Endotoxin-adapted septic shock leukocytes selectively alter production of sIL-1RA and IL-1beta.

During septic shock, circulating levels of anti-inflammatory mediators are increased relative to those of pro-inflammatory. The reduced capacity of septic shock blood leukocytes in expressing pro-inflammatory genes in response to bacterial lipopolysaccharide endotoxin (LPS) may contribute to reductions in these mediators, but the reasons for persistent increases in circulating anti-inflammatory mediators are unknown. We determined whether septic shock leukocytes that have adapted to LPS induction of the IL-1beta gene could continue to express sIL-1RA in response to LPS. Septic shock whole-blood leukocytes and neutrophils (PMNs) selectively maintained production of sIL-1RA after treatment with LPS while limiting that of IL-1beta. Repressed transcription of IL-1beta and rapid decay of IL-1beta mRNA in septic shock neutrophils correlated with reductions in levels of IL-1beta after stimulation with LPS. Transcription of sIL-1RA mRNA was also suppressed, but the ability of LPS to stimulate events that lead to efficient translation of a stable sIL-1RA mRNA appeared responsible for maintaining sIL-1RA production. We conclude that LPS adaptation of septic shock leukocytes selectively influences signaling pathways that regulate transcription, mRNA processing, and translation, leading to changes in the balance of production of pro- and anti-inflammatory mediators.

Endotoxin inducible transcription is repressed in endotoxin tolerant cells.

Stimulation of the human promonocytic cell line, THP-1, with endotoxin results in a rapid and transient increase in interleukin 1beta expression. Endotoxin pretreatment of THP-1 cells results in tolerance, characterized by decreased levels of endotoxin-induced interleukin 1beta expression due to decreased transcription of the interleukin 1beta gene. We hypothesized that tolerant cells could not activate transcription factors necessary to express the interleukin 1beta gene. This hypothesis was tested in tolerant THP-1 cells by using stable and transiently transfected reporter genes containing the interleukin 1beta promoter. We found decreased endotoxin-induced transcription of all reporter genes tested; however, individual transcription factors, such as NFkappaB, retain normal, CD14-dependent, nuclear translocation and DNA binding. Tolerance is specific for endotoxin, because phorbol ester is still able to activate transcription of the endogenous interleukin 1beta gene and transfected reporter genes. A constitutively active reporter gene that is not inducible by endotoxin is unaffected. We further show that nuclear extracts of tolerant cells show transcription inhibitor activity that is specific for promoter sequences of the interleukin 1beta gene. These results support a mechanism of endotoxin tolerance that is independent of transcription factor DNA binding and appears to be associated with the inability of DNA-bound transcription factors to activate transcription, perhaps through the activity of a repressor.

Inhibition of host RNA polymerase II-dependent transcription by vesicular stomatitis virus results from inactivation of TFIID.

During infection with vesicular stomatitis virus (VSV), host-cell mRNA synthesis is inhibited due to shut off of host-cell transcription. The transcriptional activity of nuclear extracts prepared from VSV-infected cells was compared to the activity of nuclear extracts from uninfected cells. An exogenous DNA template was used which contained an adenovirus major late promoter (AdMLP) but lacked upstream activating sequences, so that only basal transcription activity was assayed in these experiments. AdMLP-initiated transcription was decreased by 75% in nuclear extracts from infected cells as early as 3 h p.i. and by >90% by 6 h p.i. Mixing nuclear extracts from uninfected and VSV-infected cells revealed that the inhibition was caused by lack of an active form of a host factor involved in basal transcription rather than by the presence of an excess of inhibitory factor. To determine which transcription factors were lacking from nuclear extracts of infected cells, host transcription initiation factors isolated from uninfected cells by ion-exchange chromatography were added separately to nuclear extracts inactivated by VSV infection. A phosphocellulose column fraction from uninfected cells eluted with 0. 8 M KCl, which contained transcription factor IID (TFIID), overcame the inhibition. The corresponding fraction from infected cells had no detectable activity in a TFIID-dependent in vitro transcription assay. TATA-binding protein (TBP) is the DNA-binding subunit of TFIID and has been shown previously to substitute for TFIID in basal transcription. Purified recombinant TBP also reconstituted the transcription activity of nuclear extracts from infected cells, supporting the idea that TFIID is the target of virus-induced inhibition. Western blot analysis showed that the level of TBP in nuclear extracts or in the 0.8 M KCl column fraction was not changed by VSV infection. These results indicated that VSV infection leads to an inhibition of host transcription by inactivation of TFIID rather than reduction in the level of TFIID.

