Activation Of Primary Macrophages Research Articles

Cathepsin Inhibition Modulates Metabolism and Polarization of Tumor-Associated Macrophages.

Simple SummaryStroma-infiltrating tumor-associated macrophages (TAM) play an important role in regulating tumor progression and chemoresistance. Many tumor-infiltrating macrophage populations can be identified by preferential expression of distinct marker genes associated with an M2 phenotype and may execute tumor-promoting functions by enhancing tissue remodeling, facilitating angiogenesis, and suppressing immune responses. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype. For this purpose, we investigated the molecular effects of cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH2, led to a polarization shift from M2- to M1 macrophages, associated with distinct alterations in lysosomal signaling and lipid metabolism. This could be therapeutically exploited in tumors with strong infiltration of M2-macrophages, thereby possibly reverting M2 polarization, overcoming drug resistance, and improving the prognosis of our patients.Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH2, led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE2, which correlated with the upregulation of numerous NFkB-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu.

Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice

AbstractHemophagocytic syndromes comprise a cluster of hyperinflammatory disorders, including hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Overwhelming macrophage activation has long been considered a final common pathway in the pathophysiology of hemophagocytic syndromes leading to the characteristic cytokine storm, laboratory abnormalities, and organ injuries that define the clinical spectrum of the disease. So far, it is unknown whether primary macrophage activation alone can induce the disease phenotype. In this study, we established a novel mouse model of a hemophagocytic syndrome by treating mice with an agonistic anti-CD40 antibody (Ab). The response in wild-type mice is characterized by a cytokine storm, associated with hyperferritinemia, high soluble CD25, erythrophagocytosis, secondary endothelial activation with multiple organ vaso-occlusion, necrotizing hepatitis, and variable cytopenias. The disease is dependent on a tumor necrosis factor-α–interferon-γ–driven amplification loop. After macrophage depletion with clodronate liposomes or in mice with a macrophage-selective deletion of the CD40 gene (CD40flox/flox/LysMCre), the disease was abolished. These data provide a new preclinical model of a hemophagocytic syndrome and reinforce the key pathophysiological role of macrophages.

Inhibition of Transglutaminase 2 as a Potential Host-Directed Therapy Against Mycobacterium tuberculosis.

Host-directed therapies (HDTs) are emerging as a potential valid support in the treatment of drug-resistant tuberculosis (TB). Following our recent report indicating that genetic and pharmacological inhibition of transglutaminase 2 (TG2) restricts Mycobacterium tuberculosis (Mtb) replication in macrophages, we aimed to investigate the potentials of the TG2 inhibitors cystamine and cysteamine as HDTs against TB. We showed that both cysteamine and cystamine restricted Mtb replication in infected macrophages when provided at equimolar concentrations and did not exert any antibacterial activity when administered directly on Mtb cultures. Interestingly, infection of differentiated THP-1 mRFP-GFP-LC3B cells followed by the determination of the autophagic intermediates pH distribution (AIPD) showed that cystamine inhibited the autophagic flux while restricting Mtb replication. Moreover, both cystamine and cysteamine had a similar antimicrobial activity in primary macrophages infected with a panel of Mtb clinical strains belonging to different phylogeographic lineages. Evaluation of cysteamine and cystamine activity in the human ex vivo model of granuloma-like structures (GLS) further confirmed the ability of these drugs to restrict Mtb replication and to reduce the size of GLS. The antimicrobial activity of the TG2 inhibitors synergized with a second-line anti-TB drug as amikacin in human monocyte-derived macrophages and in the GLS model. Overall, the results of this study support the potential usefulness of the TG2-inhibitors cysteamine and cystamine as HDTs against TB.

Lumican is upregulated in osteoarthritis and contributes to TLR4-induced pro-inflammatory activation of cartilage degradation and macrophage polarization

Lumican (LUM) is a major extracellular matrix glycoprotein in adult articular cartilage and its expression is known to be upregulated upon cartilage degeneration. LUM is associated with the pathogen-associated molecular pattern (PAMP) activation of the TLR4 signalling cascade, with TLR4 being highly associated with inflammation in rheumatic diseases. However, the main role of the LUM structural molecule in osteoarthritis (OA) remains elusive. The aim of this study was, therefore, to understand the role of LUM during TLR4-mediated activation in OA. After measuring LUM levels in synovial fluid (SF) of OA patients and lipopolysaccharide (LPS)-induced TLR4 activation, the role of LUM in the expression of pro-inflammatory molecules and cartilage degradation was assessed invitro and exvivo in a cartilage explant model. Primary macrophage activation and polarization were studied upon LUM co-stimulation with LPS. We demonstrate that LUM is not only significantly upregulated in SF from OA patients compared to healthy controls, but also that LUM increases lipopolysaccharide (LPS)-induced TLR4 activation. Furthermore, we show that a pathophysiological level of LUM augments the LPS-induced TLR4 activation and expression of downstream pro-inflammatory molecules, resulting in extensive cartilage degradation. LUM co-stimulation with LPS also provided a pro-inflammatory stimulus, upregulating primary macrophage activation and polarization towards the M1-like phenotype. These findings strongly support the role of LUM as a mediator of PAMP-induced TLR4 activation of inflammation, cartilage degradation, and macrophage polarization in the OA joint and potentially other rheumatic diseases.

