IL-1β Promoter Research Articles

Association of Interleukin 1β and Receptor Antagonist Gene Polymorphisms with Primary Open-Angle Glaucoma

Purpose: Genetic factors are known to play a role in the etiology of glaucoma. More specifically, the role of the immune system is highly suspected. We evaluated the association between 2 polymorphisms in the interleukin (IL) 1β gene (IL-1 promoter 511, IL-1 exon 5) and 1 polymorphism in the IL-1 gene receptor antagonist (IL-1Ra) intron 2 with primary open-angle glaucoma (POAG). Patients and Methods: Fifty-eight POAG patients and 105 healthy volunteers were enrolled in this study. Analysis based on polymerase chain reaction was used to resolve the 2 IL-1β polymorphisms and the IL-1Ra intron 2 polymorphism. Results: There were significant differences in the distribution of the IL-1β exon 5 polymorphism between the POAG patients and the control subjects (p < 0.05). The E2 allele of IL-1β exon 5 was more frequently found in POAG patients than in healthy patients (odds ratio: 4.224, 95% confidence interval = 1.135–15.717). The distributions of the other 2 polymorphisms, IL-1β promoter 511 and IL-1Ra intron 2, were not significantly different between the POAG patients and the healthy control group. Conclusions: The frequency of the E2 allele of the IL-1β exon 5 polymorphism was high in POAG patients. Therefore the E2 allele can be used as a marker to predict or search for the genetic causes of glaucoma in Chinese POAG patients. Furthermore, we have concluded that the other 2 polymorphisms (IL-1 promoter 511 and IL-1Ra) are not helpful in predicting Chinese POAG.

Investigation of IL-1B (-511, +3954) and IL-1RN Gene Polymorphisms in Korean Psoriasis Patients

Background: Psoriasis is an inflammatory skin disorder that is characterized by a marked proliferation of keratinocytes, vascular dilation and leukocyte infiltration. Cytokines play important roles in the pathogenesis of inflammatory disorders. An overexpression of proinflammatory cytokines was characterized in psoriasis plaque. Among these cytokines, IL-1β is major pro-inflammatory cytokine synthesized during the infection and inflammatory process. The IL-1 receptor antagonist (IL-1Ra) competes for the same IL-1 receptor for IL-1α and -1β, which prevents activation of the target cells. Three single nucleotide polymorphisms (SNPs) in the IL-1β gene have been reported at position -31, -511 and +3954. Within the IL-1Ra gene (IL-1RN), there is a variable number of tandem repeats (VNTR) of an 86 bp length in intron 2. These polymorphisms related to cytokine production and associated with various diseases. Methods: We investigated the polymorphisms of IL-1B (promoter -511 and +3954) and IL-1RN on 114 psoriasis patients and 311 healthy normal controls in Korean. We performed PCR-RFLP on single nucleotide polymorphisms (SNPs) of IL-1B (promoter -511 and +3954) and fragment analysis on IL-1RN 86 bp VNTR polymorphism. Results: The frequency of IL-1B -511*1 allele (patients vs. controls; 50.0% vs. 42.3%, RR=1.4) was significantly increased and IL-1B -511*2 allele (patients vs. controls; 50.0% vs. 57.7%, RR=0.7) decreased in psoriasis patients compared to normal controls. We also analyzed the IL-1B -511 polymorphism according to patients' characters (age of onset, sex and family history). The IL-1B -511 alleles were significantly associated in patients with male and family history than health normal controls. There were no significant associations of IL-1B +3954 and IL-1RN polymorphisms with psoriasis patients. Conclusion: These results suggest that the polymorphism of IL-1B -511 could be genetic susceptibility to psoriasis in Koreans. (Immune Network 2003;3(3):242-247)

Interleukin-1β and interleukin-1 receptor antagonist gene polymorphism in postmenopausal women: correlation to bone mineral density and susceptibility to osteoporosis

