Functional analysis of the interleukin-1-receptor-associated kinase (IRAK-1) in interleukin-1 beta-stimulated nuclear factor kappa B (NF-kappa B) pathway activation: IRAK-1 associates with the NF-kappa B essential modulator (NEMO) upon receptor stimulation.

The interleukin-1 (IL-1)-receptor-associated kinase (IRAK-1) is essential for IL-1-stimulated nuclear factor kappa B (NF-kappa B) activation. To study the role of IRAK-1 in IL-1 beta signalling, we have generated a set of IRAK-1 variants that express distinct domains of IRAK-1 either alone or in combination and have examined their effects on an NF-kappa B-responsive reporter in HeLa cells. Unlike full-length IRAK-1, the deletion mutants were unable to activate NF-kappa B in the absence of cytokine stimulation. However, an IRAK-1 variant lacking only the N-terminal domain retained the ability of the full-length protein to potentiate both IL-1 beta and tumour necrosis factor alpha (TNF alpha)-induced NF-kappa B activation. In contrast, expression of the N-terminus or the C-terminus of IRAK-1, or a fusion protein incorporating both domains, inhibited both IL-1 beta- and TNF alpha-induced effects. Expression of an IRAK-1 variant lacking only the C-terminal domain preferentially inhibited IL-1 beta versus TNF alpha-induced NF-kappa B activation. These data suggest that the C-terminal domain may link IRAK-1 to downstream signalling components common to both the IL-1 and TNF pathways. Furthermore, we have demonstrated that endogenous IRAK-1 becomes phosphorylated upon IL-1 beta treatment and can be detected along with NF-kappa B essential modulator (NEMO) and I kappa B kinase beta (IKK beta) in high-molecular-mass complexes of 600-800 kDa. Moreover, IRAK-1 could be detected in NEMO immunoprecipitates from IL-1 beta-stimulated cells. We conclude that IRAK-1 mediates IL-1 beta signal transduction through a ligand-dependent association of IRAK-1 with the IKK complex.

A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans.

BACKGROUND: Both animals and plants respond rapidly to pathogens by inducing the expression of defense-related genes. Whether such an inducible system of innate immunity is present in the model nematode Caenorhabditis elegans is currently an open question. Among conserved signaling pathways important for innate immunity, the Toll pathway is the best characterized. In Drosophila, this pathway also has an essential developmental role. C. elegans possesses structural homologs of components of this pathway, and this observation raises the possibility that a Toll pathway might also function in nematodes to trigger defense mechanisms or to control development. RESULTS: We have generated and characterized deletion mutants for four genes supposed to function in a nematode Toll signaling pathway. These genes are tol-1, trf-1, pik-1, and ikb-1 and are homologous to the Drosophila melanogaster Toll, dTraf, pelle, and cactus genes, respectively. Of these four genes, only tol-1 is required for nematode development. None of them are important for the resistance of C. elegans to a number of pathogens. On the other hand, C. elegans is capable of distinguishing different bacterial species and has a tendency to avoid certain pathogens, including Serratia marcescens. The tol-1 mutants are defective in their avoidance of pathogenic S. marcescens, although other chemosensory behaviors are wild type. CONCLUSIONS: In C. elegans, tol-1 is important for development and pathogen recognition, as is Toll in Drosophila, but remarkably for the latter rôle, it functions in the context of a behavioral mechanism that keeps worms away from potential danger.

Formation and biochemical characterization of tube/pelle death domain complexes: critical regulators of postreceptor signaling by the Drosophila toll receptor.

