Leukocyte responsiveness to LPS is dependent upon CD14 and receptors of the Toll-like receptor (TLR) family. Neutrophils respond to LPS, but conflicting data exist regarding LPS responses of eosinophils and basophils, and expression of TLRs at the protein level in these granulocyte lineages has not been fully described. We examined the expression of TLR2, TLR4, and CD14 and found that monocytes expressed relatively high levels of cell surface TLR2, TLR4, and CD14, while neutrophils also expressed all three molecules, but at low levels. In contrast, basophils expressed TLR2 and TLR4 but not CD14, while eosinophils expressed none of these proteins. Tested in a range of functional assays including L-selectin shedding, CD11b up-regulation, IL-8 mRNA generation, and cell survival, neutrophils responded to LPS, but eosinophils and basophils did not. In contrast to previous data, we found, using monocyte depletion by negative magnetic selection, that neutrophil responses to LPS were heavily dependent upon the presence of a very low level of monocytes, and neutrophil survival induced by LPS at 22 h was monocyte dependent. We conclude that LPS has little role in the regulation of peripheral blood eosinophil and basophil function, and that, even in neutrophils, monocytes orchestrate many previously observed leukocyte LPS response patterns.

Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 μM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.

Apoptosis and clearance of neutrophils is essential for successful resolution of inflammation. Altered signaling via the Fas receptor could explain the observed prolongation of neutrophil lifespan and associated tissue injury at inflammatory sites. We therefore compared inflammatory neutrophils extracted from joints of rheumatoid arthritis patients, with peripheral blood neutrophils. Inflammatory neutrophils underwent constitutive apoptosis in culture more rapidly than peripheral blood neutrophils; this was not explained by changes in surface expression of Fas or by induction of Fas ligand. Inflammatory neutrophils remained sensitive to Fas-induced death, at levels comparable to those seen in peripheral blood neutrophils. Similarly, granulocyte-macrophage colonystimulating factor reduced apoptosis but did not abolish signaling via Fas. These data provide evidence for the rate of apoptosis in inflammatory neutrophils being continually modulated by death and survival signals in the inflammatory milieu. This allows for rapid resolution of inflammation as levels of survival factors fall, and suggests new strategies for inducing resolution of inflammation.

4D (PDUD), part of the complex CAMP-specific PDE4 family, plays a pivotal role in the regulation of airway smooth muscle relaxation. Its gene on 5q12 encodes 5 splice-variants which show tissue-dependent expression and regulation. We have demonstrated both transcriptional and functional activity upregulation of PDE4D5 in human airway smooth muscle cells (hASM) in response to increased intracellular CAMP. In silico methods were used to determine genomic arrangement and identify a putative promoter of PDE4D5. 1544 and 621 b.p. constructs of this putative promoter were ligated into the luciferase reporter vector PGL3 enhancer, and transiently transfected into a CHO cell line that stably expressed both a2 adrenoceptors and a secreted placental alkaline phosphatase (SPAP) reporter driven by a CAMP responsive promoter. The cells were then stimulated for 5 hours with isoprenaline, isoprenaline and IBMX (non-selective PDE inhibitor), lBMX alone, and forskolin at doses calculated to produce a maximal CAMP increase. SPAP output was increased by up to 4 fold from basal (p

and D. Shiokawa toxins a member of the DN& I-family. Uniik; other DNase-I family DNases, DNase gamma carries a putative NLS at the C-terminus. Among DNase I-family DNases, only DNase gamma causes DNA fragmentation when each transfectant of HeLa S3 cells is induced by several apoptotic stimuli. Interestingly, DNA fragmentation scarcely occurs in the cells expressing the C-terminal deletion mutant, DNase gamma(-C). Furthermore, the transfected wild-type DNase gamma activity increases in the nuclei during apoptosis, while DNase gamma(-C) activity is not detected in the nuclei and secreted to the extracellular medium. The analysis of intercellular localization of DNase gamma using GFP fusion reveals that it distributes in the perinuclear membranes in normal states, and locates in the nuclei undergoing apoptosis. These results suggest that the C-terminus plays an important role in DNase gamma activation during apoptosis. Thus, we suppose the possible activation mechanism of DNase gamma that it is released to the cytoplasm by apoptotic stimuli and translocated to the nucleus via the C-terminal NLS.377 Low molecular weight factors of human seminal plasma activate adenylyl cyclase and induce caspase 3 independent apoptosis in prostatic epithelial cells by decreasing mitochondrial potential Rumpold H1, Plas E2, Reiter W3, and Berger P1 and bcl2/bax ratio The majority of elderly men is affected by benign and malign diseases of the prostate which seem to be governed by endocrine, auto/paracrine and as postulated by us luminal factors as well. Prostate epithelial cells secrete numerous factors into the seminal plasma (SMP) which are thought to be responsible for nutrition and the right pH and ionic environment of sperm. We hypothesize, that they might as well have a retrograde influence on prostatic epithelial cell growth, differentiation and function. Next to cell viability/proliferation apoptosis of prostate epithelial cells was determined by caspase activity assays, by tunel, by annexinl propidiumiodide staining, changes in mitochondrial potential, p53, par4, bax and bc12. SMP was analyzed for its content of proteins and other biological active substances by size exclusion HPLC. Each fraction was tested for its effect on cell growth. Interestingly, a low molecular weight fraction (2-4 kD) was responsible for the apoptotic effect on BPH-1 cells. Signal transduction was investigated by phosphotyrosine Western blots and the production of CAMP. In summary, SMP acts on proliferating prostate epithelial cells by induction of apoptosis. Therefore, SMP might contain protective factors helping to suppress excessive epithelial cell growth and inducing cell differentiation. Age-related changes in the composition of SMP could contribute to the development of proliferative prostatic diseases, like benign hyperplasia of the prostate and prostatic cancer.Why infection with Pseudomonas aeruginosa (PSa) remains so difficult to eradicate is not we11 understood. We hypothesise that the effects of the PSa toxins pyocyanin (pyo), 1 -hydroxyph...

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