Adenovirus RID Complex Inhibits Lipopolysaccharide Signaling without Altering TLR4 Cell Surface Expression
http://www.100md.com
《病菌学杂志》
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461
Department of Pediatrics, Division of Infectious Diseases, Albert Einstein College of Medicine, Bronx, New York 10461
ABSTRACT
The transmembrane heterotrimer complex 10.4K/14.5K, also known as RID (for "receptor internalization and degradation"), is encoded by the adenovirus E3 region, and it down-regulates the cell surface expression of several unrelated receptors. We recently showed that RID expression correlates with down-regulation of the cell surface expression of the tumor necrosis factor (TNF) receptor 1 in several human cells. This observation provided the first mechanistic explanation for the inhibition of TNF alpha-induced chemokines by RID. Here we analyze the immunoregulatory activities of RID on lipopolysaccharide (LPS) and interleukin-1 beta (IL-1)-mediated responses. Although both signaling pathways are strongly inhibited by RID, the chemokines up-regulated by IL-1 stimulation are only marginally inhibited. In addition, RID inhibits signaling induced by LPS without affecting the expression of the LPS receptor Toll-like receptor 4, demonstrating that RID need not target degradation of the receptor to alter signal transduction. Taken together, our data demonstrate the inhibitory effect of RID on two additional cell surface receptor-mediated signaling pathways involved in inflammatory processes. The data suggest that RID has intracellular targets that impair signal transduction and chemokine expression without evidence of receptor down-regulation.
INTRODUCTION
For their survival, most human viruses have developed diverse strategies to overcome host defenses, including the inflammatory responses of the innate immune system (49, 55, 72). Some viruses have developed antiapoptotic functions: human herpes simplex virus type 1, for example, expresses at least seven proteins capable of modulating apoptosis in herpes simplex virus-infected cells (4). Other viruses inhibit specific signaling pathways by coding for soluble receptor mimics that block specific receptors by competing with their ligands (2, 52) or by down-regulating the expression of cell surface receptors. Components of the tumor necrosis factor alpha (TNF-) signal transduction pathway are a recurrent target for viral immune evasion because of their central roles in mounting innate and adaptive immune responses (8, 9). For instance, TNF- signaling is inhibited by the hepatitis C viral protein NS5A that binds TRAF2 (57); human cytomegalovirus infection down-regulates TNF receptor 1 (TNFR1) cell surface expression by an unknown mechanism in human monocytic and U373 astrocytoma cell lines (5); the Epstein-Barr viral protein BZLF1 represses directly the TNFR1 promoter (51); and poxvirus N1L protein blocks TNF and interleukin-1 (IL-1)-Toll signaling mainly by binding to TBK1 (22).
Adenoviruses (Ads) are important human pathogens (33) and have been used extensively as vectors for gene transduction and gene therapy (37). Their versatility has also made them candidate vectors for generating vaccines against various pathogens (15). After natural infection, some Ad serotypes persist in tonsillar tissue for years (41). Ads have thus developed several mechanisms to circumvent the host strategies of viral elimination. Most of the viral proteins involved in immunoregulatory functions are coded by the early expression cassette E3, from which at least seven proteins are expressed (for recent reviews, see references 27, 34, 48, and 74). For instance, gp19K blocks major histocompatibility complex class I antigen presentation by sequestering it in the endoplasmic reticulum (16). The 14.7K protein inhibits apoptosis-induced by TNF- in murine cells by unknown mechanisms (28, 32), and it interacts with four unrelated cellular proteins, as shown by yeast two-hybrid experiments (46). The 10.4-14.5K complex, also known as RID (for "receptor internalization and degradation"), blocks TNF--induced cytolysis (65), inhibits release of arachidonic acid induced by TNF- in murine cells (42), and down-regulates the cell surface expression of FAS (25, 62), TRAIL-R1 and -R2, all members of the TNFR superfamily (10, 68), as well as epidermal growth factor receptor (67), in several cell lines. A tyrosine-sorting motif (YXX) in RID (14.5K) was shown to be essential for FAS- and TRAIL-R1-induced internalization (47). In addition, a cytosolic dileucine transport motif in the RID subunit (10.4K) is essential for the function of RID in FAS, TRAIL-R1, and epidermal growth factor receptor cell surface down-regulation in human cell lines (30). Although there have been contradictory reports on the effect of RID on TNFR1 levels, we recently demonstrated a pronounced decrease in cell surface expression of TNFR1 in human 293 and HeLa cells (26). Furthermore, our recent data suggest that RID associates with TNFR1 at the cell surface and causes it to be internalized by a clathrin-mediated mechanism (19).
We had previously demonstrated the anti-inflammatory properties of the AdE3 region in vivo in two different murine models. First, we showed that transgenic expression of E3 genes in pancreatic islets prolonged the survival of the islets transplanted under the kidney capsule of allogeneic recipients (24). Second, in the lymphocytic choriomeningitis virus mouse model of autoimmune diabetes, the transgenic expression of E3 in islets, directed by a tissue-specific promoter (rat insulin promoter), prevented the onset of diabetes induced by infection with lymphocytic choriomeningitis virus (71). Similarly, transgenic expression of E3 in islets of NOD mice delayed the onset of autoimmune diabetes (24). In both cases, the analysis of histological specimens showed a substantial decrease in mononuclear cell infiltration surrounding the islets expressing the E3 cassette. These observations indicated that E3 expression could affect the process that drives monocytic cells to the allogeneic transplant or to the target of autoimmunity. A recent report also showed that infection of a human cell line with an adenovirus vector expressing only AdE3 genes prevented its rejection when xenotransplanted to immunocompetent mice (69).
At the site of an injury or infection, the early phase of inflammation entails the activation of signal transduction pathways mediated by cytokines, chemokines, and derived bacterial components. Increased levels of proinflammatory cytokines such as TNF- and IL-1 are hallmarks of an inflammatory response. TNF-, a proinflammatory stimulus, is produced in response to injury or infection mainly by macrophages and NK cells as well as infiltrating T lymphocytes. IL-1, also a proinflammatory stimulus, is also produced in response to injury or infection primarily by macrophages and epithelial cells. One potent inducer of these cytokines is lipopolysaccharide (LPS) or endotoxin, a component of the cell wall of gram-negative bacteria. A common downstream target activated by proinflammatory cytokines as well as LPS is the transcription factor NF-B (11, 29), which is involved in a vast array of intracellular processes in almost all cell types. AP-1 and p38 are other ubiquitous transcription factors stimulated by proinflammatory signals in some cells (36).
