to perform phagocytosis or to respond to fMLP with an appropriate increase in [Ca2+]i. The immunocytological detection of NFkappaB p65 translocation revealed that exogenous LPS led to the nuclear localization of the p65 subunit of NFkappaB, whereas only the cytoplasmic localization of p65 was observed following microinjection of LPS. These data show that one major process in macrophage activation, the NFkappaB dependent transcription of a number of genes encoding for many inflammatory mediators cannot be induced by intracellular LPS but requires the interaction of LPS with external membrane components. However intracellular LPS causes a drastic decrease in potassium currents which by keeping the cell membrane at a depolarized potential may result in changed biological answers of these cells.

Extracellular NAD+ regulates intracellular free calcium concentration in human monocytes.

Ca(2+) ions play a critical role in the biochemical cascade of signal transduction pathways, leading to the activation of immune cells. In the present study, we show that the exposure of freshly isolated human monocytes to NAD(+) results in a rapid concentration-dependent elevation of [Ca(2+)](i) (intracellular free Ca(2+) concentration) caused by the influx of extracellular Ca(2+). NAD(+) derivatives containing a modified adenine or nicotinamide ring failed to trigger a Ca(2+) increase. Treating monocytes with ADPR (ADP-ribose), a major degradation product of NAD(+), also resulted in a rise in [Ca(2+)](i). Selective inhibition of CD38, an NAD-glycohydrolase that generates free ADPR from NAD(+), does not abolish the effect of NAD(+), excluding the possibility that NAD(+) might act via ADPR. The NAD(+)-induced Ca(2+) response was prevented by the prior addition of ADPR and vice versa, indicating that both compounds share some mechanisms mediating the rise in [Ca(2+)](i). NAD(+), as well as ADPR, were ineffective when applied following ATP, suggesting that ATP controls events that intersect with NAD(+) and ADPR signalling.

Regulation of adenosine receptor subtypes during cultivation of human monocytes: role of receptors in preventing lipopolysaccharide-triggered respiratory burst.

Adenosine is a potent anti-inflammatory agent that modulates the function of cells involved in the inflammatory response. Here we show that it inhibits lipopolysaccharide (LPS)-induced formation of reactive oxygen intermediates (ROI) in both freshly isolated and cultured human monocytes. Blocking of adenosine uptake and inactivation of the adenosine-degrading enzyme adenosine deaminase enhanced the inhibitory action of adenosine, indicating that both pathways regulate the extracellular adenosine concentration. Adenosine-mediated inhibition could be reversed by XAC (xanthine amine congener), an antagonist of the adenosine receptor A(2A), and MRS 1220 [N-9-chloro-2-(2-furanyl)[1, 2, 4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide], an A(3) receptor antagonist, in both cell populations, while DPCPX (1,3-dipropyl-8-cyclopentylxanthine), an A(1) receptor antagonist, had no effect. Similar to what was seen with adenosine, CGS 21680, an A(2A) and A(3) receptor agonist, and IB-MECA, a nonselective A(1) and A(3) receptor agonist, dose dependently prevented ROI formation, indicating the involvement of A(3) and probably also A(2A) in the suppressive effect of adenosine. Pretreatment of monocytes with adenosine did not lead to changes in the LPS-induced increase in intracellular calcium levels ([Ca(2+)](i)). Thus, participation of [Ca(2+)](i) in the action of adenosine seems unlikely. The adenosine-mediated suppression of ROI production was found to be more pronounced when monocytes were cultured for 18 h, a time point at which changes in the mRNA expression of adenosine receptors were observed. Most prominent was the increase in the A(2A) receptor mRNA. These data demonstrate that cultivation of monocytes is accompanied by changes in the inhibitory action of adenosine mediated by A(3) and probably also the A(2A) receptor and that regulation of adenosine receptors is an integral part of the monocyte differentiation program.