CXCL4-induced monocyte survival, cytokine expression, and oxygen radical formation is regulated by sphingosine kinase 1.

Human monocytes respond to a variety of stimuli with a complex spectrum of activities ranging from acute defense mechanisms to cell differentiation or cytokine release. However, the individual intracellular signaling pathways related to these functions are not well understood. CXC chemokine ligand 4 (CXCL4) represents a broad activator of monocytes, which induces acute as well as delayed activities in these cells including cell differentiation, survival, or the release of ROS, and cytokines. Here, we report for the first time that CXCL4-treated monocytes significantly upregulate sphingosine kinase 1 (SphK1) mRNA and that CXCL4 induces SphK1 enzyme activity as well as its translocation to the cell membrane. Furthermore, we could show that pharmacological inhibition of SphK results in reversal of CXCL4-induced monocyte survival, cytokine expression, and release of oxygen radicals, which was confirmed by the use of SphK1-specific siRNA. CXCL4-mediated rescue from apoptosis, which is accompanied by inhibition of caspases, is controlled by SphK1 and its downstream element Erk. Taken together, these data assign SphK1 as a central regulator of acute and delayed monocyte activation and suggest SphK1 as a potential therapeutic target to suppress pro-inflammatory responses induced by CXCL4.

The cathelicidin LL-37 activates human mast cells and is degraded by mast cell tryptase: counter-regulation by CXCL4.

The cathelicidin LL-37 represents a potent antimicrobial and cell-stimulating agent, most abundantly expressed in peripheral organs such as lung and skin during inflammation. Because mast cells (MC) overtake prominent immunomodulatory roles in these organs, we wondered whether interactions exist between MC and LL-37. In this study, we show for the first time to our knowledge that physiological concentrations of LL-37 induce degranulation in purified human lung MC. Intriguingly, as a consequence LL-37 rapidly undergoes limited cleavage by a released protease. The enzyme was identified as beta-tryptase by inhibitor studies and by comparison to the recombinant protease. Examining the resulting LL-37 fragments for their functional activity, we found that none of the typical capacities of intact LL-37, i.e., MC degranulation, bactericidal activity, and neutralization of LPS, were retained. Conversely, we found that another inflammatory protein, the platelet-derived chemokine CXCL4, protects LL-37 from cleavage by beta-tryptase. Interestingly, CXCL4 did not act as a direct enzyme inhibitor, but destabilized active tetrameric beta-tryptase by antagonizing the heparin component required for the integrity of the tetramer. Altogether our results suggest that interaction of LL-37 and MC initiates an effective feedback loop to limit cathelicidin activity during inflammation, whereas CXCL4 may represent a physiological counter-regulator of beta-tryptase activity.

Animal shed Bacillus licheniformis spores possess allergy-protective as well as inflammatory properties.

BACKGROUND: Numerous epidemiologic studies have demonstrated an allergy-protective effect of farm life early in childhood. It has been hypothesized that environmental exposure to microbes may contribute to this effect. Because of their small size and thereby their potential for deposition in lower airways of small children, bacterial spores may be candidates for such allergy-protective effects. OBJECTIVE: To investigate immune responses elicited by exposure to Bacillus spores in experimental settings. METHODS: Animal shed and mattress dusts were analyzed for bacteria and fungi by aerobic and anaerobic growth. Bacillus licheniformis, the most prominent microorganism found in these samples, was investigated with respect to spore specific stimulation of pattern recognition receptors, monocyte-derived dendritic cells and T(H)-cell polarization in vitro as well as to the prevention of asthma development in a mouse model of allergic asthma. RESULTS: In vitro, B. licheniformis spores activated a T(H)1 cytokine expression profile. In vivo application of these spores resulted in less spore-specific but long-lasting immune activation preventing eosinophilia and goblet cell hyperplasia; however, they provoked an influx of neutrophils in lung tissue of asthmatic mice. CONCLUSION: Bacterial spores may contribute to the allergy-protective properties of farming environments, but their persistence in the lung causes ongoing immune activation in mouse experiments.

Platelet factor 4/CXCL4-stimulated human monocytes induce apoptosis in endothelial cells by the release of oxygen radicals.