The main cell types that respond to injury and inflammation in the brain are part of the glia, a supportive network constituted of astrocytes, microglia, and oligodendroglia. Astrocytes have diverse functions in the brain; they represent the most abundant glial constituent and play a central role in maintaining local tissue homeostasis. The proinflammatory program of astrocytes has been extensively characterized in studies focusing on specific downstream targets as well as more global transcriptional changes by microarray analysis (40, 56, 61). Astrocytes can be activated by proinflammatory stimuli, and one of the outcomes is up-regulation of chemokine production (58, 59, 64). Chemokines are small secretable proteins whose function is to direct immune effector cells to the site of inflammation (21). Astrocytes respond to cytokines such as TNF- and IL-1 but are also highly responsive to LPS (7, 14, 35). Thus, the human astrocytoma cell line U373 provides an excellent model for studying the effects of RID upon stimulation with these ligands (44, 66). In a previous report, we demonstrated that transduction of U373 cells with the entire E3 cassette prevented the expression of TNF--induced chemokines such as monocyte chemoattractant protein (MCP-1) and IL-8 (44). We subsequently showed that this activity maps to RID, and here we demonstrate that RID mediates this activity by down-regulating cell surface levels of the TNFR1. In addition, we wanted to test whether proinflammatory cascades triggered by other factors such as IL-1 and LPS are affected by RID. Both IL-1 and LPS share significant portions of the signaling pathways downstream of the respective receptors, namely, IL-1R and Toll-like receptor 4 (TLR4), including the adaptor molecules required to stimulate signaling (45). However, we find that RID expression has differential inhibitory effects on IL-1R and TLR4 signaling. In both situations, the activation of NFB and AP-1 is inhibited by RID. In contrast, while the up-regulation of chemokines MCP-1 and IL-8 induced by LPS is abolished by RID, the same chemokines up-regulated by IL-1 are only marginally affected. Interestingly, the inhibition of TLR4 signaling by RID does not involve receptor down-regulation on the cell surface, suggesting a novel mechanism of action to achieve a reduction in LPS-mediated inflammation.
(Data in this paper are from a thesis to be submitted by Fernando Delgado-Lopez in partial fulfillment of the requirements for a Ph.D. from the Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.)
MATERIALS AND METHODS
Cells and viruses. The human astrocytoma cell line U373 was grown in alpha minimal essential medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin; the same medium was used for growing the A549 human cell line. HEK293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with FBS and antibiotics. All four cell lines were obtained from the American Type Culture Collection, Rockville, MD. Adenoviral infections were performed in six-well plates with cells at 95% confluence in 1 ml of alpha minimal essential medium supplemented with antibiotics and 10% FBS. Mutant adenoviral vectors expressing, under cytomegalovirus promoter control, RID only, 14.7K protein only, RID plus 14.7K, the entire E3 cassette, or null virus (expressing none of the E3 proteins) were derived from Ad rec700 and kindly provided by W.S. Wold (70). Viruses were added directly to the plate at a concentration of 500 particles/cell (p/cell) (1 PFU equivalent to 50 particles) or at the concentrations indicated. Human recombinant TNF- (R&D Systems) at 10 ng/ml, human recombinant IL-1 (R&D Systems) at 5 ng/ml, or Escherichia coli LPS (Sigma) at 100 ng/ml was added at the times indicated in the corresponding figures. Phorbol myristate acetate (PMA) and okadaic acid were obtained from Biomol.
Virus isolation and purification. Serial dilutions in 300 μl of DMEM were adsorbed to a monolayer of 293 cells growing at about 95% confluence in 60-mm dishes. After an hour, the cells were overlaid with 5 ml of warm 0.9% agar in DMEM and kept in the incubator at 37°C. Fresh medium was added every 2 days, and plaques were isolated after 8 to 15 days postinfection. The plaque-purified virus was expanded in 293 cells. The virus was extracted from approximately 1 x 109 cells. Infected cells were pooled and resuspended in 10 mM TRIS (pH 8.1), lysate was extracted with Freon three times, and the final phase was layered on top of a linear CsCl gradient in 10 mM TRIS (pH 8.1). After 4 h of centrifugation at 23,000 rpm in a SW-28 Beckman rotor, the viral band was isolated and transferred to a fresh CsCl gradient for a second round of centrifugation and isolation. The concentration of the virus was determined by the optical density at 260 nm.
Western blotting. Cells were lysed in 250 μl of electrophoresis loading buffer (50 mM Tris-HCl, 100 mM dithiothreitol, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol), boiled at 100°C for 3 min, and sonicated. Samples of the extracts were separated using SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to a nitrocellulose membrane for 1.5 h in 20 mM Tris (pH 8)-150 mM glycine at room temperature (RT). The membrane was blocked for 30 min at RT in blocking buffer (1x phosphate-buffered saline[PBS], 5% nonfat milk), washed several times with PBS, and incubated overnight at 4°C with the appropriate antibodies diluted in blocking buffer. After several washes the blot was incubated for 1.5 h at RT with horseradish peroxidase-conjugated antibody diluted 1:3,000 in 2%milk:PBS. Proteins were detected with ECL chemoluminescence (PerkinElmer). Polyclonal antibody for RID was raised against the C terminal of RID (CEISYFNLTGGDD) and produced by Genemed Synthesis Inc. Polyclonal antibodies against TNFR1, TLR4, FAS, IB, phospho-c-Jun, and -tubulin were obtained from Santa Cruz Biotechnology. Anti-phosphoserine-536-p65 and anti-phospho-p38 were purchased from Cell Signaling Technologies. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G was purchased from Amersham Biosciences. Biotinylated mouse anti-TNFR1 and its biotinylated isotype control were purchased from BD Bioscience.
RNase protection assays (RPAs). Total RNA was extracted from U373 cells by use of TRIzol reagent (Life Technologies) per the suggested protocol. In brief, the cells were lysed with 1 ml TRIzol reagent on the plates and then mixed with 0.2 ml of chloroform in Eppendorf tubes, shaken by hand, allowed to stand at room temperature for 5 min, and then centrifuged at 10,000 rpm at 4°C for 15 min. The supernatants were collected and precipitated with isopropanol at –80°C for 15 min and centrifuged at 14,000 rpm at 4°C for 25 min. The pellet was washed with 70% ethanol at room temperature, dried, and resuspended in 10 μl of Ambion nuclease-free water. The yield of the RNA was measured by optical density at 260 nm.
