Analysis of thymocyte MHC specificity with thymocyte hybridomas.

Medullary, peanut agglutinin-negative (PNA-), thymocytes were activated in vitro with either exogenous interleukin 1 (IL-1) or accessory cells. T cell blasts from these cultures were subsequently fused to BW5147 to generate thymocyte hybridomas. Fusion frequencies similar to those obtained with peripheral T lymphocytes were observed. A high frequency of these hybrids are triggered to produce IL-2 in the presence of syngeneic accessory cells. Exogenous, nominal antigens do not appear to be required for this activation. Using accessory cells from a series of recombinant inbred mice, the specificity of this hybrid-accessory cell interaction could be mapped to either I-Ak or I-Ek or both. This was confirmed by blocking with alpha Ia monoclonal antibodies (mAbs). A high frequency of these self-reactive cells are also alloreactive. Interestingly, several clones were identified that appear to recognize public Ia determinants broadly shared by different alleles and genetic subregions. Such specificities appear to contrast with those of peripheral T lymphocytes whose specificity is dominated by the genetically polymorphic portion of the Ia molecule. These results document the clonal specificity occurring in the cultures of in vitro activated thymocytes and allow an analysis of at least a portion of the intrathymic repertoire for major histocompatibility complex (MHC) determinants. The implications of these findings are discussed.

TAP, a novel T cell-activating protein involved in the stimulation of MHC-restricted T lymphocytes.

Five mAbs have been generated and used to characterize TAP (T cell activating protein) a novel, functional murine T cell membrane antigen. The TAP molecule is a 12-kD protein that is synthesized by T cells. By antibody crossblocking, it appears to be closely associated with a 16-kD protein on the T cell membrane also identified with a novel mAb. These molecules are clearly distinct from the major well-characterized murine T cell antigens previously described. Antibody binding to TAP can result in the activation of MHC-restricted, antigen-specific inducer T cell hybridomas that is equivalent in magnitude to maximal antigen or lectin stimulation. This is a direct effect of soluble antibody and does not require accessory cells or other factors. The activating anti-TAP mAbs are also mitogenic for normal heterogeneous T lymphocytes in the presence of accessory cells or IL-1. In addition, these antibodies are observed to modulate specific immune stimulation. Thus, the activating anti-TAP mAbs synergise with antigen-specific stimulation of T cells, while a nonactivating anti-TAP mAb inhibits antigen driven activation. These observations suggest that the TAP molecule may participate in physiologic T cell activation. The possible relationship of TAP to known physiologic triggering structures, the T3-T cell receptor complex, is considered. TAP is expressed on 70% of peripheral T cells and therefore defines a major T cell subset, making it perhaps the first example of a murine subset-specific activating protein.

PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1.

Nuclear domain 10 (ND10), also referred to as nuclear bodies, are discrete interchromosomal accumulations of several proteins including promyelocytic leukemia protein (PML) and Sp100. In this study, we investigated the mechanism of ND10 assembly by identifying proteins that are essential for this process using cells lines that lack individual ND10-associated proteins. We identified the adapter protein Daxx and BML, the RecQ helicase missing in Bloom syndrome, as new ND10-associated proteins. PML, but not BLM or Sp100, was found to be responsible for the proper localization of all other ND10-associated proteins since they are dispersed in PML-/- cells. Introducing PML into this cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10 attesting to PMLs essential nature in ND10 formation. In the absence of PML, Daxx is highly enriched in condensed chromatin. Its recruitment to ND10 from condensed chromatin requires a small ubiquitin-related modifier (SUMO-1) modification of PML and reflects the interaction between the COOH-terminal domain of Daxx and PML. The segregation of Daxx from condensed chromatin in the absence of PML to ND10 by increased accumulation of SUMO-1-modified PML suggests the presence of a variable equilibrium between these two nuclear sites. Our findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML.

Activation of CPP32-like protease in tumor necrosis factor-induced apoptosis is dependent on mitochondrial function.