MicroRNA‑33 regulates the NLRP3 inflammasome signaling pathway in macrophages.

The nucleotide binding domain and leucine-rich repeat pyrin3 domain (NLRP3) inflammasome/interleukin(IL)-1β axis serves an essential role in regulating the development of rheumatoid arthritis (RA). The dysregulation of cellular metabolism, such as mitochondrial dysfunction, results in the activation of the NLRP3 inflammasome. microRNA (miR)‑33 has previously been identified to be a regulator of lipid metabolism and mitochondrial function. However, whether miR‑33 regulates the NLRP3 inflammasome/IL‑1β axis remains unknown. In the present study, it was observed that an miR‑33 mimic or anti‑miR‑33 markedly stimulated or inhibited, respectively, IL‑1β protein expression levels in mouse peritoneal macrophages. Mechanistically, miR‑33 upregulated the expression of NLRP3 mRNA and protein as well as caspase‑1 activity in primary macrophages. In addition, the results demonstrated that miR‑33 impaired mitochondrial oxygen consumption rates, resulting in the accumulation of cellular reactive oxygen species, which stimulated NLRP3 expression, caspase‑1 activity and IL‑1β secretion. The results of the present study demonstrated that miR‑33 levels and NLRP3 inflammasome activity were increased in peripheral blood monocytes from patients with RA patients compared with healthy donors. In conclusion, the present study identified miR‑33 to be a positive regulator of the NLRP3 inflammasome in macrophages. The miR‑33/NLRP3 inflammasome pathway may therefore be involved in RA development.

Commercial strain-derived clinical Saccharomyces cerevisiae can evolve new phenotypes without higher pathogenicity.

Saccharomyces cerevisiae is one of the most important microbes in food industry, but there is growing evidence on its potential pathogenicity as well. Its status as a member of human mycobiome is still not fully understood. In this study, we characterize clinical S. cerevisiae isolates from Hungarian hospitals along with commercial baking and probiotic strains, and determine their phenotypic parameters, virulence factors, interactions with human macrophages, and pathogenicity. Four of the clinical isolates could be traced back to commercial strains based on genetic fingerprinting. Our observations indicate that the commercial-derived clinical isolates have evolved new phenotypes and show similar, or in two cases, significantly decreased pathogenicity. Furthermore, immunological experiments revealed that the variability in human primary macrophage activation after coincubation with yeasts is largely donor and not isolate dependent. Isolates in this study offer an interesting insight into the potential microevolution of probiotic and food strains in human hosts. These commensal yeasts display various changes in their phenotypes, indicating that the colonization of the host does not necessarily impose a selective pressure toward higher virulence/pathogenicity.

MiR-155 targets Caspase-3 mRNA in activated macrophages

ABSTRACTTo secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScanTM we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation.

Alternative and classic activation of primary macrophages – a systems biology approach

Alternative and classic activation of primary macrophages – a systems biology approach

Alternative and classic activation of primary macrophages – a systems biology approach

Alternative and classic activation of primary macrophages – a systems biology approach

Functional Imaging of Legumain in Cancer Using a New Quenched Activity-Based Probe

Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.

Cultured Primary Macrophage Activation by Lipopolysaccharide Depends on Adsorbed Protein Composition and Substrate Surface Chemistry