RPAs were performed using an RPA III kit (Ambion) and probes labeled using a Maxiscript T7 kit (Ambion). The multiprobe template set hCK-5 (Pharmingen) was transcribed using T7 RNA polymerase with [32P]UTP (Amersham-Pharmacia), phenol chloroform extracted, precipitated with ethanol and ammonium acetate, and resuspended in 70 μl hybridization buffer. Five micrograms of sample RNA was hybridized with 1.5 μl of probe overnight at 44°C. Samples were then digested with RNase A/T1 for 30 min at 37°C. RNase was inactivated, and the samples were precipitated with proprietary buffer and ethanol. Protected samples were resuspended in 1x loading buffer and separated by 5% acrylamide-8 M urea gel electrophoresis. After drying, the gel was exposed to Kodak film (mass spectrometry).
FACS analysis. Cells were placed on ice for 10 min before washing twice with cold PBS and then incubated with 1% BSA for 10 min, followed by a 10 min incubation with FB (2% FBS in PBS), always on ice. Biotin-anti-TNFR antibody was used at 0.5 μg/ml for 30 min, cells were washed three times, and then phycoerythrin-streptavidin (Molecular Probes) was added at 1 μg/ml in FB for 30 min on ice. After three washes with FB, cells were fixed with 2% paraformaldehyde in PBS. Fluorescence-activated cell sorting (FACS) analysis was done in a FACSCalibur machine.
Cell surface biotinylation. U373 cells growing in 10-cm dishes at 90% confluence were infected with 500 p/cell of various adenoviral vectors and incubated for 20 h before labeling. We followed the instructions for use of a cell surface biotinylation kit from Pierce. Essentially, the monolayer was transferred to a bed of ice for 10 min and remained there during the length of the labeling. Cells were washed twice with ice-cold PBS, and 10 ml of EZ-Link Sulfo-NHS-SS-Biotin in PBS was added for 30 min. The reaction was stopped with quenching solution and the reaction mixture was washed three times; cells were then scraped and lysed for 30 min on ice with the provided lysis buffer. The lysate was clarified by centrifugation, and the supernatant was incubated for 1 h at RT with NeutrAvidin gel in a column. The beads were washed several times and finally eluted under reducing conditions with loading buffer. The eluate was concentrated 5x by ultrafiltration, and 25% of the eluate was loaded on an SDS gel.
RESULTS
Proinflammatory stimuli activate NFB and AP-1 pathways and induce the expression of MCP-1 and IL-8 in U373 cells. Four human cell lines were evaluated for responsiveness to four different potent proinflammatory stimuli. Three of these stimuli, TNF-, IL-1, and LPS, mediate their action through cell surface receptors, whereas PMA represents a cell-permeative stimulus with an intracellular site of action. All four factors are known to stimulate NF-B and AP-1 signal transduction. The assay used for testing NF-B activation examined degradation of IB, which normally binds the p50/p65 NF-B heterodimer and prevents its translocation to the nucleus. AP-1 activation was assayed by detecting phosphorylation of c-Jun. The four human cell lines tested were U373, 293, A549, and HeLa, which differ significantly in terms of their response to proinflammatory stimuli as well as in the extent to which AP-1 is activated (Fig. 1A). HeLa cells responded weakly and only to TNF- stimulation, as evidenced by minimal IB degradation and c-Jun phosphorylation. In 293 cells, TNF and PMA elicited a strong response in both NF-B and AP-1 activation. In A549 cells, TNF- and IL-1 stimulation degraded IB efficiently, while c-Jun was only marginally phosphorylated. The astrocytoma cell line U373 is the only cell line—among the four tested—which showed consistent activation of the NF-B as well as AP-1 pathways in response to all four stimuli.
The transcription of many chemokines is regulated by NF-B- and AP-1-responsive elements in the promoters of their genes (31, 60). We used RPA, a quantitative method of measuring steady-state levels of specific mRNAs, to analyze various chemokine transcripts. U373 cells were stimulated with TNF-, IL-1, LPS, and PMA for 4 h. Exposure to all four factors up-regulates IL-8 and MCP-1 messages to similar levels (Fig. 1B).
Expression of RID correlate with efficient suppression of TNF- signaling via down-regulation of TNFR1 in U373 cells. Adenoviruses infect cells at various efficiencies; some cells require very high multiplicities of infection (MOI) before effective transduction is achieved, while others are very permissive. We analyzed the kinetics of RID expression in U373 cells as a function of MOI and time. The kinetics of RID expression after infection with 500 p/cell of AdRID showed that the extent of NF-B and AP-1 inhibition correlated with expression of RID; this inhibition was detected as early as 8 h postinfection (Fig. 2A).
As shown previously in a study using human 293 cells (26), FACS analysis confirmed that RID down-regulates the cell surface expression of TNFR1 in U373 cells and that the levels of cell surface TNFR1 decreased in a manner that is dose dependent on RID expression levels (Fig. 2B). Furthermore, this down-regulation is saturable, since infection at MOIs above 1,000 particles per cell makes RID activity less efficient. A dose-response experiment showed that infection for 20 h with as few as 50 p/cell is sufficient for detection of RID and inhibition of TNF- signaling (Fig. 2C). Infection with AdNull (lacking the E3 domain) had no inhibitory effect. In contrast to cell surface expression results, the total cellular levels of TNFR1 remained the same over the course of the infection.
LPS activation of the NF-B and AP-1 pathways is slower than the rapid activation induced by IL-1 and TNF-. In addition to IB degradation, we utilized p65 phosphorylation to assess NF-B activation (p65 conveys the transactivation activity in the p50/p65 heterodimer complex of NF-B). To measure AP-1 and p38 activation, we analyzed c-Jun phosphorylation and p38 phosphorylation, respectively. Both TNF- and IL-1 showed rapid kinetics of NF-B, AP-1, and p38 activation, reaching a maximum level at 10 min postinitiation of stimulation (Fig. 3A) consistent with previous reports (6). The downstream signaling events mediated by LPS were significantly slower, reaching complete activation only after 60 min of stimulation (Fig. 3B). Infection with AdRID (500 p/cell) for 20 h prior to LPS stimulation completely inhibited the LPS-induced IB degradation as well as c-Jun, p65, and p38 phosphorylation, while infection with AdNull (at the same MOI) had no effect on LPS-stimulated cells. Furthermore, experiments conducted to determine the induction rates of IL-8 and MCP-1 mRNA showed that TNF- and IL-1 up-regulated the expression of chemokines with faster kinetics than did LPS (Fig. 3C).