Objective: Osteoporosis is a common disorder with a strong genetic component. Our aim was to investigate the correlations of the interleukin-1β (IL-1β) and interleukin-1 receptor antagonist (IL-1Ra) gene polymorphisms with bone mineral density (BMD) and their relationship to osteoporosis. Methods: The IL-1β (promoter and exon 5) and IL-1Ra (intron 2) gene polymorphisms were determined using polymerase chain reaction. BMD of the lumbar spine and proximal femur were measured using dual-energy X-ray absorptiometry. Results: The prevalence of each genotype of the interleukin-1 related genes in the study population was: (1) 14% C/C, 71.5% C/T, and 14.5% T/T in IL-1β promoter; (2) 95.3% E1/E1 and 4.7% E1/E2 in IL-1β exon 5; (3) 92.4% I/I, 6.4% I/II, and 1.2% II/II in IL-1Ra intron 2. After adjustment for potential confounding factors such as age, height, weight, years since menopause, and daily calcium intake, subjects with genotype E1/E2 ( n=8) in IL-1β exon 5 had lower BMD values and a significantly greater risk for osteoporosis (OR 10.6, 95% CI 1.3–83.8) at the lumbar spine when compared with subjects with genotype E1/E1 ( n=164) in IL-1β exon 5. Conclusion: The Taq I IL-1β exon 5 gene polymorphism is associated with reduced BMD and predisposes women to osteoporosis at the lumbar spine, but our results should be interpreted with caution because of the small number of subjects with the unfavorable E1/E2 genotype.

A promoter polymorphism in the gene encoding interleukin-12 p40 (IL12B) is associated with mortality from cerebral malaria and with reduced nitric oxide production.

Interleukin-12 (IL-12) is an important regulatory cytokine in infection and immunity. Administration of IL-12 may reduce complications of severe malaria in rodents. Polymorphisms in IL12B, the gene encoding the IL-12 p40 subunit, influence the secretion of IL-12 and susceptibility to Type 1 diabetes. We therefore investigated whether IL12B polymorphisms may affect the outcome of severe malaria. Homozygosity for a polymorphism in the IL12B promoter was associated with increased mortality in Tanzanian children having cerebral malaria but not in Kenyan children with severe malaria. Furthermore, homozygotes for the IL12B promotor polymorphism had decreased production of nitric oxide, which is in part regulated by IL-12 activity. These studies suggest that IL12B polymorphisms, via regulation of IL-12 production, may influence the outcome of malaria infection in at least one African population.

Polymorphisms of the Interleukin-1 Gene Cluster and Ovarian Cancer

Interleukin-1 (IL-1) is known to be critically involved in ovarian carcinogenesis. We investigated a panel of polymorphisms of the IL-1 and the IL-1 receptor antagonist (IL-1 RA) genes in patients with ovarian cancer. One hundred thirty-four patients with surgically staged ovarian cancer, 27 patients with borderline ovarian cancer, and 134 healthy controls were genotyped for three polymorphisms of the IL-1 gene (-889 C/T polymorphism of the IL-1alpha gene [IL1A], -511 C/T polymorphism of the IL-1beta promoter [IL1B promoter], a polymorphism of IL-1beta exon 5 [IL1B exon 5]), and an 86-base pair repeat in intron 2 of the IL-1 RA gene [IL1RN]) using polymerase chain reaction and pyrosequencing. Allelic frequencies did not differ between patients with ovarian cancer and controls. In the ovarian cancer group, polymorphism did not correlate with any of the investigated clinicopathologic variables, including tumor stage, lymph node, and histologic grade. In univariate and multivariate models, there was no correlation between any polymorphism and patients' overall and disease-free survival. We investigated interleukin polymorphisms in ovarian cancer but did not find any association between common polymorphisms of IL1A, IL1B, and IL1RN and the occurrence of ovarian cancer. Allelic variation within the IL-1 gene clusters does not seem to play a role in ovarian carcinogenesis and does not appear to be a useful tool for possible screening and risk evaluation.

Association of IL12B promoter polymorphism with severity of atopic and non-atopic asthma in children.