Interleukin-1beta expression after inhibition of protein phosphatases in endotoxin-tolerant cells.

Endotoxin (lipopolysaccharide [LPS]) is a potent activator of a number of inflammatory genes in blood leukocytes, including interleukin-1 (IL-1). Blood leukocytes isolated from patients with septic shock fail to produce IL-1 in response to LPS, a phenomenon known as endotoxin tolerance. To study the regulation of IL-1 expression in endotoxin-tolerant cells, the protein phosphatase inhibitor okadaic acid was used to examine the effects of protein phosphorylation on IL-1beta gene expression. We found that endotoxin-tolerant cells produced normal levels of IL-1beta when protein phosphatases were inhibited. In the human pro-monocytic cell line THP-1, okadaic acid increased mRNA accumulation and synthesis of IL-1beta protein. Normal and endotoxin-tolerant THP-1 cells accumulated IL-1beta mRNA and protein with similar delayed kinetics. Okadaic acid stabilization of IL-1beta mRNA appears to be the primary mechanism through which endotoxin-tolerant cells accumulate IL-1beta mRNA and protein. Endotoxin-tolerant cells were unable to activate transcription in response to okadaic acid. However, the transcription factor NF-kappaB, which is known to be involved in IL-1beta expression, was translocated to the nucleus in both normal and endotoxin-tolerant cells after treatment with okadaic acid. These studies revealed that protein phosphorylation can affect gene expression on at least two distinct levels, transcription factor activation and mRNA stability. Endotoxin-tolerant cells have decreased transcription activation potential, while IL-1beta mRNA stability remains responsive to protein phosphorylation.

Protein-tyrosine kinase activation is required for lipopolysaccharide induction of interleukin 1beta and NFkappaB activation, but not NFkappaB nuclear translocation.

In human monocytes, interleukin 1beta protein production and steady state mRNA levels are increased in response to lipopolysaccharide, predominantly as a result of increased transcription of the interleukin 1beta gene. Expression of interleukin 1beta and other cytokines, such as interleukin 6 and tumor necrosis factor alpha, has been shown to be dependent on the activation of the transcription factor, NFkappaB. Since recent studies have shown that lipopolysaccharide-induced tyrosine kinase activation is not required for NFkappaB nuclear translocation, we sought to determine whether NFkappaB translocated in the absence of tyrosine kinase activity was active in stimulating transcription. We have found that, in the human pro-monocytic cell line, THP-1, the lipopolysaccharide-induced expression of interleukin 1beta is dependent on tyrosine kinase activation. Tyrosine kinases are not required for lipopolysaccharide-mediated nuclear translocation of NFkappaB. However, in the absence of tyrosine kinase activity, the ability of NFkappaB to stimulate transcription is impaired. This inhibition of transcription is specific for NFkappaB; in the absence of tyrosine kinase activity, AP-1-dependent transcription is enhanced. These results suggest that, while lipopolysaccharide-induced expression of inflammatory mediators requires tyrosine kinase activity, tyrosine kinase activity is not obligatory for lipopolysaccharide signal transduction.

Interleukin 1 activation of the AP-1 transcription complex in murine T cells is regulated at the level of Jun B protein accumulation.

We have examined the regulation of the AP-1 DNA transcription complex during T cell activation in response to interleukin 1 (IL-1) and phorbol ester (TPA) treatment of the IL-1 responsive murine thymoma T cell line, EL4 6.1 C 10. IL-1 synergistically enhances the stimulatory effect of TPA on AP-1-mediated gene expression in this cell line. To elucidate the mechanism(s) by which IL-1 enhances AP-1-mediated gene expression, we examined the effect of IL-1 on the synthesis and turnover of Jun B, the member of the jun gene family that is present in AP-1 complexes in EL4 cells. We found that IL-1 + TPA-treated cells contain significantly higher Jun B protein levels than cells treated with TPA alone. IL-1 promotes the prolonged accumulation of Jun B, whereas the cellular content of Jun B decreases dramatically after 6 hr in cells treated with only TPA. IL-1 enhancement of Jun B protein levels is not the result of a change in the turnover rate of the Jun B protein, but rather results from the maintenance of sufficient jun B mRNA to support continued accumulation of newly synthesized protein. In addition to Jun B, we found that the T cell AP-1 complex contains the Fra-1 protein, a member of the fos gene family. Although IL-1 dramatically increases Jun B accumulation, it does not enhance TPA-induced Fra-1 protein levels in EL4 cells. Thus, the stimulation of AP-1-mediated gene expression by IL-1 in EL4 cells is due to the promotion of Jun B protein accumulation that, in turn, facilitates Jun B heterodimerization with TPA-induced Fra-1 protein, thereby forming an active AP-1 complex.

