Lipopolysaccharide (LPS) from Gram-negative bacteria is one of the most potent innate immune-activating stimuli known. Here we review the current understanding of LPS effects on human monocyte and macrophage function. We provide an overview of LPS signal transduction with attention given to receptor cooperativity and species differences in LPS responses, as well as the role of tyrosine phosphorylation and lysine acetylation in signalling. We also review LPS-regulated transcription, with emphasis on chromatin remodeling and primary versus secondary transcriptional control mechanisms. Finally, we review the regulation and function of LPS-inducible cytokines produced by human monocytes and macrophages including TNFα, the IL-1 family, IL-6, IL-8, the IL-10 family, the IL-12 family, IL-15 and TGFβ.

Latterly, nicotinamide adenine dinucleotide (NAD+) has emerged as a molecule with versatile functions and of enormous impact on the maintenance of cell integrity. Besides playing key roles in almost all major aspects of energy metabolism, there is mounting evidence that NAD+ and its degradation products affect various biological activities including calcium homeostasis, gene transcription, DNA repair, and intercellular communication. This review is aimed at giving a brief insight into the life cycle of NAD+ in the cell, referring to synthesis, action and degradation aspects. With respect to their immunological relevance, the importance and function of the major NAD+ metabolizing enzymes, namely CD38/CD157, ADP-ribosyltransferases (ARTs), poly-ADP-ribose-polymerases (PARPs), and sirtuins are summarized and roles of NAD+ and its main degradation product adenosine 5'-diphosphoribose (ADPR) in cell signaling are discussed. In addition, an outline of the variety of immunological processes depending on the activity of nicotinamide phosphoribosyltransferase (Nampt), the key enzyme of the salvage pathway of NAD+ synthesis, is presented. Taken together, an efficient supply of NAD+ seems to be a crucial need for a multitude of cell functions, underlining the yet only partly revealed potency of this small molecule to influence cell fate.

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Candida albicans (CA) increasingly causes septic shock, acute lung injury, and multiple organ damage during immunosuppression-related neutropenia. However, the effects of neutrophil (PMN) depletion on induction of tumor necrosis factor-alpha (TNF) by CA and its potential mediation of Candida septic shock are unknown. We hypothesized that reduced CA uptake by circulating PMNs during cyclophosphamide (CY)-related neutropenia sensitizes to TNF-mediated shock from enhanced cytokine production after phagocytosis by tissue macrophages. Absolute or relative neutropenia (PMNs < or = 500/microliters or 2,500/microliters) was modeled in rats by intraperitoneal CY 4-8 days before 10(9) yeast-phase CA (acute studies < or = 24 h, n = 81 animals) or 10(6) CA (subacute studies < or = 72 h, n = 25). Compared with neutrophil-sufficient rats, absolute neutropenia accelerated hemodynamic collapse and respiratory distress after 10(9) CA, and pulmonary microvascular permeability was amplified. These changes evolved without increased candidemia or elevations in bioactive or antigenic serum TNF, which remained low even at death (42.3 +/- 14.8 U/ml vs. 12.6 +/- 2.9 U/ml for CY + saline, means +/- SE, P = NS). In contrast, significant TNF in lung tissue and bronchoalveolar lavage fluid (BALF) was evident within 6 h in CY + 10(9) CA rats. Electron microscopy confirmed hyphal proliferation into alveoli from yeast within mononuclear cells in lung capillaries. Subacute disseminated candidiasis after 10(6) CA was not associated with elevated serum, lung, or BALF TNF. We conclude that differential systemic and intrapulmonary TNF production occur in CA septic shock during preexisting neutropenia, with compartmentalized TNF production in the lower respiratory tract accompanying yeast-mycelial transformation. Thus TNF is not an obligate mediator of acute candidemic shock or subacute disseminated candidiasis during CY-induced immunosuppression but may initiate pulmonary injury accompanying high-grade candidemia.

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD + ) induces a transient rise in [Ca 2+ ] i in human monocytes caused by an influx of extracellular calcium. The NAD + -induced Ca 2+ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X 1 , P2X 4 , and P2X 7 receptors being engaged in the NAD + -induced rise in [Ca 2+ ] i . Among the P2X receptor subtypes, the P2X 7 receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X 7 receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD + , we found that NAD + unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD + at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD + and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.

Recent studies have identified enzymes that use NAD as a substrate, thus contributing to its net consumption. To maintain the intracellular pool, NAD is re-synthesized by a salvage pathway using nicotinamide, the by-product generated by the enzymatic cleavage of NAD. Enzymes involved in NAD re-synthesis include nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase. Our studies show, that NAMPT was substantially up-regulated by LPS in primary human monocytes, suggesting that it may be especially required during the process of monocyte activation. To evaluate the contribution of the NAD rescue pathway to LPS-induced biological responses in human monocytes, we used APO866, a well-characterized inhibitor of NAMPT. Concomitant with the inhibition of NAMPT, LPS-induced TNF-α protein synthesis declined, while TNF-α mRNA levels were minimally affected. Moreover, APO866 strongly decreased the production of reactive oxygen species (ROS), increased surface expression of the NAD-consuming enzyme CD38, and modified the production of selective eicosanoids. We further demonstrate that protein ADP-ribosylation was strongly reduced, indicating a possible link between this post-translational protein modification and human monocyte inflammatory responses. Despite a substantial reduction in intracellular NAD levels, activated monocytes were resistant to apoptosis, while resting monocytes were not. Taken together, our data suggest that activated monocytes strongly depend on the NAD salvage pathway to mount an appropriate inflammatory response. Their survival is not affected by NAD-depletion, probably as a result of LPS-mediated anti-apoptotic signals.

We analyzed differences in host regulation of tumor necrosis factor-alpha (TNF-alpha) production and pathophysiological responses in conscious rats after infection with two strains of pathogenic Candida albicans spp. (CA-1 and CA-2) compared with Escherichia coli serotype 055:B5 (EC). The hypothesis was tested that, in contrast to EC, hypotension, organ injury, and mortality after candidemia are not obligatorily dependent on TNF-alpha or TNF-alpha-induced cyclooxygenase pathway metabolites. Dose, viability, and strain-specific dependencies were established after intravenous 10(6) or 10(9) viable CA, as well as heat-killed (HK) or Formalin-inactivated (FI) CA blastospores, compared with live EC at the 24-h LD25 [10(9) colony-forming units (CFU)] and LD100 (10(10) CFU). Shock without endotoxemia developed 4-8 h after 10(9) live CA-1 or CA-2 (LD100 at 24 h) with disseminated yeast-mycelial transformation and increased microvascular permeability in multiple organs but not after HK or FI CA-1. Peak serum TNF-alpha after an LD100 of CA-1 or CA-2 was < 3% of LD25 EC values and was < 1% of peak values during lethal bacteremia. Similar pathogen-specific differences were found in liver- and lung-associated TNF. Production of functionally inactive TNF-alpha during candidemia was excluded by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western blotting. Passive immunization against TNF-alpha 2 h before microbial challenge was not protective against CA but prevented otherwise lethal EC sepsis. Cyclooxygenase inhibition also failed to attenuate candidemic shock. We conclude that the magnitude and kinetics of TNF-alpha production and TNF-alpha-dependent immunophysiological responses are differentially regulated after lethal fungal vs. gram-negative bacterial infection. Thus TNF-alpha is not a pivotal mediator of the acute Candida septic shock syndrome with disseminated candidiasis.

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