Severe asthma is a frequent cause of hospital admission, especially among children. The main environmental triggers of airway inflammation in asthma are viruses and aeroallergens. These agents elicit reciprocal immune responses, characterised by production of T helper 1 and T helper 2 cytokines, respectively. There is no genetic explanation for how hyper-responsiveness to these disparate environmental stimuli develops among individuals with asthma. Our aim was to assess relation between an IL12B promoter polymorphism and asthma. We did a cohort study in which we initially genotyped 411 6-year olds for the IL12B promoter polymorphism. We then assessed the relation between this polymorphism and asthma severity. A further 85 asthmatic children in an additional sample of 433 children from the same cohort were then assessed to confirm these findings. We also examined in-vitro interleukin-12 responses in a subgroup of individuals. Heterozygosity for the IL12B promoter polymorphism was observed in 76% (16) of atopic and non-atopic individuals with severe asthma in the initial sample. By comparison, heterozygotes comprised only 31% (17) of the moderate asthma group, and 48% (20) of individuals with mild asthma were heterozygous, as were unaffected controls. These findings were confirmed in the second sample (overall p<0.0001). Our data suggest that IL12B promoter heterozygosity contributes to asthma severity rather than susceptibility per se. The severity-predisposing genotype was associated with reduced interleukin 12 p40 gene transcription and decreased interleukin 12 p70 secretion. Interleukin 12 plays a key part in antagonism of T helper 2 differentiation, and in induction of antiviral host defense. Genetically determined attenuation of interleukin-12 response capacity would, therefore, provide a plausible common immunological pathway to disease severity for the two major forms of asthma.

Autocrine induction of the human pro-IL-1beta gene promoter by IL-1beta in monocytes.

IL-1beta is produced primarily by activated monocytes/macrophages. We report in this study that IL-1beta induces the human pro-IL-1beta (IL1B) gene promoter in human THP-1 monocytic cells. The -131 to +12 minimal IL1B promoter was induced by IL-1beta in a dose-dependent manner. The promoter possesses two important transcription factor binding motifs, one for an ETS family transcription factor Spi-1 (PU.1), and the other a binding site for NF-IL6 (CCAAT/enhancer binding protein beta). Autocrine promoter activity was completely inhibited by mutation of the Spi-1 site. Mutation of the NF-IL6 binding motif caused partial loss of activity. EMSAs using THP-1 cell nuclear extracts indicated that IL-1beta significantly induced Spi-1 binding to its target site within the IL1B promoter that was maximal at 1 h after stimulation, correlating with the kinetics of IL-1beta induction. The importance of Spi-1 was supported by our observation that Spi-1-deficient EL4 thymocytes exhibited IL-1beta-induced activity only after transfection with a Spi-1 expression vector. Moreover, TNFR-associated factor 6 also required Spi-1 to activate the promoter. Transfection studies using Spi-1 mutant constructs showed that the TATA-binding protein binding and glutamine-rich domains of Spi-1 were important for IL-1beta induction, whereas LPS induction required the proline, glutamic acid, serine, and threonine-rich domain containing serine 148 as well as the TATA-binding protein and glutamine-rich domains. We conclude that the IL1B promoter is an IL-1beta-responsive sequence as a result of its ability to bind Spi-1 in response to IL-1beta.

Lack of association of interleukin-1beta gene polymorphisms in Chinese patients with systemic lupus erythematosus.

The purpose of this study was to examine whether interleukin-1beta (IL-1beta) (promoter and exon 5) gene polymorphisms were markers of susceptibility or clinical manifestation in Chinese patients with systemic lupus erythematosus (SLE). The study included 52 Chinese patients with SLE. In addition, 103 unrelated healthy individuals living in central Taiwan served as control subjects. From genomic DNA, polymorphisms of the gene for IL-1beta promoter and exon 5 were typed. Allelic frequencies were compared between SLE patients and control subjects. The relationship between allelic frequencies and clinical manifestations of SLE was evaluated. No significant differences were observed in the allelic frequencies of the IL-1beta promoter and IL-1beta exon 5 between patients with SLE and healthy control subjects. Additionally, we did not detect any association of IL-1beta promoter and the exon 5 genotype with the clinical and laboratory profiles in SLE patients. The results from the present study suggest that the polymorphisms of the IL-1beta promoter and IL-1beta exon 5 are not related to SLE patients in Taiwan.

Inflamed glomeruli-specific gene activation that uses recombinant adenovirus with the Cre/loxP system.