Induction of AP-1 transcription factor components during T-cell activation by interleukin 1 and phorbol esters.

We have examined the effect of interleukin 1 (IL-1) and phorbol esters [12-O-tetradecanoylphorbol-13-acetate (TPA)] on the expression of various components of the AP-1 transcription factor complex during T-cell activation. We previously found that a chloramphenicol acetyltransferase reporter gene driven by the collagenase TPA responsive element was expressed upon stimulation of T-cells by TPA and that this expression was enhanced when IL-1 was added as a costimulant; IL-1 alone had no effect on TPA responsive element-chloramphenicol acetyltransferase expression. In this study, we have found that stimulation of T-cells by IL-1 and TPA is accompanied by activation of a subset of immediate early genes that comprise the AP-1 transcription factor complex. junB and fosB were rapidly induced following stimulation with TPA. Although the levels of other fos-related mRNAs were also elevated, their maximal induction was delayed by approximately 5 h. IL-1 alone had little or no effect, but enhanced TPA induced transcription and steady-state levels of these mRNAs. The expression of fos and jun during T-cell activation was accompanied by increased specific binding of JunB, FosB, and fos-related antigen containing complexes to the TPA responsive element. These findings indicate that the synergistic effect of IL-1 and TPA on AP-1 mediated gene expression is due, in part, to the ability of IL-1 to enhance the expression of genes encoding specific AP-1 transcription factor components.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of AP-1 by IL-1 and phorbol esters in T cells. Role of protein kinase A and protein phosphatases.

We have examined the regulation of the AP-1 transcription complex in the IL-1-responsive murine T cell thymoma cell line EL-4 6.1 C10. Our results demonstrate that AP-1-mediated gene expression in T cells may be regulated by several signaling pathways and factors, including IL-1, protein kinase C, protein kinase A (PKA), and one or more serine/threonine-specific protein phosphatases. The activation of protein kinase C results in an increase in nuclear AP-1 DNA binding activity, as well as enhanced gene expression. IL-1 and agents that elevate intracellular cAMP levels do not, by themselves, induce AP-1 activation, but they synergize with phorbol esters. IL-1 and forskolin may enhance AP-1 function by different mechanisms, because forskolin enhanced gene expression without producing an increase in nuclear AP-1 DNA binding, whereas IL-1 increased AP-1-binding activity and gene expression. These observations, in conjunction with the lack of a demonstrable effect of IL-1 on cAMP production in EL-4 cells, are consistent with the view that IL-1 enhances AP-1 activation by a pathway that does not directly involve cAMP and PKA. However, the induction of AP-1 activity by IL-1 and phorbol esters is dependent upon the presence of PKA, as evidenced by the loss of AP-1 inducibility in cells transfected with a cDNA encoding protein kinase inhibitor, a specific inhibitor of PKA. The effect of protein kinase inhibitor on AP-1 activation in response to IL-1 and tetradecanoyl-phorbol-13-acetate was reversed in the presence of the serine/threonine protein phosphatase inhibitor okadaic acid. Thus, the level of AP-1 activity in T cells may be determined by the balance between the activities of several serine/threonine protein kinases and phosphatases.

Identification of a novel factor that interacts with an immunoglobulin heavy-chain promoter and stimulates transcription in conjunction with the lymphoid cell-specific factor OTF2.