RID inhibits IL-1-mediated signaling. Although the receptors and upstream adaptors of the TNF- and IL-1 pathways are different, they both lead to the activation of the canonical NF-B and AP-1 pathways. While AdNull or Ad14.7K-expressing viruses did not have any effect on either prevention of IB degradation or phosphorylation of c-Jun, p65, or p38 induced by either TNF- or IL-1, AdRID infection prevented IB degradation and c-Jun phosphorylation induced by the same cytokines. Although RID efficiently inhibited p65 and p38 phosphorylation induced by TNF-, it only partially inhibited the phosphorylation of p65 or p38 induced by IL-1 (Fig. 4).
Non-receptor-mediated activation of NF-B and AP-1 by PMA or okadaic acid is not affected by RID. The activation of NFB by phorbol esters and okadaic acid has been previously described (43, 63). These small cell-permeative molecules activate signaling without the mediation of a cell surface receptor. PMA activates protein kinase C (20), while okadaic acid inhibits the PP2 family members of serine/threonine phosphatases (23). We found that RID does not have an impact on PMA- or okadaic acid-mediated activation of the NFB and AP-1 pathways, as judged by the fact that IB degradation and c-Jun phosphorylation are unaffected (Fig. 5). This supports the notion that the target for RID action is a receptor proximal to PMA and okadaic acid action.
RID strongly inhibits induction of chemokines by LPS and TNF- but not that by IL-1. Transduction of U373 cells with the entire AdE3 cassette inhibits chemokines induced by TNF- stimulation (44). Further mapping studies of the astrocytoma cell line as well as other human cell lines showed that this effect was specific to RID (data not shown). The E3 cassette and the mutant vectors expressing individual E3 proteins were also evaluated in the context of IL-1, LPS, or PMA stimulation. In contrast to the robust inhibition by RID of signaling and chemokine expression triggered by TNF- and LPS and the significant inhibition of IB degradation and c-Jun activation triggered by IL-1, the chemokines up-regulated by IL-1 were not affected to the same extent (Fig. 6). Specifically, while IL-8 and MCP-1 messages were completely suppressed by RID after TNF- or LPS stimulation, the same chemokines, when stimulated by IL-1, were only marginally inhibited. On the other hand, the fact that RID significantly disrupts IL-1 signaling leading to NFB and AP-1 activation but only marginally affects chemokine expression suggests that the cell surface expression of IL-1R is not affected.
The differential effects of RID on three proinflammatory challenges are independent of each other. There are several alternative explanations for the observed differential effects of RID, the most likely being that IL-1 activates pathways in addition to NFB and AP-1 that can lead to chemokine synthesis, especially since the inhibition of IL-1 signaling by RID is not complete (Fig. 4A). However, it is also possible that RID targets only one molecule or protein complex common to the receptors for those three ligands. This component could be more accessible after TNFR1 or TLR4 stimulation compared to its accessibility after IL-1R stimulation. To test this, we simultaneously stimulated U373 cells with various combinations of TNF-, IL-1, and LPS and compared the effects of RID under these circumstances (Fig. 7). In order to appreciate the slight effect of RID on IL-1-induced chemokine expression, gels were underexposed (20 min). As shown in Fig. 7, every time the cells were exposed to IL-1 stimulation, RID was unable to completely inhibit chemokine expression. This indicates that IL-1 has other mechanisms, unaffected by RID, to signal induction of chemokine expression.
Biotinylated human TLR4 is not down-regulated from the cell surface by RID. Despite the availability of a few commercial antibodies against TLR4, we were unable to assess the endogenous levels of this receptor in astrocytoma cells by FACS analysis, probably because of low levels of TLR4 expression on these cells or low affinity of the antibodies (73). Therefore, we used a non-cell-permeative cross-linker to biotinylate and isolate the cell surface proteins by affinity chromatography for Western blotting experiments with specific antibodies. As shown in Fig. 8, cell surface expression of TLR4 is not affected by RID. We measured cell surface expression of FAS, a molecule known to be down-regulated by RID, as a positive control. In addition, RID was also biotinylated and isolated by the affinity protocol (Fig. 8).
DISCUSSION
There is increasing evidence that TLRs play crucial roles in diverse pathogenic scenarios, including sepsis, asthma, transplant rejection, and several inflammatory diseases (3). Therefore, the identification of novel inhibitors of these signaling pathways will have direct clinical implications. Here, we provide evidence that the adenovirus-derived RID can block signaling induced by LPS, the ligand of TLR4, as well as signaling induced by IL-1. Because the intracellular domains of TLR4 and the IL-1R are highly homologous and share many downstream effectors, it was expected that RID would inhibit IL-1 and LPS signaling comparably. However, we found significant variations between the two. While RID is able to inhibit equivalently IB degradation and c-Jun phosphorylation after IL-1 or LPS stimulation, the expression of IL-8 and MCP-1 chemokines induced by LPS is completely abrogated by RID but only marginally inhibited following IL-1 stimulation. The observation that RID does inhibit IB degradation but does not completely inhibit phosphorylation of p65 induced by IL-1 may be explained by the fact that p65 can be phosphorylated by kinases in pathways other than the classical NF-B pathway. Its activity can also be modulated by phosphorylations at other serine residues as well as acetylation of lysine residues (18). Likewise, the versatility of the ways in which p65 activity is modified may help explain the small effect that RID has on the chemokine expression induced by IL-1. Because RID expression down-regulates the cell surface expression of several receptors, it seemed likely that the mechanism for inhibiting LPS-induced signaling and chemokine expression might involve the cell surface down-regulation of TLR4. However, cell surface biotinylation experiments showed that RID does not down-regulate the cell surface expression of TLR4. The fact that IL-1-induced chemokines are only marginally affected by RID provides indirect supporting evidence that the cell surface expression of the IL-1 receptor is also not targeted by RID.
One possibility to be considered is whether U373 cells have mechanisms of response to LPS that do not involve TLR4 signaling. For example, an alternative LPS receptor might be down-regulated by RID. Others have shown that in U373 cells, LPS transduces signaling only through TLR4, specifically through Bruton's tyrosine kinase, as an essential component of the signal transduction machinery (39). However, since there is a report showing that LPS can also signal through TLR2 (54), we did stimulate the cells with zymosan (a known ligand for TLR2) and observed neither NF-B and AP-1 activation nor chemokine up-regulation (data not shown). In addition, we also determined that TLR2 cell surface levels are not affected by RID (data not shown).