Mitochondria have been implicated in apoptosis, however, the precise mechanisms whereby mitochondria exert their effect are not clear. To gain further insights, we generated a panel of cells from ML-1a cells that were rendered respiration deficient by ethidium bromide treatment. Two respiration-deficient clones were subsequently reconstituted by fusion with platelets. Respiration-deficient clones were resistant to TNF-induced apoptosis, whereas ML-1a and reconstituted clones were sensitive. In contrast, inhibition of proliferation and induction of differentiation by TNF were still observed in respiration deficient clones, suggesting a selective requirement of respiration in TNF-induced apoptosis. Furthermore the apoptosis machinery is not completely altered in respiration-deficient cells because they underwent apoptosis after staurosporine treatment. Next, we showed that apoptosis induced by TNF and staurosporine were blocked by z-DEVD-CH2F, an inhibitor of CPP32-like cysteine protease, suggesting the involvement of CPP32-like protease in both apoptosis signaling pathways. Interestingly, TNF activated CPP32-like protease in the parental and reconstituted clones but not in respiration-deficient clones, and staurosporine in all clones. Thus, the apoptosis signaling block in respiration-deficient clones is located at a step before CPP32-like protease activation, which can be bypassed by staurosporine.

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

p47phox is required for atherosclerotic lesion progression in ApoE(-/-) mice.

NADPH oxidase is upregulated in smooth muscle cells (SMCs) in response to growth factor stimulation, concomitant with increased reactive oxygen species (ROS) production. We investigated the role of ROS production by NADPH oxidase in SMC responses to growth factors and in atherosclerotic lesion formation in ApoE(-/-) mice. SMCs from wild-type, p47phox(-/-), and gp91phox(-/-) mice differed markedly with respect to growth factor responsiveness and ROS generation. p47phox(-/-) SMCs had diminished superoxide production and a decreased proliferative response to growth factors compared with wild-type cells, whereas the response of gp91phox(-/-) SMCs was indistinguishable from that of wild-type SMCs. The relevance of these in vitro observations was tested by measuring atherosclerotic lesion formation in genetically modified (wild-type, p47phox(-/-), ApoE(-/-), and ApoE(-/-)/p47phox(-/-)) mice. ApoE(-/-)/p47phox(-/-) mice had less total lesion area than ApoE(-/-) mice, regardless of whether mice were fed standard chow or a high-fat diet. Together, these studies provide convincing support for the hypothesis that superoxide generation in general, and NADPH oxidase in particular, have a requisite role in atherosclerotic lesion formation, and they provide a rationale for further studies to dissect the contributions of ROS to vascular lesion formation.

The potential role of antiplatelet agents in modulating inflammatory markers in atherothrombosis.

Atherothrombosis is the process that links atherosclerotic lesion development with unpredictable and life-threatening ischemic vascular events such as angina, myocardial infarction, transient ischemic attack, and stroke. Atherothrombosis is triggered when an unstable atherosclerotic lesion is ruptured, leading to platelet activation and thrombus formation. Inflammatory mediators are responsible for lesion instability leading to rupture, and in recent years atherothrombosis and its underlying condition of atherosclerosis have come to be recognized as manifestations of inflammatory disease. Inflammatory mediators may therefore serve as early markers of atherothrombosis. Measurement of early markers may be used to predict future ischemic events and improve risk stratification in patients following diagnosis of atherothrombotic disease. In addition, detection of such markers may help to optimize the use of current therapies to manage atherothrombosis. Molecules that may serve as early markers of atherothrombotic disease include C-reactive protein, CD40 ligand, myeloperoxidase, pregnancy-associated plasma protein and plasminogen activator inhibitor-1. Early indications are that levels of these markers are influenced by therapies currently in use in the treatment of atherothrombotic conditions, including antiplatelet agents. Ongoing studies will provide further insight into routine assessment of inflammatory markers as a guide to the management of patients with atherothrombosis.

SUMOylation and SENP3 regulate STAT3 activation in head and neck cancer.