The tissue-specific expression of the MOPC 141 immunoglobulin heavy-chain gene was studied by using in vitro transcription. B-cell-specific transcription of this gene was dependent on the octamer element 5'-ATGCAAAG-3', located in the upstream region of this promoter and in the promoters of all other immunoglobulin heavy- and light-chain genes. The interaction of purified octamer transcription factors 1 and 2 (OTF1 and OTF2) with the MOPC 141 promoter was studied by using electrophoretic mobility shift assays and DNase I footprinting. Purified OTF1 from HeLa cells and OTF1 and OTF2 from B cells bound to identical sequences within the heavy-chain promoter. The OTF interactions we observed extended over the heptamer element 5'-CTCAGGA-3', and it seems likely that the binding of the purified factors involves cooperation between octamer and heptamer sites in this promoter. In addition to these elements, we identified a second regulatory element, the N element with the sequence 5'-GGAACCTCCCCC-3'. The N element could independently mediate low levels of transcription in both B-cell and HeLa-cell extracts, and, in conjunction with the octamer element, it can promote high levels of transcription in B-cell extracts. The N element bound a transcription factor, NTF, that is ubiquitous in cell-type distribution, and NTF was distinct from any of the previously described proteins that bind to similar sequences. Based on these results, we propose that NTF and OTF2 interactions (both with their cognate DNA elements and possibly at the protein-protein level) may be critical to B-cell-specific expression and that these interactions provide additional pathways for regulating gene expression.

Functional cooperativity between protein molecules bound at two distinct sequence elements of the immunoglobulin heavy-chain promoter.

Immunoglobulin heavy-chain gene promoters contain two conserved upstream sequence elements, octamer and heptamer, both of which are required for normal cell type-specific promoter function in vivo. The octamer sequence motif 5'-ATGCAAAT-3', and its precise inverse, are strongly conserved in heavy- and light-chain gene promoters and are important determinants for the lymphoid-specific function of these promoters and of the heavy-chain enhancer. The heptameric sequence element with the consensus 5'-CTCATGA-3' (refs 3 and 4) is also required in addition to the octamer for full lymphoid-specific activity of heavy-chain promoters. Although these two elements have no sequence similarity, they are both recognized in vitro by the ubiquitous octamer transcription factor OTF-1 (reviewed refs 13 and 14) and the lymphoid-specific OTF-2 (reviewed in refs 15 and 16). Here we show that purified OTF-2 binds cooperatively to the immunoglobulin heptamer and octamer elements so that interaction with the octamer element facilitates binding of OTF-2 to the heptamer motif. More important, cooperativity in OTF-2 binding is closely mirrored by functional cooperation between the heptamer and octamer elements in activating transcription from the heavy-chain promoter in vitro.

Accurate in vitro transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters.

The mitochondrial RNA polymerase from Xenopus laevis oocytes was partially purified by heparin-Sepharose chromatography and phosphocellulose chromatography. This RNA polymerase preparation specifically initiated the transcription of X. laevis mitochondrial DNA (mtDNA) from two bidirectional promoters contained within a 123-base-pair segment of the mtDNA between the heavy-strand replication origin and the rRNA cistrons. Transcription in vitro initiated from precisely the same start sites previously mapped as initiation sites for transcription in vivo. At each of the four sites, initiation occurred within a conserved nucleotide sequence, ACPuTTATA. This consensus sequence is not related to promoters for transcription of human mtDNA.

Identification of initiation sites for transcription of Xenopus laevis mitochondrial DNA.

The sites of transcription initiation for Xenopus laevis mtDNA have been mapped using in vitro capping and primer extension analysis of RNA isolated from oocytes. Transcription of the heavy strand initiates predominantly at a site 33 nucleotides upstream of the tRNAPhe gene. This promoter is bidirectional, with transcription of the light strand initiating only one base pair away. A second, more predominant light strand promoter is located 70 nucleotides away from the major heavy strand promoter. The overall organization of the transcription initiation sites with respect to the tRNAPhe gene and the 5' termini of major stable D-loop DNA strands resembles that of the human mtDNA genome. Analysis of the sequences surrounding the Xenopus start sites suggests that the occurrence of a conserved sequence element, ACPuTTATA, around the start sites may be important for promoter recognition and transcription initiation. This sequence is not found in human mtDNA promoters.

Identification of a promoter for transcription of the heavy strand of human mtDNA: in vitro transcription and deletion mutagenesis.