Recent efforts show that significantly reducing implant-adsorbed proteins does not avoid the foreign body response. Fluorinated surfaces are commonly used to passivate cell-mediated inflammatory responses to implanted materials but adsorb host proteins and facilitate the attachment and proliferation of macrophages. This study considers in vitro macrophage activation to fluorinated TeflonAF® compared to tissue-culture polystyrene using pre-adsorbed proteins (fibrinogen, BSA, collagen and elastin). Primary macrophage cultures adhere on all pre-adsorbed protein surfaces in a protein concentration-dependent manner and activate to the same extent after 72 h, regardless of surface chemistry. However, macrophages alter their cultured adherent morphology depending on which protein is pre-adsorbed to these surfaces. Macrophages cultured on TeflonAF® on all pre-adsorbed proteins produced overall higher levels of the pro-inflammatory cytokines — TNF-α, IL-6, IL-1β or MCP-1 — than those cultured on tissue-culture polystyrene and those cultured in serum-free media. However, at 72 h, macrophages adherent on BSA or fibrinogen pre-adsorbed surfaces failed to exhibit increased amounts of TNF-a, IL-6 or IL-1/S on either TeflonAF® or TCPS, as well as MCP-1 on TCPS, in the presence of activating lipopolysaccharide. Different cell responses to pre-adsorbed proteins reflect substrate-specific regulation of macrophage cytokine secretion, indicative of LPS tolerance distinct from secondary macrophage cultures, and also distinct from macrophages adherent to surfaces in the absence of proteins. This result has bearing on connecting macrophage adhesion via adsorbed proteins on (fluorinated) biomaterials, and their resulting chronic activation that yields the FBR and possibly reduces effective macrophage clearance of microbes around implanted materials.

The fungal T-2 toxin alters the activation of primary macrophages induced by TLR-agonists resulting in a decrease of the inflammatory response in the pig

T-2 toxin is known to be one of the most toxic trichothecene mycotoxins. Exposure to T-2 toxin induces many hematologic and immunotoxic disorders and is involved in immuno-modulation of the innate immune response. The objective of this work was to evaluate the effects of T-2 toxin on the activation of macrophages by different agonists of Toll-like receptors (TLR) using an in vitro model of primary porcine alveolar macrophages (PAM). Cytotoxic effects of T-2 toxin on PAM were first evaluated. An IC50 of 19.47 ± 0.9753 nM was determined for the cytotoxicity of T-2 toxin. A working concentration of 3 nM of T-2 toxin was chosen to test the effect of T-2 toxin on TLR activation; this dose was not cytotoxic and did not induce apoptosis as demonstrated by Annexin/PI staining. A pre-exposure of macrophages to 3 nM of T-2 toxin decreased the production of inflammatory mediators (IL-1 beta, TNF-alpha, nitric oxide) in response to LPS and FSL1, TLR4 and TLR2/6 agonists respectively. The decrease of the pro-inflammatory response is associated with a decrease of TLR mRNA expression. By contrast, the activation of TLR7 by ssRNA was not modulated by T-2 toxin pre-treatment. In conclusion, our results suggest that ingestion of low concentrations of T-2 toxin affects the TLR activation by decreasing pattern recognition of pathogens and thus interferes with initiation of inflammatory immune response against bacteria and viruses. Consequently, mycotoxins could increase the susceptibility of humans and animals to infectious diseases.

Characterization of Trex1 Induction by IFN-γ in Murine Macrophages

3' Repair exonuclease (Trex1) is the most abundant mammalian 3' → 5' DNA exonuclease with specificity for ssDNA. Trex1 deficiency has been linked to the development of autoimmune disease in mice and humans, causing Aicardi-Goutières syndrome in the latter. In addition, polymorphisms in Trex1 are associated with systemic lupus erythematosus. On the basis of all these observations, it has been hypothesized that Trex1 acts by digesting an endogenous DNA substrate. In this study, we report that Trex1 is regulated by IFN-γ during the activation of primary macrophages. IFN-γ upregulates Trex1 with the time course of an early gene, and this induction occurs at the transcription level. The half-life of mRNA is relatively short (half-life of 70 min). The coding sequence of Trex1 has only one exon and an intron of 260 bp in the promoter in the nontranslated mRNA. Three transcription start sites were detected, the one at -580 bp being the most important. In transient transfection experiments using the Trex1 promoter, we have found that two IFN-γ activation site boxes, as well as an adaptor protein complex 1 box, were required for the IFN-γ-dependent induction. By using EMSA assays and chromatin immune precipitation assays, we determined that STAT1 binds to the IFN-γ activation site boxes. The requirement of STAT1 for Trex1 induction was confirmed using macrophages from Stat1 knockout mice. We also establish that c-Jun protein, but not c-Fos, jun-B, or CREB, bound to the adaptor protein complex 1 box. Therefore, our results indicate that IFN-γ induces the expression of the Trex1 exonuclease through STAT1 and c-Jun.