The authors previously reported that bone marrow-derived CD11b(+)CD18(+) cells could be used as a vehicle to deliver foreign genes into inflamed glomeruli and that this vehicle cell (v-cell) could retard the progression of nephritis by delivering anti-inflammatory molecules. As a next step, the authors tried to establish a switching system by which v-cells are activated only at the inflamed glomeruli. A recombinant adenovirus (Ad) that expressed Cre recombinase under the control of the interleukin-1 beta (IL-1 beta) promoter (AxIL-1pr/Cre) was constructed and transfected into v-cells. After confirming that AxIL-1pr/Cre expresses Cre by lipopolysaccharide (LPS) treatment, AxIL-1pr/Cre was infected together with another Ad bearing a switching reporter unit in which the LacZ gene is activated under the control of the CAG promoter by the Cremediated excisional deletion of interposed stuffer DNA. Only a negligible number of double-infected (Cre/loxPCAG) cells expressed LacZ. This number, however, was significantly increased by LPS, which suggests that LPS-induced Cre effectively deletes the stuffer DNA, which allows for a complete CAG promoter. DBA/2j mice were then transplanted with Cre/loxPCAG cells via a tail vein and treated with anti-glomerular basement membrane (GBM) serum. To trace the transplanted cells, marker v-cells, infected with AxCANLacZ to constitutively express the LacZ gene, were also used. Although transplanted cells expressing LacZ collected in the spleen independent of anti-GBM treatment, they did not express the LacZ gene in the mice transplanted with Cre/loxPCAG cells. On the other hand, transplanted cells were recruited in the glomeruli and expressed the LacZ gene upon anti-GBM treatment. These results suggested that only the v-cells recruited in the glomeruli could be switched on and activate foreign genes.

NEGATIVE REGULATION OF IL-1β PRODUCTION AT THE LEVEL OF TRANSCRIPTION IN MACROPHAGES STIMULATED WITH LPS

The IL-1β gene is rapidly and transiently expressed in LPS-stimulated macrophages. While several studies have addressed the molecular basis of LPS-induced transcriptional activity, the mechanisms which underlie the subsequent decrease in IL-1β gene expression have not been as extensively examined. In this regard, we found that the characteristic decrease in IL-1β production after LPS stimulation could be abrogated by treatment of macrophages with the protein kinase inhibitor staurosporine. This inhibitor mediated an enhancement of IL-1β production which was first evident 8–12h after LPS stimulation and continued at peak levels for the rest of the incubation period (24h). IL-1β production was correlated with the level of mRNA specific for the cytokine. Staurosporine also mediated an enhancement of LPS-induced IL-1β promoter activity measured in RAW 264.7 cells transiently transfected with an IL-1β reporter plasmid. This increase paralleled the enhancement of IL-1β mRNA by staurosporine both in intensity and time after LPS stimulation, suggesting that the negative regulation of IL-1β is exerted primarily at the level of transcription. This regulation may be at least partially due to an observed inhibition of nitric oxide production by staurosporine in LPS-activated macrophages, which was correlated with enhanced IL-1β production. However, the intensity of the observed effects suggested that additional staurosporine-sensitive regulatory mechanisms are in operation at the level of promoter activity.

Influence of interleukin-1β gene polymorphism on age-at-onset of spinocerebellar ataxia 6 (SCA6) in Japanese patients

An inverse correlation is observed between the expanded CAG repeat number and age-at-onset of spinocerebellar ataxia 6 (SCA6). To detect another modifying genetic factor for SCA6, we studied polymorphisms in the genes for interleukin (IL)-1β and tumor necrosis factor in 122 Japanese patients with SCA6. No contribution of these polymorphisms to the variance in disease onset was observed by regression analysis or by ANOVA. The IL-1β promoter polymorphism, however, significantly affected the age-at-onset, when adjusted for the CAG repeat number as a covariate ( P=0.0004, by ANCOVA), suggesting that IL-1β may be a genetic factor other than the SCA6 gene that modifies the age-at-onset of the disease.

PU.1 and multiple IFN regulatory factor proteins synergize to mediate transcriptional activation of the human IL-1 beta gene.