Plasmids containing the origin region of human mtDNA are specifically transcribed by the partially purified homologous mtRNA polymerase in vitro. Transcription of both the light and heavy strands initiates at the same sites previously identified as in vivo transcription start sites. The sequences responsible for initiation of heavy strand transcription are investigated in detail, since this transcript includes the rRNA cistrons and the majority of tRNA genes and potential mRNAs. Deletion mutagenesis is employed to delimit the sequences responsible for heavy strand transcription to a region of less than 35 bp surrounding the transcription start site. This region contains a repetitive sequence, AAACCCC, and shows homology to other regions of the human mtDNA that have been implicated as transcription initiation sites or that show unusual homology to other mammalian mtDNA genomes.

Identification and in vitro capping of a primary transcript of human mitochondrial DNA.

A discrete primary transcript of HeLa mitochondrial DNA containing 12 S rRNA, tRNAPhe, and 19 +/- 2 nucleotides upstream has been identified. The 5' ends of heavy strand encoded RNAs adjacent to the D-loop region of mitochondrial DNA were mapped by primer extension and S1 nuclease protection to a site 19 +/- 2 nucleotides upstream from the start of tRNAPhe. Hybridization of a tRNAPhe-specific probe to a blot of mitochondrial RNA detected a discrete species with a molecular weight consistent with an RNA containing 12 S rRNA, tRNAPhe, and a short upstream leader sequence. The di- or triphosphate nature of the 5' end of this polycistronic RNA was demonstrated by in vitro capping with [alpha-32P]GTP and guanylyltransferase. S1 nuclease protection of the purified in vitro capped RNA established that the 5' end, 19 +/- 2 nucleotides upstream from tRNAPhe, is an initiation site for heavy strand transcription.

Changes in responsiveness of the beta-adrenergic and serotonergic pathways of the rabbit corneal epithelium.

Adrenergic agonists stimulate the synthesis of cyclic AMP by incubated rabbit corneas with the following order of potency: isoproterenol greater than epinephrine greater than norepinephrine. These agonists have the same order of potency when displacing the specific, beta-adrenergic radioligand, 3H-dihydroalprenolol, from beta-adrenergic receptors on membranes prepared from corneal epithelium. At another locus, serotonin stimulates cyclic AMP synthesis. Inhibition of stimulation in vitro by lysergic acid diethylamide, methysergide, cyproheptadine, and spiroperidol demonstrates the specificity of this pathway for serotonin. Topical epinephrine causes subsensitivity or decreased responsiveness of the beta-adrenergic pathway. There is loss of approximately half the beta-adrenergic receptors from the cornea and a similar loss of epinephrine-stimulated cyclic AMP synthesis, both of which return to control levels in 96 hrs. There is no change in affinity for catecholamines and no loss of responsiveness to prostaglandin E2 or serotonin. Pretreatment with nialamide or subsequent treatment with additional epinephrine does not cause further loss of responsiveness. Supersensitivity or increased responsiveness of this pathway occurs following superior cervical ganglionectomy. Topical serotonin causes decreased responsiveness of the serotonergic pathway. When potentiated by nialamide, serotonin causes almost complete loss of serotonin-stimulated cyclic AMP synthesis for 24-48 hrs. There is no loss of responsiveness to epinephrine. Increased responsiveness of this pathway does not occur following superior cervical ganglionectomy. The authors conclude that the corneal epithelium has both beta 2-adrenergic and serotonin-2 pathways, and each pathway exhibits altered responsiveness by similar mechanisms. In response to exogenous or endogenous stimulation, the beta-adrenergic responsive cells and the serotonergic responsive cells apparently regulate the total number of pathway-specific receptors on their surfaces. Furthermore, the authors postulate that two populations of beta-adrenergic responsive cells exist; those on the apical surface of the epithelium that respond to catecholamine in the tears and those near the basal surface that respond to neuronal catecholamine.

Diurnal rhythm of mitosis in rabbit corneal epithelium.

Incorporation of 3H-thymidine by rabbit corneal epithelium during the course of a one-hour incubation in vitro varies according to the time of day, suggesting a diurnal rhythm of mitotic activity. Adrenergic decentralization of the cornea does not affect this rhythm. Furthermore, there is no diurnal variation in the basal or sympathomimetically-stimulated cyclic AMP production by freshly excised rabbit corneas, incubated in vitro. Therefore, the diurnal rhythm of corneal epithelial mitotsis in the rabbit is not paced by catecholamines.