Adenoviruses have been reported to trigger NF-B activation and chemokine production (12, 13, 53). These observations were made in the context of utilizing adenovirus as a vector for gene therapy, using very high titers of viral vectors that lack RID (most adenoviral vectors have the whole E3 cassette deleted). Under these conditions, structural components of viral capsids may induce the production of proinflammatory molecules. In our experiments, relatively low viral titers were used (in general, 500 p/cell, equivalent to 10 PFU/cell), conditions in which activation of NF-B or chemokine production is not induced by the virus itself. Likewise, we did not observe any change in cell viability or morphology under any of our conditions. In continued use of adenoviruses as vector platforms for gene delivery, it will be important to assess viral input carefully to minimize the intrinsic proinflammatory properties of the vector. It might also be beneficial to include RID (or some component of RID) in the vectors as a means to diminish the intrinsic proinflammatory characteristics of the vectors.
When combinations of the various proinflammatory challenges were tested, the levels of inhibition by RID were not altered in comparisons of its effects after stimulation with one stimulus to those resulting from stimulation with a mixture of them, which implies that RID blocks signaling from the three stimuli by targeting each of the individual pathways in a specific manner. Several strategies have been used to identify the molecular targets of RID. By comparing human astrocytoma cell lines that either overexpress or are deficient in caveolin-1, we determined that RID does not require caveolin-1 for its inhibitory effects (F. Delgado-Lopez and M. Horwitz, unpublished data). In addition, disrupting clathrin-mediated endocytosis by overexpressing a dominant-negative form of the clathrin heavy chain (50) did not affect the ability of RID to down-regulate chemokine expression (Delgado-Lopez and Horwitz, unpublished). However, in 293 cells the μ2 subunit of adaptor protein 2 is involved in clathrin-dependent TNFR1 down-regulation by RID (19), a distinction that could be explained by cell type differences. Here it is pertinent to note that 293 cells do not express significant levels of chemokines regardless of the stimuli, presumably because this cell line is transformed by the adenovirus E1 expression cassette (1). The targets of RID, other than cell surface receptors, are likely to be upstream of the IB kinase (IKK) complex, a nodal point where several kinases—activated by diverse stimuli—converge to signal IKK to phosphorylate IB, a reaction that signals IB for degradation. This notion is supported by the inability of RID to block the signaling at the level of IB in response to PMA and okadaic acid. On the other hand, upstream of IKK, the forced expression of the adaptor molecule MyD88 inhibits IL-1- and LPS-induced NFB activation by preventing IRAK-1 phosphorylation by IRAK-4 (17, 38). RID, therefore, probably does not function at the level of MyD88, since it does not inhibit equivalently IL-1 and LPS signaling. Furthermore, since TAK-1, a mitogen-activated protein kinase kinase kinase, is the molecule in the IL-1 and LPS pathways where the p38 signaling and the AP-1 signaling diverge, it is possible that RID may be working downstream of TAK-1.
In conclusion, the data presented here indicate that RID targets molecules other than cell surface receptors, and this implies there are novel mechanisms for RID function yet to be resolved. Further mapping of pathway components intersecting with the RID complex should provide important information on specificity that could facilitate the use of RID for therapeutic purposes.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grant 1PO1DK52956.
Special gratitude is extended to Todd Evans, Tsoline Kojaoghlanian, and Philipp Scherer for the time they spent reviewing the manuscript.
Dedicated to the memory of Marshall S. Horwitz.
Deceased.
REFERENCES
Aiello, L., R. Guilfoyle, K. Huebner, and R. Weinmann. 1979. Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad-5 or 293). Virology 94:460-469.
Alcami, A. 2003. Viral mimicry of cytokines, chemokines and their receptors. Nat. Rev. Immunol. 3:36-50.
Andreakos, E., B. Foxwell, and M. Feldmann. 2004. Is targeting Toll-like receptors and their signaling pathway a useful therapeutic approach to modulating cytokine-driven inflammation Immunol. Rev. 202:250-265.
Aubert, M., and J. A. Blaho. 2001. Modulation of apoptosis during herpes simplex virus infection in human cells. Microbes Infect. 3:859-866.
Baillie, J., D. A. Sahlender, and J. H. Sinclair. 2003. Human cytomegalovirus infection inhibits tumor necrosis factor alpha (TNF-) signaling by targeting the 55-kilodalton TNF- receptor. J. Virol. 77:7007-7016.
Barchowsky, A., D. Frleta, and M. P. Vincenti. 2000. Integration of the NF-B and mitogen-activated protein kinase/AP-1 pathways at the collagenase-1 promoter: divergence of IL-1 and TNF-dependent signal transduction in rabbit primary synovial fibroblasts. Cytokine 12:1469-1479.
Basu, A., J. K. Krady, and S. W. Levison. 2004. Interleukin-1: a master regulator of neuroinflammation. J. Neurosci. Res. 78:151-156.
Benedict, C. A. 2003. Viruses and the TNF-related cytokines, an evolving battle. Cytokine Growth Factor Rev. 14:349-357.
Benedict, C. A., T. A. Banks, and C. F. Ware. 2003. Death and survival: viral regulation of TNF signaling pathways. Curr. Opin. Immunol. 15:59-65.
Benedict, C. A., P. S. Norris, T. I. Prigozy, J. L. Bodmer, J. A. Mahr, C. T. Garnett, F. Martinon, J. Tschopp, L. R. Gooding, and C. F. Ware. 2001. Three adenovirus E3 proteins cooperate to evade apoptosis by tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2. J. Biol. Chem. 276:3270-3278.
Bonizzi, G., and M. Karin. 2004. The two NF-B activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25:280-288.
Borgland, S. L., G. P. Bowen, N. C. Wong, T. A. Libermann, and D. A. Muruve. 2000. Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-B. J. Virol. 74:3941-3947.
Bowen, G. P., S. L. Borgland, M. Lam, T. A. Libermann, N. C. Wong, and D. A. Muruve. 2002. Adenovirus vector-induced inflammation: capsid-dependent induction of the C-C chemokine RANTES requires NF-kappa B. Hum. Gene Ther. 13:367-379.
Bowman, C. C., A. Rasley, S. L. Tranguch, and I. Marriott. 2003. Cultured astrocytes express toll-like receptors for bacterial products. Glia 43:281-291.