In Drosophila, the Toll receptor signaling pathway is required for embryonic dorso-ventral patterning and at later developmental stages for innate immune responses. It is thought that dimerization of the receptor by binding of the ligand spätzle causes the formation of a postreceptor activation complex at the cytoplasmic surface of the membrane. Two components of this complex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thought that pelle is bound by tube during formation of the activation complexes, and that this interaction is mediated by the death domains. In this paper, we show using the yeast two-hybrid system that the wild-type tube and pelle death domains bind together. Mutant tube proteins which do not support signaling in the embryo are also unable to bind pelle in the 2-hybrid assay. We have purified proteins corresponding to the death domains of tube and pelle and show that these form corresponding heterodimeric complexes in vitro. Partial proteolysis reveals a smaller core consisting of the minimal death domain sequences. We have studied the tube/pelle interaction with the techniques of surface plasmon resonance, analytical ultracentrifugation and isothermal titration calorimetry. These measurements produce a value of K(d) for the complex of about 0.5 microM.

A search within the IL-1 type I receptor reveals a peptide with hydropathic complementarity to the IL-1beta trigger loop which binds to IL-1 and inhibits in vitro responses.

In previous research, we were able to demonstrate that a seven amino acid residue peptide (VITFFSL), designed as an antisense peptide of the beta-bulge trigger loop region of interleukin 1beta (IL-1beta) (QGEESND; residues 48-54 [mature protein sequence]), was able to interact with IL-1 specifically and inhibit the response to IL-1 in an in vitro bioassay. The evidence was consistent with a specific interaction ocurring between antisense peptide and the trigger loop region. On the basis that antisense peptides are able to interact with their corresponding sense peptide sequences as a result of their mutually complementary hydropathic profiles (Fassina G., Verdoliva, A., Cassani, G., Melli, M., 1994. Binding of type I IL-1 receptor fragment 151-162 to IL-1. Growth Factors 10, 99-106; Maier, C.C., Moseley, H.N.B., Zhou, S., Whitaker, J.N., Blalock, J.E., 1994. Indentification of interactive determinants on idiotypic-anti-idiotypic antibodies through comparison of their hydropathic profiles. Immunomethods 5, 107-113), we devised a computer program (FINDH) to search the amino acid residue sequence of interleukin-1 type 1 receptor (IL-1 R1) for peptide motifs possessing hydropathic complementarity to the trigger loop sequence. The most complementary "best-fit peptide" motif (LITVLNI) was located in the third extracellular domain of IL-1 R1. A best-fit peptide corresponding to this motif was synthesised and found to bind to IL-1beta as well as inhibit the response to IL-1 in two independent in vitro bioassays (monitoring IL-1 dependent serum amyloid A synthesis and IL-1 dependent alkaline phosphatase activity, respectively). A second peptide motif (VIEFITL) was identified and the corresponding peptide synthesised along with a reordered version (LTILINV) of the best fit peptide. Both failed to bind measurably with IL-1beta or inhibit the response to IL-1 in the two bioassays. This best fit peptide behaved very similarly, in terms of IL-1 binding and inhibition behaviour, to the original trigger loop antisense peptide. Reference to the recently released X-ray crystal structure of IL-1beta and the IL1-R1 extracellular domain shows that the best fit peptide motif in IL-1 R1 is not apparantly interacting with the IL-1 trigger loop, although both are close in space. The intriguing possibility exists that the best fit peptide motif could represent an alternative site for IL-1beta receptor interaction which has not thus far been identified.

Induction of the E-selectin promoter by interleukin 1 and tumour necrosis factor alpha, and inhibition by glucocorticoids.

