Control of pathogens by formation of abscesses and granulomas is a major strategy of the innate immune system, especially when effector mechanisms of adaptive immunity are insufficient. We show in human listeriosis that DCs expressing indoleamine 2,3-dioxygenase (IDO), together with macrophages, are major cellular components of suppurative granulomas in vivo. Induction of IDO by DCs is a cell-autonomous response to Listeria monocytogenes infection and was also observed in other granulomatous infections with intracellular bacteria, such as Bartonella henselae. Reporting on our use of the clinically applied anti-TNF-α antibody infliximab, we further demonstrate in vitro that IDO induction is TNF-α dependent. Repression of IDO therefore might result in exacerbation of granulomatous diseases observed during anti-TNF-α therapy. These findings place IDO + DCs not only at the intersection of innate and adaptive immunity but also at the forefront of bacterial containment in granulomatous infections.

Immune defense capacity differs between men and women. Whereas men are more prone to infection and sepsis, women more commonly develop autoimmune diseases. We investigated the difference in cytokine secretion between males and females in response to different immune stimuli. Whole blood from 154 healthy volunteers (age 24 +/- 5.2; 82 females, 72 males) was collected within 2 h on 2 consecutive days. Blood from males produced significantly more tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and IL-8 than blood from females in response to a high concentration of either lipopolysaccharide (LPS) or lipoteichoic acid (LTA), whereas IL-10 and interferon-gamma (IFN-gamma) secretion did not differ. Normalization of cytokine measurement to individual monocyte counts cancelled these differences for all parameters except TNF-alpha. Stimulation with a lower concentration of LPS (100 pg/mL) produced even stronger differences in cytokine release, which were not cancelled by normalization to the producing cells. The coefficients of variation (CV) of the LPS-induced and LTA-induced cytokine responses were higher in blood from women than men for all parameters and stimuli measured. Thus, the stronger innate immune response of males in comparison to females appears to stem not only from a difference in monocyte counts but also from the steepness of the response curve.

To investigate the circadian rhythm of inducible cytokine release and a potential pacemaker role of endogenous cortisol, cortisol levels as well as cytokine release from ex vivo LPS-stimulated blood were assessed at 4-h intervals over 24 h in 11 volunteers. We found a significant diurnal variation for IFN-c and IL-8, and a tendency for TNF, all inversely correlated to the serum cortisol levels, but no evidence for such a rhythm for IL-1b and IL-6. In vitro IC 50 values for cytokine inhibition by hydrocortisone (HC) corresponded to the observed rank order for circadian rhythmicity. mRNA analyses revealed that this was due to a reduction of gene transcription. These effects of HC were significantly reversed by the glucocorticoid receptor antagonist RU486. Supplementation of HC in vivo to maintain morning cortisol levels throughout the day blunted the circadian rhythm of ex vivo LPS-induced cytokines. Surprisingly, no significant diurnal variation for any investigated cytokine was found in the same volunteer group upon stimulation with lipoteichoic acid (LTA), the gram-positive counterpart to LPS. Furthermore, 10-50-fold higher HC concentrations as compared to LPS were required to block LTA-induced cytokine release. LTA, in contrast to LPS, failed to activate Jun kinase, a reported target for HC action.

More than 90% of all publications on endotoxin were carried out with endotoxins (lipopolysaccharide, LPS) from enterobacteriaceae. We compared the immune stimulatory potency of 11 different LPSs using human whole blood incubations. While the majority of LPSs induced cytokine release equipotently, a 1,000-fold more LPS from Pseudomonas aeruginosa or Vibrio cholerae was still less potent in inducing TNF, IL-1 beta, IL-10 and IFN-gamma though it potently induced nanogram quantities IL-8. All LPSs tested, regardless of the micro-organism, showed Toll-like receptor (TLR)4-dependence, except for the LPSs from P. aeruginosa and V. cholerae, which were both TLR4- and TLR2-dependent. Interestingly, UV-inactivated P. aeruginosa bacteria, although Gram-negative, also showed TLR2- and TLR4-dependence. Re-purification of commercial LPS preparations by phenol re-extraction led to a complete loss of the TLR2 dependency, indicating contamination with lipoproteins. In the Limulus amebocyte lysate assay, often performed to exclude contamination in purified water likely to originate from P. aeruginosa, P. aeruginosa LPS was only 2-fold less potent than LPS from S. abortus equi or the assay standard LPS from E. coli. This results in an overestimation of pyrogenic burden by a factor of 500 in the sample when compared with the biological activity of highly purified P. aeruginosa LPS in human whole blood.

In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.