Boyer, J. L., G. Kobinger, J. M. Wilson, and R. G. Crystal. 2005. Adenovirus-based genetic vaccines for biodefense. Hum. Gene Ther. 16:157-168.
Burgert, H. G., and S. Kvist. 1985. An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell 41:987-997.
Burns, K., S. Janssens, B. Brissoni, N. Olivos, R. Beyaert, and J. Tschopp. 2003. Inhibition of interleukin 1 receptor/Toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4. J. Exp. Med. 197:263-268.
Chen, L. F., and W. C. Greene. 2004. Shaping the nuclear action of NF-B. Nat. Rev. Mol. Cell Biol. 5:392-401.
Chin, Y. R., and M. S. Horwitz. 2005. Mechanism for removal of tumor necrosis factor receptor 1 from the cell surface by the adenovirus RID/ complex. J. Virol. 79:13606-13617.
Crabos, M., R. Imber, T. Woodtli, D. Fabbro, and P. Erne. 1991. Different translocation of three distinct PKC isoforms with tumor-promoting phorbol ester in human platelets. Biochem. Biophys. Res. Commun. 178:878-883.
Cyster, J. G. 2005. Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu. Rev. Immunol. 23:127-159.
DiPerna, G., J. Stack, A. G. Bowie, A. Boyd, G. Kotwal, Z. Zhang, S. Arvikar, E. Latz, K. A. Fitzgerald, and W. L. Marshall. 2004. Poxvirus protein N1L targets the I-B kinase complex, inhibits signaling to NF-B by the tumor necrosis factor superfamily of receptors, and inhibits NF-B and IRF3 signaling by toll-like receptors. J. Biol. Chem. 279:36570-36578.
Dounay, A. B., and C. J. Forsyth. 2002. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor. Curr. Med. Chem. 9:1939-1980.
Efrat, S., G. Fejer, M. Brownlee, and M. S. Horwitz. 1995. Prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes. Proc. Natl. Acad. Sci. USA 92:6947-6951.
Elsing, A., and H. G. Burgert. 1998. The adenovirus E3/10.4K-14.5K proteins down-modulate the apoptosis receptor Fas/Apo-1 by inducing its internalization. Proc. Natl. Acad. Sci. USA 95:10072-10077.
Fessler, S. P., Y. R. Chin, and M. S. Horwitz. 2004. Inhibition of tumor necrosis factor (TNF) signal transduction by the adenovirus group C RID complex involves downregulation of surface levels of TNF receptor 1. J. Virol. 78:13113-13121.
Fessler, S. P., F. Delgado-Lopez, and M. S. Horwitz. 2004. Mechanisms of E3 modulation of immune and inflammatory responses. Curr. Top. Microbiol. Immunol. 273:113-135.
Gooding, L. R., I. O. Sofola, A. E. Tollefson, P. Duerksen-Hughes, and W. S. Wold. 1990. The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis. J. Immunol. 145:3080-3086.
Hayden, M. S., and S. Ghosh. 2004. Signaling to NF-B. Genes Dev. 18:2195-2224.
Hilgendorf, A., J. Lindberg, Z. Ruzsics, S. Honing, A. Elsing, M. Lofqvist, H. Engelmann, and H. G. Burgert. 2003. Two distinct transport motifs in the adenovirus E3/10.4-14.5 proteins act in concert to down-modulate apoptosis receptors and the epidermal growth factor receptor. J. Biol. Chem. 278:51872-51884.
Hoffmann, E., O. Dittrich-Breiholz, H. Holtmann, and M. Kracht. 2002. Multiple control of interleukin-8 gene expression. J. Leukoc. Biol. 72:847-855.
Horton, T. M., T. S. Ranheim, L. Aquino, D. I. Kusher, S. K. Saha, C. F. Ware, W. S. Wold, and L. R. Gooding. 1991. Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis. J. Virol. 65:2629-2639.
Horwitz, M. S. 2001. Adenoviruses, p. 2301-2326. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.
Horwitz, M. S. 2004. Function of adenovirus E3 proteins and their interactions with immunoregulatory cell proteins. J. Gene Med. 6(Suppl. 1):S172-S183.
Hua, L. L., and S. C. Lee. 2000. Distinct patterns of stimulus-inducible chemokine mRNA accumulation in human fetal astrocytes and microglia. Glia 30:74-81.
Huang, Q., J. Yang, Y. Lin, C. Walker, J. Cheng, Z. G. Liu, and B. Su. 2004. Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3. Nat. Immunol. 5:98-103.
Imperiale, M. J., and S. Kochanek. 2004. Adenovirus vectors: biology, design, and production. Curr. Top. Microbiol. Immunol. 273:335-357.
Janssens, S., K. Burns, J. Tschopp, and R. Beyaert. 2002. Regulation of interleukin-1- and lipopolysaccharide-induced NF-B activation by alternative splicing of MyD88. Curr. Biol. 12:467-471.
Jefferies, C. A., S. Doyle, C. Brunner, A. Dunne, E. Brint, C. Wietek, E. Walch, T. Wirth, and L. A. O'Neill. 2003. Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor B activation by Toll-like receptor 4. J. Biol. Chem. 278:26258-26264.
John, G. R., S. C. Lee, X. Song, M. Rivieccio, and C. F. Brosnan. 2005. IL-1-regulated responses in astrocytes: relevance to injury and recovery. Glia 49:161-176.
Kojaoghlanian, T., P. Flomenberg, and M. S. Horwitz. 2003. The impact of adenovirus infection on the immunocompromised host. Rev. Med. Virol. 13:155-171.
Krajcsi, P., T. Dimitrov, T. W. Hermiston, A. E. Tollefson, T. S. Ranheim, S. B. Vande Pol, A. H. Stephenson, and W. S. Wold. 1996. The adenovirus E3-14.7K protein and the E3-10.4K/14.5K complex of proteins, which independently inhibit tumor necrosis factor (TNF)-induced apoptosis, also independently inhibit TNF-induced release of arachidonic acid. J. Virol. 70:4904-4913.
Krappmann, D., A. Patke, V. Heissmeyer, and C. Scheidereit. 2001. B-cell receptor- and phorbol ester-induced NF-B and c-Jun N-terminal kinase activation in B cells requires novel protein kinase C's. Mol. Cell. Biol. 21:6640-6650.
Lesokhin, A. M., F. Delgado-Lopez, and M. S. Horwitz. 2002. Inhibition of chemokine expression by adenovirus early region three (E3) genes. J. Virol. 76:8236-8243.