Both lymphoid and myeloid cells express two related members of the IFN regulatory factor (IRF) family of transcription factors, specifically IRF-4 and IFN consensus binding protein (ICSBP or IRF-8). We previously reported that macrophages express IRF-4 and in combination with the ETS-like protein PU.1 can synergistically activate a human IL-1beta reporter gene. Here we report that this synergy is mediated by a composite PU.1/IRF element located within an upstream enhancer known to confer cytokine- and LPS-inducible expression. In macrophages, synergistic activation of IL-1beta reporter gene expression was preferentially mediated by IRF-4, whereas IRF-4 and ICSBP were equally capable of synergizing with PU.1 when coexpressed in fibroblasts. Furthermore, coexpression of IRF-1 and IRF-2 dramatically increased the capacity of both PU.1/IRF-4 and PU.1/ICSBP to induce IL-1beta reporter gene expression in fibroblasts. The additional synergy observed with IRF-1 and IRF-2 coexpression is mediated by a region of DNA distinct from either the IL-1beta enhancer or promoter. We also assessed the capacity of these transcription factors to activate endogenous IL-1beta gene when overexpressed in human embryonic kidney 293 cells. Although ectopic expression of PU.1 alone was sufficient to activate modest levels of IL-1beta transcripts, endogenous IL-1beta expression was markedly increased following coexpression of additional IRF proteins. Thus, maximal expression of both a human IL-1beta reporter gene and the endogenous IL-1beta gene was observed in cells that coexpressed PU.1, IRF-4 (or ICSBP), IRF1, and IRF2. Together, our observations suggest that these factors may function together as an enhanceosome.

Interleukin-1ß and interleukin-1 receptor antagonist gene polymorphisms in rheumatoid arthritis

The purpose of this study was to evaluate if IL-1β (IL-1β promoter and IL-1β exon 5) and IL-1 receptor antagonist (IL-1Ra) gene polymorphisms act as markers of susceptibility to or severity of RA. The study included 104 RA patients and 103 normal controls. No significant difference was observed in the cytokine allelic frequencies of IL-1β promoter and IL-1β exon 5 between patients with RA and healthy controls. In addition, there was no significant association in the cytokine carriage rates of I and II allele of IL-1Ra between RA patients and healthy controls. In contrast, the IV allele of IL-1Ra was significantly increased in RA patients with low inflammatory activity (P = 0.03). This study indicated that allelic frequency and carriage rate of IL-1β (IL-1β promoter and IL-1β exon 5) and IL-1Ra (I and II allele) do not differ significantly between normal controls and RA patients in Taiwan. However, the carriage rate of IV allele of IL-1Ra was high in the RA patients with low inflammatory activity.

TRANSCRIPTIONAL REGULATION OF THE HUMAN INTERLEUKIN 1β GENE BY FIBRONECTIN: ROLE OF PROTEIN KINASE C AND ACTIVATOR PROTEIN 1 (AP-1)

Interleukin 1β (IL-1β) is a multifunctional polypeptide considered a key cytokine during inflammation. Fibronectin (FN), a matrix glycoprotein highly expressed in injured tissues, can induce expression of IL-1β in human blood monocytic cells. Herein, we explore the intracellular signals and transcriptional mechanisms responsible for IL-1β induction by FN using human promonocytic U937 cells transfected with the human IL-1β promoter connected to a reporter gene. Exposure of transfected U937s to FN resulted in increased expression of the full-length IL-1β promoter. This effect, mediated via the α5β1 integrin, was associated with activation of mitogen-activated protein kinases (MAPKs) and was abolished by pre-treatment of cells with Calphostin C, a specific inhibitor of protein kinase C (PKC) activation. Deletion analysis and co-transfection studies using consensus activator protein 1 (AP-1) oligonucleotides suggested that an AP-1 site present in the 5′ end of the IL-1β promoter was involved in the FN-induced response. Finally, electrophoretic mobility shift assays showed that FN induced binding of AP-1, but not NF-κB. Together, these experiments demonstrate that FN binding to the α5β1 integrin activates MAPK-dependent signal pathways, and results in the transcription of the IL-1β promoter in U937 cells by activating PKC and inducing AP-1.

T Helper 1 (Th1) Immunity to Trophoblast in Women with Recurrent Pregnancy Loss (RPL) Is Associated with a Variant in the IL1B Promoter Region

Objectives: Biased Th1 immunity to trophoblast and defective Th2 cytokine production by decidual T cells have been associated with RPL. The underlying genetic mechanisms remain to be elucidated. Our preliminary studies did not show association of polymorphisms of the IFNG gene and the TNFA promoter region with Th1 immunity to trophoblast. Because IL-1 may influence either Th1 or Th2 immune response, in the present study, we investigated whether polymorphisms of the IL1B and IL1RN genes influence Th1 immunity to trophoblast in women with RPL.