Cytokine-induced expression of the endothelial cell surface adhesion molecule E-selectin is inhibited by glucocorticoids (GCs). To investigate possible mechanisms for steroid inhibition, a reporter gene (ESAP) was constructed, comprising the cytokine responsive region of the E-selectin gene (nt -383 to +81) coupled to alkaline phosphatase (AP). In A549 cells stably transfected with the ESAP gene, AP production was highly responsive to the cytokines interleukin 1beta (IL-1beta) and tumour necrosis factor alpha, with ED50 values of 3 pM and 1000 pM respectively. Furthermore the cytokine-induced AP responses were inhibited by GCs, indicating that both transcriptional activation and GC suppression of the E-selectin gene were mediated via regulatory elements within the same region of the promoter. The relative potencies of GC drugs as inhibitors of IL-1beta (10 pM)-stimulated ESAP-gene activation were fluticasone> beclomethasone>dexamethasone, with IC50 values of 0.13, 1.1 and 2.7 nM respectively. Inhibition by fluticasone was blocked by the GC receptor (GR) antagonist drug mifepristone (Ru486), which is consistent with the suppressive effects of GCs being mediated via the GR. However, because the E-selectin promoter lacks a consensus glucocorticoid responsive element, mechanisms for inhibition independent of GR-DNA binding were investigated. Evidence that GCs also inhibited cytokine activation of a synthetic nuclear factor kappaB (NFkappaB)-driven reporter gene transiently transfected into A549 cells suggested that interference with the activation and/or function of this transcription factor was important for GC inhibition of ESAP. However, in A549-ESAP cells, fluticasone (100 nM) did not affect IL-1beta (10 pM)-induced IkBalpha degradation, NFkappaB-p65 nuclear translocation or the DNA-binding capacity of nuclear NFkappaB complexes, over a period during which cytokine-induced ESAP-gene activation was inhibited. Finally, there was no evidence to suggest that GC enhancement of IkBalpha gene expression contributed to the suppression of the cytokine response. We conclude that interference by GR with the transcriptional activation potential of DNA-bound NFkappaB complexes might contribute to mechanisms underlying the anti-inflammatory effects of GCs.

Reduction by inhibitors of mono(ADP-ribosyl)transferase of chemotaxis in human neutrophil leucocytes by inhibition of the assembly of filamentous actin.

1. Chemotaxis of human neutrophils is mediated by numerous agents [e.g. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet activating factor (PAF)] whose receptors are coupled to phospholipase C. However, the subsequent transduction pathway mediating cell movement remains obscure. We now propose involvement of mono(ADP-ribosyl)transferase activity in receptor-dependent chemotaxis. 2. Human neutrophils were isolated from whole blood and measurements were made of FMLP or PAF-dependent actin polymerization and chemotaxis. The activity of cell surface Arg-specific mono(ADP-ribosyl)transferase was also measured. Each of these activities was inhibited by vitamin K3 and similar IC50 values obtained (4.67 +/- 1.46 microM, 2.0 +/- 0.1 microM and 4.7 +/- 0.1 microM respectively). 3. There were similar close correlations between inhibition of (a) enzyme activity and (b) actin polymerization or chemotaxis by other known inhibitors of mono(ADP-ribosyl)transferase, namely vitamin K1, novobiocin, nicotinamide and the efficient pseudosubstrate, diethylamino(benzylidineamino)guanidine (DEA-BAG). 4. Intracellular Ca2+ was measured by laser scanning confocal microscopy with two fluorescent dyes (Fluo-3 and Fura-Red). Exposure of human neutrophils to FMLP or PAF was followed by transient increases in intracellular Ca2+ concentration, but the inhibitors of mono(ADP-ribosyl)transferase listed above had no effect on the magnitude of the response. 5. A panel of selective inhibitors of protein kinase C, tyrosine kinase, protein kinases A and G or phosphatases 1 and 2A showed no consistent inhibition of FMLP-dependent polymerization of actin. 6. We conclude that eukaryotic Arg-specific mono(ADP-ribosyl)transferase activity may be implicated in the transduction pathway mediating chemotaxis of human neutrophils, with involvement in the assembly of actin-containing cytoskeletal microfilaments.

Microelectronics effects as seen on CRRES.

A MicroElectronics Test Package (MEP) measured total dose degradation and single event upsets (SEUs) on 60 device types on the Combined Release and Radiation Effects Satellite (CRRES) in an 18 degrees inclination orbit between 350 km and 36000 km from July 1990 to October 1991. Simultaneous measurements of the high energy particle environment were used to make a direct cause and effect comparison of the energetic particle backgrounds and microelectronic performance characteristics. The galactic cosmic ray background for the period of the CRRES mission was at a minimum. The SEUs experienced from the cosmic ray background were correspondingly few in number, but surprisingly produced an equal probability of upset over an L-shell range of 8.5 Earth radii (RE) down to less than 3.0 RE. Cosmic ray induced upset frequencies in proton sensitive chips were over 2 orders of magnitude lower than those produced by protons in the heart of the inner proton radiation belts. Multiple upsets, those produced when a single particle upsets more than one memory location, were just as common from protons as from cosmic rays.