Hyperphosphorylation of signal transducer and activator of transcription 3 (STAT3) has been found in various types of human cancers, including head and neck cancer (HNC). Although smoking is critical in the development and progression of HNC, how tobacco components activate STAT3 is unclear. We demonstrated that exposure of HNC cell lines to a tobacco extract induced a rapid Y705 phosphorylation of STAT3 and a rapid increase in the SUMO protease SENP3 that depended on a simultaneous increase in reactive oxygen species. We identified that SUMOylation at the lysine 451 site facilitated STAT3 binding to the phosphatase TC45 through an SUMO-interacting motif of TC45. SENP3 could thus enhance STAT3 phosphorylation by de-conjugating the SUMO2/3 modification of STAT3. Knocking-down of SENP3 greatly impaired basal and induced STAT3 phosphorylation by tobacco extract or interleukin 6. A correlation between SENP3 protein levels and STAT3 Y705 phosphorylation levels in human laryngeal carcinoma specimens was found, which was more significant in the specimens derived from the smoker patients and with poor clinicopathological parameters. Our data identified SUMOylation as a previously undescribed post-translational modification of STAT3 and SENP3 as a critical positive modulator of tobacco- or cytokine-induced STAT3 activation. These findings provide novel insights into the hyperphosphorylation of STAT3 in development of HNC.

Inhibition of mitochondrial respiratory chain complex I by TNF results in cytochrome c release, membrane permeability transition, and apoptosis.

Mitochondria have been shown to play a key role in apoptosis induction. However, the sequence of changes that occur in the mitochondria in the initial step of apoptosis has not been clearly elucidated. Here, we showed that mitochondrial respiratory chain (MRC) complex I was inhibited during the early phase of TNF- or serum withdrawal apoptosis. The importance of complex I inhibition in apoptosis is also supported by the observation that rotenone, an inhibitor of complex I but not that of other complexes, could induce apoptosis in a manner comparable to TNF. We hypothesized that inhibition of complex I could affect electron flow through other complexes leading to cytochrome c release by an antioxidant-sensitive pathway and caspase 3 activation followed by the induction of membrane permeability transition (MPT). This hypothesis is supported by the following observations: (1) TNF and rotenone induced MPT and cytochrome c release; (2) TNF-induced complex I inhibition was observed prior to cytochrome c release and MPT induction; (3) MPT induction was inhibited by a caspase 3 inhibitor, z-DEVD-CH2F, and an antioxidant pyrrolidine dithiocarbamate (PDTC), whereas cytochrome c release was only inhibited by PDTC. Thus, these results suggest that MRC complex I plays a key role in apoptosis signalings.

Regulation of reactive oxygen species-induced apoptosis and necrosis by caspase 3-like proteases.

Reactive oxygen species (ROS) and caspases have been implicated as potential mediators of cell death. However, their mechanistic relationship remains to be elucidated. Here we investigated the roles of caspases in apoptosis and necrosis induced by ROS, generated by the mixture of xanthine and xanthine oxidase (X/XO). A low concentration of XO (0.025 U/ml) induced DNA fragmentation with little cellular membrane damage 3 h after treatment, suggesting the induction of apoptosis. The same treatment induced membrane blebbing, a morphological change typical of apoptosis, 15 min after treatment. A high concentration of XO (0.1 U/ml) damaged cell membranes with little concomitance of DNA fragmention, suggesting the induction of necrosis. ROS also activated caspase 3-like proteases and caspase 3 itself together with the release of cytochrome c which might be the cause of caspase activation. Apoptosis induced by low concentrations of XO and necrosis induced by high concentrations of XO was inhibited by z-DEVD-CH2F, an irreversible inhibitor of caspase 3. However, rapid induction of membrane blebbing was not inhibited by z-DEVD-CH2F. These results suggest that both apoptosis and necrosis could be induced by ROS through the activation of caspase 3-like protease; however, caspase 3 activation is not needed for ROS-induced membrane blebbing.

GNB1L, a gene deleted in the critical region for DiGeorge syndrome on 22q11, encodes a G-protein beta-subunit-like polypeptide.

CATCH 22 syndromes, which include DiGeorge syndrome and Velocardiofacial syndrome, are the most common cause of congenital heart disease which involve microdeletion of 22q11. Using a strategy including EST searching, PCR amplification and 5'-RACE, we have cloned a 1487 bp cDNA fragment from human heart cDNA library. The cloned GNB1L cDNA encodes a G-protein beta-subunit-like polypeptide, and the GNB1L gene is located in the critical region for DiGeorge syndrome. A comparison of GNB1L cDNA sequence with corresponding genomic DNA sequence revealed that this gene consists of seven exons and spans an approximately 60 kb genomic region. Northern blot analysis revealed GNB1L is highly expressed in the heart.