The generation of reactive oxygen species (ROS) represents a pivotal element of phagocyte defense against microbial invaders. However, oxidative stress also participates in pathophysiological processes of vascular damage leading to cell death of endothelial cells (EC). Currently, ROS-producing cells involved in this process as well as the corresponding extracellular signals required for their activation are ill-defined. In this study, we investigate the impact of the platelet-derived CXC chemokine platelet factor 4 (PF4/CXCL4) on the interaction of human monocytes and EC. We can show for the first time that PF4-activated monocytes become cytotoxic for EC but not epithelial cells. Cytotoxicity was time- and dose-dependent, and earliest effects were seen after 15 h of culture and at a concentration from 0.125 microM PF4 up. By performing transwell experiments and by using specific inhibitory antibodies, we could show that direct cell contact between effector and target cells, mediated by beta(2)integrins as well as their corresponding ligand ICAM-1, is essential for the cytotoxic effect. Investigations of the cellular mechanisms of cytotoxicity revealed that in the presence of EC, PF4-activated monocytes are capable of releasing high amounts of ROS for more than 2 h following stimulation. This causes programmed cell death in EC, as inhibitors of the NADPH oxidase (diphenyleneiodonium and apocynin) effectively blocked PF4-induced monocyte oxidative burst and protected EC from undergoing apoptosis. Taken together, our data suggest a role for platelet-derived PF4 in oxidative stress-mediated vascular disorders, as observed during atherosclerosis or ischemia/reperfusion injury.

Capsular arabinomannans from Mycobacterium avium with morphotype-specific structural differences but identical biological activity.

The capsules of two colony morphotypes of Mycobacterium avium strain 2151 were investigated, i.e. the virulent smooth-transparent (SmT1) and the nonvirulent smooth-opaque (SmO) types. From both morphotypes we separated a nonacylated arabinomannan (AM) from an acylated polysaccharide fraction by affinity chromatography, of which the AMs were structurally characterized. The AMs from the virulent morphotype, in contrast to that from the nonvirulent form, possessed a larger mannan chain and a shorter arabinan chain. Incubation of murine bone marrow-derived macrophages and human dendritic cells showed that the acylated polysaccharide fractions were potent inducers of tumor necrosis factor-alpha, interleukin-12, and interleukin-10 compared with nonacylated AMs, which led to only a marginal cytokine release. Further in vitro experiments showed that both the acylated polysaccharide fractions and the nonacylated AMs were able to induce in vitro anti-tumor cytotoxicity of human peripheral blood mononuclear cells. Thus, morphotype-specific structural differences in the capsular AMs of M. avium do not correlate with biological activity; however, their acylation is a prerequisite for effective stimulation of murine macrophages and human dendritic cells.

Mycobacteria induce IFN-gamma production in human dendritic cells via triggering of TLR2.

IFN-gamma is of central importance for the induction of robust cell-mediated immunity and for the activation of APC. Recent studies using experimental murine systems have now suggested a fundamental role for APC-derived IFN-gamma during infection with intracellular pathogens. It is currently unknown whether human dendritic cells (DC) can respond to bacterial stimulation with production of IFN-gamma. To test this question, we used human monocyte-derived DC stimulated by Mycobacterium bovis bacillus Calmette-Guérin as a model system. We demonstrate production of IFN-gamma mRNA and protein on the single cell level. IFN-gamma in DC cultures was not simply produced by contaminating lymphocytes because production of DC-IFN-gamma could also be demonstrated in highly purified DC cultures containing virtually no T, B, and NK cells. TLR2 was identified as a key receptor involved in triggering production of DC-IFN-gamma. Interestingly, DC-IFN-gamma seems to participate in an autocrine DC activation loop, and production of DC-IFN-gamma could be enhanced by costimulation of DC with IL-12/IL-15/IL-18. In conclusion, we have demonstrated production of IFN-gamma by human DC on the single cell level, identified TLR2 as a pattern recognition receptor involved in this process, and elucidated some of the functional consequences of autocrine IFN-gamma production by human DC.

Assembly of plant Shaker-like K(out) channels requires two distinct sites of the channel alpha-subunit.

SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K(+) channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K(out) channels. Deletion mutants and chimeric proteins generated from SKOR and the K(in) channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K(T) domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K(out) alpha-subunits did not assemble with K(in) alpha-subunits because of the absence of interaction between their assembly sites.