Li, X., and J. Qin. 2005. Modulation of Toll-interleukin 1 receptor mediated signaling. J. Mol. Med. 83:258-266.
Li, Y., J. Kang, J. Friedman, L. Tarassishin, J. Ye, A. Kovalenko, D. Wallach, and M. S. Horwitz. 1999. Identification of a cell protein (FIP-3) as a modulator of NF-B activity and as a target of an adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis. Proc. Natl. Acad. Sci. USA 96:1042-1047.
Lichtenstein, D. L., P. Krajcsi, D. J. Esteban, A. E. Tollefson, and W. S. Wold. 2002. Adenovirus RID subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis. J. Virol. 76:11329-11342.
Lichtenstein, D. L., K. Toth, K. Doronin, A. E. Tollefson, and W. S. Wold. 2004. Functions and mechanisms of action of the adenovirus E3 proteins. Int. Rev. Immunol. 23:75-111.
Lybarger, L., X. Wang, M. Harris, and T. H. Hansen. 2005. Viral immune evasion molecules attack the ER peptide-loading complex and exploit ER-associated degradation pathways. Curr. Opin. Immunol. 17:71-78.
Molinete, M., S. Dupuis, F. M. Brodsky, and P. A. Halban. 2001. Role of clathrin in the regulated secretory pathway of pancreatic -cells. J. Cell Sci. 114:3059-3066.
Morrison, T. E., A. Mauser, A. Klingelhutz, and S. C. Kenney. 2004. Epstein-Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor alpha-induced signaling and apoptosis by downregulating tumor necrosis factor receptor 1. J. Virol. 78:544-549.
Murphy, P. M. 2001. Viral exploitation and subversion of the immune system through chemokine mimicry. Nat. Immunol. 2:116-122.
Muruve, D. A., M. J. Barnes, I. E. Stillman, and T. A. Libermann. 1999. Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo. Hum. Gene Ther. 10:965-976.
Muta, T., and K. Takeshige. 2001. Essential roles of CD14 and lipopolysaccharide-binding protein for activation of toll-like receptor (TLR)2 as well as TLR4 reconstitution of TLR2- and TLR4-activation by distinguishable ligands in LPS preparations. Eur. J. Biochem. 268:4580-4589.
Naniche, D., and M. B. Oldstone. 2000. Generalized immunosuppression: how viruses undermine the immune response. Cell. Mol. Life Sci. 57:1399-1407.
Pang, Y., Z. Cai, and P. G. Rhodes. 2001. Analysis of genes differentially expressed in astrocytes stimulated with lipopolysaccharide using cDNA arrays. Brain Res. 914:15-22.
Park, K. J., S. H. Choi, S. Y. Lee, S. B. Hwang, and M. M. Lai. 2002. Nonstructural 5A protein of hepatitis C virus modulates tumor necrosis factor alpha-stimulated nuclear factor kappa B activation. J. Biol. Chem. 277:13122-13128.
Parri, R., and V. Crunelli. 2003. An astrocyte bridge from synapse to blood flow. Nat. Neurosci. 6:5-6.
Rezaie, P., G. Trillo-Pazos, I. P. Everall, and D. K. Male. 2002. Expression of beta-chemokines and chemokine receptors in human fetal astrocyte and microglial co-cultures: potential role of chemokines in the developing CNS. Glia 37:64-75.
Roebuck, K. A., L. R. Carpenter, V. Lakshminarayanan, S. M. Page, J. N. Moy, and L. L. Thomas. 1999. Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-B. J. Leukoc. Biol. 65:291-298.
Schwamborn, J., A. Lindecke, M. Elvers, V. Horejschi, M. Kerick, M. Rafigh, J. Pfeiffer, M. Prullage, B. Kaltschmidt, and C. Kaltschmidt. 2003. Microarray analysis of tumor necrosis factor alpha induced gene expression in U373 human glioblastoma cells. BMC Genomics 4:46. [Online.] http://www.biomedcentral.com/1471-2164/4/46.
Shisler, J., C. Yang, B. Walter, C. F. Ware, and L. R. Gooding. 1997. The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis. J. Virol. 71:8299-8306.
Sonoda, Y., T. Kasahara, Y. Yamaguchi, K. Kuno, K. Matsushima, and N. Mukaida. 1997. Stimulation of interleukin-8 production by okadaic acid and vanadate in a human promyelocyte cell line, an HL-60 subline. Possible role of mitogen-activated protein kinase on the okadaic acid-induced NF-B activation. J. Biol. Chem. 272:15366-15372.
Speth, C., M. P. Dierich, and S. Sopper. 2005. HIV-infection of the central nervous system: the tightrope walk of innate immunity. Mol. Immunol. 42:213-228.
Stewart, A. R., A. E. Tollefson, P. Krajcsi, S. P. Yei, and W. S. Wold. 1995. The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane. J. Virol. 69:172-181.
Tanaka, C., H. Kamata, H. Takeshita, H. Yagisawa, and H. Hirata. 1997. Redox regulation of lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) gene expression mediated by NF kappa B and AP-1 in human astrocytoma U373 cells. Biochem. Biophys. Res. Commun. 232:568-573.
Tollefson, A. E., A. R. Stewart, S. P. Yei, S. K. Saha, and W. S. Wold. 1991. The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus form a complex and function together to down-regulate the epidermal growth factor receptor. J. Virol. 65:3095-3105.
Tollefson, A. E., K. Toth, K. Doronin, M. Kuppuswamy, O. A. Doronina, D. L. Lichtenstein, T. W. Hermiston, C. A. Smith, and W. S. Wold. 2001. Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins. J. Virol. 75:8875-8887.
Toth, K., K. Doronin, M. Kuppuswamy, P. Ward, A. E. Tollefson, and W. S. Wold. 2005. Adenovirus immunoregulatory E3 proteins prolong transplants of human cells in immunocompetent mice. Virus Res. 108:149-159.
Toth, K., M. Kuppuswamy, K. Doronin, O. Doronina, D. Lichtenstein, A. Tollefson, and W. Wold. 2002. Construction and characterization of E1-minus replication-defective adenovirus vectors that express E3 proteins from the E1 region. Virology 301:99-108.
von Herrath, M. G., S. Efrat, M. B. Oldstone, and M. S. Horwitz. 1997. Expression of adenoviral E3 transgenes in beta cells prevents autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94:9808-9813.