Identification of novel isoforms of human RAD52.

In yeast, RAD52 has been shown to be essential for homologous recombination of DNA and to be involved in the repair of double-stranded DNA breaks. Recently, the human homologue of yeast RAD52, a 418-amino-acid protein, has been identified. In this study, we report three different isoforms of human RAD52 isolated from brain and testis cDNA libraries. cDNAs of these isoforms contain distinct insertions and encode truncated proteins due to translational frame-shifts. The three isoforms consist of 226-, 139-, and 118-amino-acid residues, and are designated as RAD52beta, gamma, and delta, respectively. The original RAD52 is termed as RAD52alpha in this paper. Messages of these isoforms have been detected in various human tissues. We found that the RAD52 isoforms were unable to interact with RAD52alpha because of partial defect of the self-interaction domain. Furthermore, like RAD52alpha, the isoforms have been shown to bind to both single-stranded and double-stranded DNA. These results suggest that RAD52beta, gamma, and delta might affect RAD52alpha function through their DNA-binding property and their inability to bind to RAD52alpha. Thus, these isoforms might act as dominant negative mutants or negative regulators of RAD52alpha.

Direct proinflammatory effect of C-reactive protein on human endothelial cells.

BACKGROUND: The acute-phase reactant C-reactive protein (CRP) is an important risk factor for coronary heart disease. However, the possible effects of CRP on vascular cells are not known. METHODS AND RESULTS: We tested the effects of CRP on expression of adhesion molecules in both human umbilical vein and coronary artery endothelial cells. Expression of vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and E-selectin was assessed by flow cytometry. Incubation with recombinant human CRP (10 microg/mL) for 24 hours induced an approximately 10-fold increase in expression of ICAM-1 and a significant expression of VCAM-1, whereas a 6-hour incubation induced significant E-selectin expression. Adhesion molecule induction was similar to that observed in endothelial cells activated with interleukin-1beta. In coronary artery endothelial cells, induction of ICAM-1 and VCAM-1 was already present at 5 microg/mL and reached a maximum at 50 microg/mL, at which point a substantial increase in expression of E-selectin was also evident. The CRP effect was dependent on presence of human serum in the culture medium, because no effect was seen in cells cultured with serum-free medium. In contrast, interleukin-1beta was able to induce adhesion molecule expression in the absence of human serum. CONCLUSIONS: CRP induces adhesion molecule expression in human endothelial cells in the presence of serum. These findings support the hypothesis that CRP may play a direct role in promoting the inflammatory component of atherosclerosis and present a potential target for the treatment of atherosclerosis.

Modulation of vascular inflammation in vitro and in vivo by peroxisome proliferator-activated receptor-gamma activators.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is expressed in atherosclerotic plaques and in endothelial cells. The possible effects of PPARgamma activators on endothelial activation and inflammatory response within the plaque are currently unknown. METHODS AND RESULTS: We tested the hypothesis that PPARgamma activators inhibit vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-1) expression in cultured endothelial cells (evaluated by flow cytometry) and homing of monocyte/macrophages to atherosclerotic plaques in vivo. In endothelial cells, the PPARgamma agonists troglitazone at 100 micromol/L and 15-deoxy-(Delta12,14)-prostaglandin J(2) (15d-PGJ2) at 20 micromol/L markedly attenuated the tumor necrosis factor-induced expression of VCAM-1 and ICAM-1. A significant inhibition of VCAM-1 expression was also evident at 5 and 10 micromol/L 15d-PGJ2 and 20 micromol/L troglitazone. Expression of E-selectin and PECAM-1 was not altered. To confirm the biological relevance of these results, we assessed the effects of troglitazone on monocyte/macrophage homing to atherosclerotic plaques in apoE-deficient mice. A 7-day treatment with troglitazone (400 mg/kg) significantly reduced monocyte/macrophage homing to atherosclerotic plaques (236+/-77 versus 177+/-43 macrophages, P=0.03); an even more striking inhibition was found at 3200 mg/kg troglitazone (344+/-76 versus 172+/-83 macrophages, P=0.005). CONCLUSIONS: PPARgamma activators inhibit expression of VCAM-1 and ICAM-1 in activated endothelial cells and significantly reduce monocyte/macrophage homing to atherosclerotic plaques. These findings suggest that PPARgamma activators, currently used in treatment of type II diabetes, may have beneficial effects in modulating inflammatory response in atherosclerosis.

Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs.

BACKGROUND: C-reactive protein (CRP) induces adhesion molecule expression by endothelial cells. However, the effects of CRP on chemokine expression by endothelial cells are not known. METHODS AND RESULTS: We tested the effects of CRP on the production of the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES in cultured human umbilical vein endothelial cells. The secretion of chemokines was assessed by ELISA. Incubation with 100 microgram/mL recombinant human CRP induced a 7-fold increase in MCP-1 but no change in RANTES secretion. We showed that the effect of CRP on MCP-1 was present even at 5 microgram/mL CRP, with stepwise increases as the CRP concentration was increased to 10, 50, and 100 microgram/mL. The effect of CRP on MCP-1 induction was not influenced by aspirin (at concentrations up to 1 mmol/L), but it was significantly inhibited by 5 micromol/L simvastatin. The peroxisome proliferator-activated receptor-alpha activators fenofibrate (100 micromol/L) and Wy-14649 (100 micromol/L) almost completely abolished the induction of MCP-1, but the peroxisome proliferator-activated receptor-gamma activator ciglitazone had only a moderate effect. CONCLUSIONS: These results further strengthen the role of CRP in the pathogenesis of vascular inflammation and, likely, atherosclerosis and provide a crucial insight into a novel mechanism of action of anti-atherosclerosis drugs such as simvastatin and fenofibrate.

Ubiquitin-like proteins: new wines in new bottles.

Ubiquitin is a small polypeptide that covalently modifies other cellular proteins and targets them to the proteasome for degradation. In recent years, ubiquitin-dependent proteolysis has been demonstrated to play a critical role in the regulation of many cellular processes, such as cell cycle progression, cell signaling, and immune recognition. The recent discovery of three new ubiquitin-like proteins, NEDD8, Sentrin/SUMO, and Apg12, has further broadened the horizon of this type of post-translational protein modification. This review will focus on the biology and biochemistry of the Sentrin/SUMO and NEDD8 modification pathways, which are clearly distinct from the ubiquitination pathway and have unique biological functions.

Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin-activating enzyme complex.

Sentrin-1/SUMO-1 is a novel ubiquitin-like protein, which can covalently modify a limited number of cellular proteins. Here we report the identification of the sentrin-activating enzyme complex, which consists of two proteins AOS1 and UBA2. Human AOS1 is homologous to the N-terminal half of E1, whereas human UBA2 is homologous to the C-terminal half of E1. The human UBA2 gene is located on chromosome 19q12. Human UBA2 could form a beta-mercaptoethanol-sensitive conjugate with members of the sentrin family, but not with ubiquitin of NEDD8, in the presence of AOS1. Identification of human UBA2 and AOS1 should allow a more detailed analysis of the enzymology of the activation of ubiquitin-like proteins.

No evidence of perinatal transmission of HTLV-II.

Two hundred twenty-eight plasma specimens collected over a 4-year period from 185 children aged 4 days to 10 years at risk for human immunodeficiency virus (HIV) infection were tested for antibody to human T-cell lymphotropic virus type II (HTLV-II) to determine perinatal transmission of this agent. One hundred three of the specimens were from 68 children whose mothers were intravenous drug users (IVDU). None of the children in the study were breast fed. No HTLV antibody was detected in any of the samples from children of non-IVDU mothers. However, HTLV-II antibody was found in samples from 15 of 38 infants < 6 months old born to IVDU mothers. Later specimens taken at 8-16 months from six of these children were negative, suggesting passive maternal antibody, not infant infection. The 62 samples from 30 children 6 months to 7 years old from IVDU mothers were negative for HTLV antibody. The lack of HTLV antibody in the older children indicates that no vertical transmission had occurred. Based on the prevalence rate of maternal HTLV-II antibody found in the children < 6 months old, an HTLV-II infection rate of approximately 36% was projected for the IVDU mothers in the study.