Interleukin-1 induces a nuclear form of transcription factor NF kappa B in human lung epithelial cells.

Activation of the transcription factor NF kappa B by the proinflammatory cytokine interleukin-1 (IL-1) has been investigated in the human lung epithelial cell line (A549). Nuclear NF kappa B activity was not detectable in unstimulated A549 cells, but was rapidly (within 15 min) and potently (100 fM to 1 pM) induced in the presence of IL-1. Activation of NF kappa B by IL-1 (1 pM) was prevented when incubations were performed in the presence of the IL-1 receptor antagonist protein (1 nM). However, a number of protein kinase inhibitors failed to block this response. The results suggest that a novel kinase pathway may be involved in the activation of NF kappa B by IL-1 in these cells.

Paracrine control of differentiation in the alveolar carcinoma, A549, by human foetal lung fibroblasts.

Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts.

Occurrence of rare somatomammotrophs in ovine anterior pituitary tissue studied by immunogold labelling and electron microscopy.

Prolactin and GH are distinct hormones that have been conventionally thought to be produced and secreted by separate cells in the anterior pituitary gland. Recently it has been suggested that some cells (somatomammotrophs) may secrete both hormones. We have examined the occurrence of somatomammotrophs in sheep anterior pituitary tissue using immunogold labelling. Of a number of procedures used, double labelling using first antibodies raised in different species proved the least susceptible to apparent co-localization of hormones due to artifacts. Using this approach it was shown that a large proportion of the cells in the sheep anterior pituitary glands examined were mammotrophs or somatotrophs, showing no significant co-localization of GH and prolactin. Of 1800 cells examined, only two were somatomammotrophs. One of these, from a female animal, contained GH and prolactin in different granules within the same cell. The other, from a male animal, showed co-localization of these two hormones within the same granules.

Actions of pertussis toxin on the inhibitory effects of dopamine and somatostatin on prolactin and growth hormone release from ovine anterior pituitary cells.

Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulate prolactin and GH release from ovine anterior pituitary cells cultured in vitro. Dopamine and somatostatin inhibit release of prolactin and GH respectively, after stimulation by these agents, but without effects on intracellular cyclic AMP concentrations. In each case the inhibitory effects were reversed by pretreatment of cells with pertussis toxin, in a dose-related fashion (1-100 ng/ml), again without affecting cyclic AMP levels. The results suggest that the inhibitory effects of dopamine and somatostatin in this system are mediated by one or more pertussis toxin-sensitive G proteins, and that these act by a mechanism which does not involve inhibition of adenylate cyclase.

A local network for sharing resources and technical support: BACS/PHILNET.

Within the framework of large regional and national networks, local libraries can benefit by working together. We report the establishment of an online health sciences library network to share resources and technical support in the greater St. Louis area. BACS/PHILNET has evolved beyond a local automated interlibrary loan network to pilot off-site integrated library and information management systems in hospitals.

Regulation of growth hormone secretion and cyclic AMP metabolism in ovine pituitary cells: interactions involved in activation induced by growth hormone-releasing hormone and phorbol esters.