Immunodystrophism: evidence for a novel alloimmune hypothesis for recurrent pregnancy loss involving Th1-type immunity to trophoblast.

Pregnancy loss is the most common complication of pregnancy. Recurrent pregnancy loss occurs in approximately 1% of pregnant women. Many immunologic theories have been proposed but have not withstood rigorous analysis. A novel alloimmune hypothesis involving T helper (Th) 1-type immunity to trophoblast is the latest theory proposed for recurrent pregnancy loss. The basic hypothesis is, in the decidua there are myriad of antigen presenting cells and other immune response cells. In response to trophoblast invasion, these cells may become activated. A by-product of this activation is the secretion by these cells of either a predominant Th1 or Th2 cytokine profile. In cases where a Th1 cytokine profile predominates, chiefly, interferon-gamma, tumor necrosis factor, or interleukin-12, these cytokines may directly or indirectly be detrimental to early placental cell differentiation and growth and toxic to embryo development. Further evidence for this novel hypothesis comes from recent findings of a genetic predisposition for a vigorous Th1 cytokine response in these women due to a polymorphism in the IL1B promoter region. Further studies are needed to substantiate definitive causative links between Th1-immunity to trophoblast and recurrent pregnancy loss. Clinical trials are also needed to determine the best therapy for this disorder.

Endotoxin-mediated nitric oxide synthesis inhibits IL-1beta gene transcription in ANA-1 murine macrophages.

On the basis of previous work demonstrating nitric oxide (NO)-mediated inhibition of nuclear factor-kappaB (NF-kappaB) DNA binding, we hypothesized that NO downregulates NF-kappaB-dependent interleukin-1beta (IL-1beta) production in an ANA-1 macrophage model of lipopolysaccharide (LPS) stimulation. In the presence of LPS (100 ng/ml), levels of IL-1beta protein and mRNA were significantly upregulated with NO synthase inhibition. Using nuclear run-on analysis and transient transfection studies, IL-1beta gene transcription and IL-1beta promoter activity were also found to be increased with inhibition of NO production. Parallel transfection studies using an NF-kappaB long terminal repeat-reporter plasmid exhibited similar findings, suggesting an NO-mediated effect on NF-kappaB activity. Gel shift studies showed that LPS-associated NF-kappaB DNA binding was increased, both in the setting of NO synthase inhibition and in a reducing environment. Repletion of NO by addition of an S-nitrosothiol restored IL-1beta protein synthesis, mRNA levels, gene transcription, promoter activity, and NF-kappaB DNA binding to levels noted in the presence of LPS alone. Our studies indicate that NO may regulate LPS-associated inflammation by downregulating IL-1beta gene transcription through S-nitrosation of NF-kappaB.

Differential Expression and Distinct Functions of IFN Regulatory Factor 4 and IFN Consensus Sequence Binding Protein in Macrophages

IFN regulatory factor 4 (IRF4) and IFN consensus sequence binding protein (ICSBP) are highly homologous members of the growing family of IRF proteins. ICSBP expression is restricted to lymphoid and myeloid cells, whereas IRF4 expression has been reported to be lymphoid-restricted. We present evidence that primary murine and human macrophages express IRF4, thereby extending its range of expression to myeloid cells. Here, we provide a comparative analysis of IRF4 and ICSBP expression and function in distinct cell types. These IRF proteins can form specific complexes with the Ets-like protein PU.1, and can activate transcription via binding to PU.1/IRF composite sequences. EMSA analysis revealed that murine macrophages contained both IRF4/PU.1 and ICSBP/PU.1 complexes, analogous to B cells. Over-expression of ICSBP in these macrophages activated transcription of a PU.1/IRF-dependent promoter, whereas over-expression of IRF4 had no effect on this promoter. In contrast, over-expression of either IRF4 or ICSBP in both macrophages and NIH-3T3 fibroblasts suppressed transcription of the PU.1-independent H-2Ld MHC class I promoter. In NIH-3T3 fibroblasts, IRF4 and ICSBP also synergized with exogenous PU.1 to activate transcription of a PU.1/IRF-dependent promoter. Furthermore, both IRF4 and ICSBP activated transcription of the IL-1beta promoter in both cell types. While this promoter is PU.1-dependent, it lacks any known PU.1/IRF composite binding sites. Synergistic activation of the IL-1beta promoter by these IRF proteins and PU.1 was found to require PU.1 serine 148. Together, these data demonstrate that IRF4 and ICSBP are dichotomous regulators of transcription in macrophages.