Quantitative PCR-Based Competitive Index for High-Throughput Screening of Salmonella Virulence Factors

Salmonella enterica serovar Typhimurium is an intracellular pathogen and a main cause of food-borne illness. In this study, a quantitative PCR (qPCR)-based competitive index (CI) method was developed to simultaneously compare the growth of multiple Salmonella strains. This method was applied to a mixture of 17 Salmonella mutants lacking regulator genes, and their survival ratios were compared based on expression of natural resistance-associated macrophage protein 1 (Nramp1). Nramp1, as a major host innate immune component, controls the intracellular replication of pathogens. Deletion strains containing unique DNA barcodes in place of regulator genes were mixed with the parental control, and the bacteria were inoculated into congenic mice differing only at Nramp1. Most of the deletion strains were outcompeted by wild-type bacteria in either mouse strain, and the lack of Nramp1 didn't increase the tested strain/parent control replication ratios. When the same collection of mutants was tested in congenic mouse-derived primary macrophages, a major Nramp1-expressing cell type, six strains (ΔhimD, ΔphoP/phoQ, ΔrpoE, ΔrpoS, ΔompR/envZ, and Δhfq strains) grew better in Nramp1(-/-) than in Nramp1(+/+) macrophages, suggesting that these six regulators may play roles in overcoming Nramp1-mediated bactericidal activity in primary macrophages. The discrepancy in survival of macrophages and that of mice suggests either that there are differences in macrophage populations or that other cell types expressing Nramp1 control Salmonella proliferation in the host. The method described allows competitive infection analysis to be carried out on complex mixtures of bacteria and provides high reproducibility from independent biological replicates.

Type 2 Diabetes Impairs Insulin Receptor Substrate-2-Mediated Phosphatidylinositol 3-Kinase Activity in Primary Macrophages to Induce a State of Cytokine Resistance to IL-4 in Association with Overexpression of Suppressor of Cytokine Signaling-3

Chronic elevation of proinflammatory markers in type 2 diabetes (T2D) is well defined, but the role of anti-inflammatory cytokines in T2D is less clear. In this study, we report that normal IL-4-dependent elaboration of IL-1 receptor antagonist (IL-1RA) requires IRS-2-mediated PI3K activity in primary macrophages. We also show that macrophages isolated from obese/diabetic db/db mice have impaired IRS-2-mediated PI3K activity and constitutively overexpress suppressor of cytokine signaling (SOCS)-3, which impairs an important IL-4 anti-inflammatory function. Peritoneal proinflammatory cytokine levels were examined in diabese (db/db) mice, and IL-6 was found to be nearly 7-fold higher than in nondiabese (db/+) control mice. Resident peritoneal macrophages were isolated from db/db mice and were found to constitutively overexpress IL-6 and were unable to elaborate IL-1RA in response to IL-4-like db/+ mouse macrophages. Inhibition of PI3K with wortmannin or blockage of IRS-2/PI3K complex formation with a cell permeable IRS-2-derived tyrosine phosphopeptide inhibited IL-4-dependent IL-1RA production in db/+ macrophages. Examination of IL-4 signaling in db/db macrophages revealed that IL-4-dependent IRS-2/PI3K complex formation and IRS-2 tyrosine phosphorylation was reduced compared with db/+ macrophages. SOCS-3/IL-4 receptor complexes, however, were increased in db/db mouse macrophages compared with db/+ mice macrophages as was db/db mouse macrophage SOCS-3 expression. These results indicate that in the db/db mouse model of T2D, macrophage expression of SOCS-3 is increased, and impaired IL-4-dependent IRS-2/PI3K formation induces a state of IL-4 resistance that disrupts IL-4-dependent production of IL-1RA.

Distinct functions of IRF-3 and IRF-7 in IFN-alpha gene regulation and control of anti-tumor activity in primary macrophages

Type I IFN (IFN-α/β) have important biological functions ranging from immune cell development and activation, to tumor cell killing and most importantly inhibition of virus replication. Following viral infection or activation of Toll-like receptors (TLRs) via distinct ligands, IFN-α/β are produced. Two members of the interferon regulatory factor (IRF) family – IRF-3 and IRF-7 – are the major modulators of IFN gene expression. Activation of IRF-3 and IRF-7 by TBK1/IKKɛ mediated phosphorylation promotes IFN gene expression and potentiates the production of IFN responsive genes important to the development of an effective antiviral immune response. IFN treatment can augment anti-tumor properties and they are potentially key players in cancer therapy. For example, adoptive transfer of IFN-γ-activated macrophages can mediate tumor cell killing via direct cell–cell contact, as well as release of soluble cytotoxic pro-inflammatory molecules. A recent study investigated whether IRF-3 and IRF-7 could mediate the acquisition of new anti-tumor effector functions in macrophages. Adenovirus mediated transduction of the active form of IRF-7 into primary macrophages resulted in the production of type I IFN, upregulation of target genes including TRAIL and increased tumoricidal activity of macrophages; in contrast, the active form of IRF-3 led to induction of cell death. These studies indicate that IRF-7 transduced macrophages may be an attractive candidate for in vivo adoptive therapy of cancer.