Growth hormone-releasing hormone (GHRH) and the phorbol ester tetradecanoylphorbol acetate (TPA) each stimulated a rapid and extensive (up to 15-fold) increase in the secretion of growth hormone from cultured ovine anterior pituitary cells. Effects of the releasing hormone on growth hormone secretion were associated with a concurrent, large increase in cellular cyclic AMP accumulation. TPA induced a much smaller (26-78%), though still significant, increase in cellular cyclic AMP levels. Forskolin and isobutylmethylxanthine (IBMX) also stimulated growth hormone secretion and cyclic AMP accumulation. When combined with a maximally effective concentration of GHRH these compounds did not further elevate growth hormone secretion even though they induced further increases in cyclic AMP concentration; this is consistent with activation occurring via a common cyclic AMP-dependent pathway. In contrast TPA when combined with maximally effective concentrations of either GHRH, forskolin or IBMX caused additional release of growth hormone, suggesting that the TPA-induced secretion involved a cyclic AMP-independent process. However, TPA also markedly potentiated the cellular cyclic AMP accumulation due to each of these agents. That TPA induced stimulation of basal and GHRH-stimulated cyclic AMP levels measured in the presence of IBMX suggests an action affecting cyclic AMP synthesis. Carbachol had no effect on basal or GHRH-stimulated growth hormone secretion or cyclic AMP levels. The two actions of TPA, one on secretion and one on cyclic AMP metabolism, may result from activation of some common event possibly involving protein kinase C. Our results suggest that GHRH and TPA activate independent pathways regulating growth hormone secretion.

Mechanisms involved in the effects of TRH on GHRH-stimulated growth hormone release from ovine and bovine pituitary cells.

Incubation of cultured ovine pituitary cells with growth hormone-releasing hormone (GHRH) (10(-12)-10(-7) M) stimulated growth hormone secretion up to 3-fold. At a maximal stimulatory concentration of GHRH (10(-10) M), thyrotropin-releasing hormone (TRH) (10(-7) M) caused an inhibition of growth hormone release to approx. 50% of the response obtained with GHRH alone (during a 15 min incubation period). TRH also caused a small inhibition of the GHRH-stimulated cellular cyclic AMP level but this effect was only significant at a relatively high concentration of GHRH (10(-9) M). Incubation of cultured bovine pituitary cells with GHRH (10(-11)-10(-8) M) plus TRH (10(-7) M) caused a significant stimulation of growth hormone release by up to 40%, compared with the response obtained with GHRH alone (at all concentrations of GHRH). TRH (10(-7) M) had no effect on GHRH (10(-8) M)-stimulated cellular cyclic AMP levels in a partially purified bovine pituitary cell preparation. The effects of varying extracellular [Ca2+] (0.1-10 mM) on intracellular [Ca2+] and on the responsiveness to releasing hormones were also determined using ovine pituitary cells. GHRH (10(-10) M)-stimulated growth hormone release was inhibited when cells were incubated at both high (10 mM) and low (0.1 mM) [Ca2+] (compared with 1 mM or 3 mM Ca2+) with or without TRH (10(-7) M). At 1 mM Ca2+, TRH produced a synergistic effect with GHRH to stimulate growth hormone release. However, at 3 mM Ca2+ TRH inhibited GHRH-stimulated growth hormone release.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of ovine somatotrophs using a combination of density gradient centrifugation and short-term culture.

Freshly dispersed or cultured (18 h) ovine anterior pituitary cells were fractionated on 55% sigmoidal Percoll gradients. This resulted in the separation of two sub-populations of cells at densities of 1.075 g/ml (peak I) and 1.055 g/ml (peak II). Radioimmunoassay of fractions from a gradient on which cells were separated before culture showed the profiles of GH and prolactin to be virtually superimposable. After culture, however, the lactotroph population exhibited an apparent shift in its density profile, the lighter population increasing at the expense of the denser one. Immunocytochemistry of the cells remaining in the denser peak (peak I) showed that it consisted of about 65% somatotrophs which was approximately a three-fold enrichment over the proportion of somatotrophs present in the original cell preparation (24%). Refractionation studies indicated that the change in the density profile of the lactotrophs was due to an actual reduction in their density rather than a loss of viability of the denser sub-population. Secretion data showed the purified somatotrophs to be more responsive than the crude cell preparation to the regulatory factors, GH-releasing hormone and somatostatin. This enriched cell population should prove useful in the detailed study of GH secretion.