Modulation of Substance P Release in Primary Sensory Neurons by Misoprostol and Prostaglandins.

Pain is one of the cardinal signs of inflammation. A number of inflammatory mediators have been shown in animal models to induce or augment pain. Of particular interest are the prostaglandins (PGs), which are arachidonic acid metabolites and can be pharmacologically regulated by cyclooxygenase inhibitors, the nonsteroidal anti-inflammatory drugs (NSAIDs). Indeed, NSAIDs are potent modulators of pain sensitivity. They are thought to mediate their hypoalgesic action through inhibition of prostaglandin production. However, indiscriminate inhibition of prostaglandin synthesis also creates a significant number of clinical side effects, among them gastrointestinal toxicity. With the introduction of misoprostol, a PGE(1) analog, a large number of investigative possibilities are opened. We have proposed to study the effect of misoprostol in an in vitro pain model. Our model, created by culturing primary sensory neurons isolated from dorsal root ganglions, was then differentiated by nerve growth factor and subjected to electrical stimulation. Substance P release following electrical stimulation was quantitated by radioimmunoassay. We found that misoprostol augmented substance P release in a dose-related manner. With 100 ng/ml of misoprostol added, there was a 45% increase in substance P release as compared to control. PGE(1) and PGE(2) addition at similar concentration caused a similar degree of increase in substance P release. Thus, acute addition of misoprostol to cultured sensory neurons appears to sensitize them to release more substance P. Our result does not necessarily imply that misoprostol will cause pain clinically. In our study, misoprostol at 10 ng/ml has no effect on substance P release. Because the plasma concentration of misoprostol is in the picogram per milliliter range, misoprostol most likely does not have a pain potentiation effect at the recommended therapeutic dose. Our data indicated that misoprostol at therapeutic dose has an insignificant effect on substance P release from primary sensory neuron.

Dietary marine lipids suppress continuous expression of interleukin-1 beta gene transcription.

n-3 Polyunsaturated fatty acids abundant in marine lipids suppress certain inflammatory and immune reactions, and dietary marine lipid supplements have antiinflammatory effects in experimental and human autoimmune disease. Previous work by other investigators demonstrated that dietary marine lipid supplements suppressed production of cytokines from stimulated human peripheral blood mononuclear cells ex vivo. The present study further documents the ability of n-3 fatty acids to inhibit cytokine formation, and in part defines the mechanism of the inhibition of production of interleukin-1 beta (IL-1 beta) by dietary n-3 fatty acid. Female BALB/c mice were each fed a fat-free balanced diet to which was added either a refined fish oil (FO) preparation as a source of n-3 fatty acid, or beef tallow (BT), which consisted primarily of saturated and monoenoic fatty acids. After ingesting the experimental diets for periods ranging from 3 to 12 wk. spleen cell preparations were stimulated ex vivo with either lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and proIL-1 beta mRNA (IL-1 beta mRNA) was measured by northern analysis. Levels of IL-1 beta mRNA in both LPS- and PMA-stimulated cells from BT-fed mice were elevated to a greater extent than in cells from FO-fed mice, at most concentrations of LPS and PMA. Stability of LPS-stimulated mRNA levels after actinomycin D was similar for BT and FO groups, indicating that lower levels of IL-1 mRNA with FO groups was related to suppressed IL-1 gene transcription and not due to accelerated transcript degradation. Nuclear run-on transcription assays revealed a more transient expression of the IL-1 beta gene in LPS-stimulated spleen cells from FO-fed mice compared to cells from BT-fed mice. We conclude that dietary marine lipids reduce transient expression of the IL-1 beta gene in stimulated splenic monocytic cells. Preliminary results from nuclear run-on transcription assays indicate that n-3 fatty acids may not change the initial rate of gene transcription but may promote more rapid shutting down of transcription of this gene after induction than do alternative lipids.