NON-STEROIDAL ANTI-INFLAMMATORY DRUGS INHIBIT THE EXPRESSION OF CYTOKINES AND INDUCE HSP70 IN HUMAN MONOCYTES

Recent studies have shown that the non-steroidal anti-inflammatory drugs (NSAIDs) activate heat shock transcription factor (HSF1) from a latent cytoplasmic form to a nuclear, DNA binding state. As HSF1 can function as both an activator of heat shock genes and a repressor of non-heat shock genes such as IL1B and c- fos , we have examined the potential role of HSF1 in the effects of NSAIDs on gene expression in a human monocytic cell line THP-1. We found that two members of the NSAIDs, sodium salicylate and sulindac repress the IL1B promoter to similar degree to heat shock or HSF1 overexpression. In addition, sodium salicylate and additional NSAIDs used at concentrations that activate HSF1 also inhibited the expression of other monocytic genes (TNF-α, IL-1β, IL-6, IL-8, IL-10, ICAM-1) activated by exposure to a pro-inflammatory stimulus (lipopolysaccharide, LPS). At least in the case of the IL1B promoter, repression did not seem to involve another factor whose activity is affected by the NSAIDs, NFκB as the IL1B promoter fragment used in our studies is not NFκB responsive and binds specifically to HSF1. Exposure to NSAIDs had a complex effect on HSP gene expression and while sulindac activated the stress responsive HSP70B promoter, sodium salicylate did not. In addition, only a subset of the NSAIDs induced HSP70 mRNA species. These findings reflect the properties of HSF1 which can be activated to at least two DNA binding forms only one of which activates heat shock promoters and suggest that individual NSAID family members may differentially induce one or other of these forms. Overall therefore, exposure to NSAIDs leads to a profound switch in gene expression in monocytic cells, with suppression of genes involved in macrophage activation and induction of stress genes and HSF1 appears to play a regulatory role in these effects.

HTLV-I TAX TRANSACTIVATES THE PROMOTER OF HUMAN PROINTERLEUKIN 1β GENE

P014 HTLV-I, which infects a wide variety of mammalian cells, encodes a transactivating protein designated as Tax. We now report that Tax induces the human proIL-1 β (IL1B) gene promoter. In our transient transfection assays using Jurkat CD4+ T and THP-1 monocytic cells, a chloramphenicol acetyltransferase (CAT) construct containing the IL1B promoter sequence between positions −131 and +12 showed significant increases in activity following cotransfection of a Tax expression vector in both CD4+ T and monocytic cells. In addition, Tax synergistically transactivated the IL1B promoter with LPS in monocytes. Analyses of specific nucleotide substitutions further indicated that the Tax-induced transcriptional activation requires two transcription factor binding motifs within the IL1B promoter in monocytes; One is a binding site for NF-IL6 (CCAAT/enhancer binding protein β, C/EBP β), which belongs to basic region-leucine zipper (bZIP) family and the other for Spi-1 (PU.1), which is an Ets family protein found principally in monocytes, macrophages and B lymphocytes. On the other hand, in Jurkat T cells, the NF-IL6 binding site but not the Spi-1 site is required for Tax induction of the IL1B promoter. In gel shift assays (EMSA), Tax expression in cells increased binding of the two factors to their target IL1B promoter sequences. Additional EMSA using in vitro translated proteins also showed that recombinant Tax enhances DNA binding activities of both of recombinant NF-IL6 and Spi-1 proteins. These data were further supported by our glutathione S-transferase (GST)-pulldown data, which indicated that Tax physically interacts with the two proteins. Based upon the results obtained from the present study, we conclude that the IL1B promoter is a Tax-responsive sequence in human T cells and monocytes.