An adenoviral vector for probing promoter activity in primary immune cells

Functional analysis of the DNA regulatory regions that control gene expression has largely been performed through transient transfection of promoter–reporter constructs into transformed cells. However, transformed cells are often poor models of primary cells. To directly analyze DNA regulatory regions in primary cells, we generated a novel adenoviral luciferase reporter vector, pShuttle- luciferase- GFP (pSLUG) that contains a promoterless luciferase cassette (with an upstream cloning site) for probing promoter activity, and a GFP expression cassette that allows for the identification of transduced cells. Recombinant adenoviruses generated from this vector can transduce a wide range of primary immune cells with high efficiency, including human macrophages, dendritic cells and T cells; and mouse T cells transgenic for the coxsackie and adenoviral receptor (CAR). In primary T cells, we show inducible nuclear factor of activated T cells (NF-AT) activity using a recombinant pSLUG adenovirus containing a consensus NF-AT promoter. We further show inducible IL-12/23 p40 promoter activity in primary macrophages and dendritic cells using a recombinant pSLUG adenovirus containing the proximal human IL-12/23 p40 promoter. The pSLUG system promises to be a powerful tool for the analysis of DNA regulatory regions in diverse types of primary immune cells.

PU.1 binding to ets motifs within the equine infectious anemia virus long terminal repeat (LTR) enhancer: regulation of LTR activity and virus replication in macrophages.

Binding of the transcription factor PU.1 to its DNA binding motif regulates the expression of a number of B-cell- and myeloid-specific genes. The long terminal repeat (LTR) of macrophage-tropic strains of equine infectious anemia virus (EIAV) contains three PU.1 binding sites, namely an invariant promoter-proximal site as well as two upstream sites. We have previously shown that these sites are important for EIAV LTR activity in primary macrophages (W. Maury, J. Virol. 68:6270-6279, 1994). Since the sequences present in these three binding motifs are not identical, we sought to determine the role of these three sites in EIAV LTR activity. While DNase I footprinting studies indicated that all three sites within the enhancer were bound by recombinant PU.1, reporter gene assays demonstrated that the middle motif was most important for basal levels of LTR activity in macrophages and that the 5' motif had little impact. The impact of the 3' site became evident in Tat transactivation studies, in which the loss of the site reduced Tat-transactivated expression 40-fold. In contrast, elimination of the 5' site had no effect on Tat-mediated activity. Binding studies were performed to determine whether differences in PU.1 binding affinity for the three sites correlated with the relative impact of each site on LTR transcription. While small differences were observed in the binding affinities of the three sites, with the promoter-proximal site having the strongest binding affinity, these differences could not account for the dramatic differences observed in the transcriptional effects. Instead, the promoter-proximal position of the 3' motif appeared to be critical for its transcriptional impact and suggested that the PU.1 sites may serve different roles depending upon the location of the sites within the enhancer. Infectivity studies demonstrated that an LTR containing an enhancer composed of the three PU.1 sites was not sufficient to drive viral replication in macrophages. These findings indicate that while the promoter-proximal PU.1 site is the most critical site for EIAV LTR activity in the presence of Tat, other elements within the enhancer are needed for EIAV replication in macrophages.

Activity of two different silencer elements of the chicken lysozyme gene can be compensated by enhancer elements.

The chicken lysozyme gene is constitutively expressed in macrophages. Transfection of recombinant genes containing different portions of the lysozyme 5' upstream region revealed the existence of two negative transcriptional elements within 1 kb upstream of the start sites. Both elements placed upstream or downstream of a heterologous promoter-gene unit repress transcription independent of their orientation and are therefore called silencer elements, although their repressing activities 3' of the gene are reduced. One silencer (N-1.0 kb) at position -1 kb consists of the central region of the chicken middle repetitive sequence element CR1 and can be divided into two functional domains. N-1.0 kb is active in all cell types tested. The other silencer (N-0.25 kb) at position -0.25 kb shows reduced activity in primary macrophages. Despite their different specificities, the activity of both silencer elements can be influenced similarly. An inverse linear relationship between the transcriptional activity of the tested constructs and the potential inhibition by the silencer elements was found: weak transcription units can be strongly repressed, whereas strong transcription units can be only weakly repressed. Such a mechanism may help to turn off completely a particular gene in situations or tissues where strong positive regulators are inactive.