Vossen, M. T., E. M. Westerhout, C. Soderberg-Naucler, and E. J. Wiertz. 2002. Viral immune evasion: a masterpiece of evolution. Immunogenetics 54:527-542.
Wang, R., J. Stephens, and M. J. Lacy. 2003. Characterization of monoclonal antibody HTA125 with specificity for human TLR4. Hyb. Hybridomics 22:357-365.
Windheim, M., A. Hilgendorf, and H. G. Burgert. 2004. Immune evasion by adenovirus E3 proteins: exploitation of intracellular trafficking pathways. Curr. Top. Microbiol. Immunol. 273:29-85.(Fernando Delgado-Lopez an)
Department of Pediatrics, Division of Infectious Diseases, Albert Einstein College of Medicine, Bronx, New York 10461
ABSTRACT
The transmembrane heterotrimer complex 10.4K/14.5K, also known as RID (for "receptor internalization and degradation"), is encoded by the adenovirus E3 region, and it down-regulates the cell surface expression of several unrelated receptors. We recently showed that RID expression correlates with down-regulation of the cell surface expression of the tumor necrosis factor (TNF) receptor 1 in several human cells. This observation provided the first mechanistic explanation for the inhibition of TNF alpha-induced chemokines by RID. Here we analyze the immunoregulatory activities of RID on lipopolysaccharide (LPS) and interleukin-1 beta (IL-1)-mediated responses. Although both signaling pathways are strongly inhibited by RID, the chemokines up-regulated by IL-1 stimulation are only marginally inhibited. In addition, RID inhibits signaling induced by LPS without affecting the expression of the LPS receptor Toll-like receptor 4, demonstrating that RID need not target degradation of the receptor to alter signal transduction. Taken together, our data demonstrate the inhibitory effect of RID on two additional cell surface receptor-mediated signaling pathways involved in inflammatory processes. The data suggest that RID has intracellular targets that impair signal transduction and chemokine expression without evidence of receptor down-regulation.
INTRODUCTION
For their survival, most human viruses have developed diverse strategies to overcome host defenses, including the inflammatory responses of the innate immune system (49, 55, 72). Some viruses have developed antiapoptotic functions: human herpes simplex virus type 1, for example, expresses at least seven proteins capable of modulating apoptosis in herpes simplex virus-infected cells (4). Other viruses inhibit specific signaling pathways by coding for soluble receptor mimics that block specific receptors by competing with their ligands (2, 52) or by down-regulating the expression of cell surface receptors. Components of the tumor necrosis factor alpha (TNF-) signal transduction pathway are a recurrent target for viral immune evasion because of their central roles in mounting innate and adaptive immune responses (8, 9). For instance, TNF- signaling is inhibited by the hepatitis C viral protein NS5A that binds TRAF2 (57); human cytomegalovirus infection down-regulates TNF receptor 1 (TNFR1) cell surface expression by an unknown mechanism in human monocytic and U373 astrocytoma cell lines (5); the Epstein-Barr viral protein BZLF1 represses directly the TNFR1 promoter (51); and poxvirus N1L protein blocks TNF and interleukin-1 (IL-1)-Toll signaling mainly by binding to TBK1 (22).
Adenoviruses (Ads) are important human pathogens (33) and have been used extensively as vectors for gene transduction and gene therapy (37). Their versatility has also made them candidate vectors for generating vaccines against various pathogens (15). After natural infection, some Ad serotypes persist in tonsillar tissue for years (41). Ads have thus developed several mechanisms to circumvent the host strategies of viral elimination. Most of the viral proteins involved in immunoregulatory functions are coded by the early expression cassette E3, from which at least seven proteins are expressed (for recent reviews, see references 27, 34, 48, and 74). For instance, gp19K blocks major histocompatibility complex class I antigen presentation by sequestering it in the endoplasmic reticulum (16). The 14.7K protein inhibits apoptosis-induced by TNF- in murine cells by unknown mechanisms (28, 32), and it interacts with four unrelated cellular proteins, as shown by yeast two-hybrid experiments (46). The 10.4-14.5K complex, also known as RID (for "receptor internalization and degradation"), blocks TNF--induced cytolysis (65), inhibits release of arachidonic acid induced by TNF- in murine cells (42), and down-regulates the cell surface expression of FAS (25, 62), TRAIL-R1 and -R2, all members of the TNFR superfamily (10, 68), as well as epidermal growth factor receptor (67), in several cell lines. A tyrosine-sorting motif (YXX) in RID (14.5K) was shown to be essential for FAS- and TRAIL-R1-induced internalization (47). In addition, a cytosolic dileucine transport motif in the RID subunit (10.4K) is essential for the function of RID in FAS, TRAIL-R1, and epidermal growth factor receptor cell surface down-regulation in human cell lines (30). Although there have been contradictory reports on the effect of RID on TNFR1 levels, we recently demonstrated a pronounced decrease in cell surface expression of TNFR1 in human 293 and HeLa cells (26). Furthermore, our recent data suggest that RID associates with TNFR1 at the cell surface and causes it to be internalized by a clathrin-mediated mechanism (19).
We had previously demonstrated the anti-inflammatory properties of the AdE3 region in vivo in two different murine models. First, we showed that transgenic expression of E3 genes in pancreatic islets prolonged the survival of the islets transplanted under the kidney capsule of allogeneic recipients (24). Second, in the lymphocytic choriomeningitis virus mouse model of autoimmune diabetes, the transgenic expression of E3 in islets, directed by a tissue-specific promoter (rat insulin promoter), prevented the onset of diabetes induced by infection with lymphocytic choriomeningitis virus (71). Similarly, transgenic expression of E3 in islets of NOD mice delayed the onset of autoimmune diabetes (24). In both cases, the analysis of histological specimens showed a substantial decrease in mononuclear cell infiltration surrounding the islets expressing the E3 cassette. These observations indicated that E3 expression could affect the process that drives monocytic cells to the allogeneic transplant or to the target of autoimmunity. A recent report also showed that infection of a human cell line with an adenovirus vector expressing only AdE3 genes prevented its rejection when xenotransplanted to immunocompetent mice (69).
At the site of an injury or infection, the early phase of inflammation entails the activation of signal transduction pathways mediated by cytokines, chemokines, and derived bacterial components. Increased levels of proinflammatory cytokines such as TNF- and IL-1 are hallmarks of an inflammatory response. TNF-, a proinflammatory stimulus, is produced in response to injury or infection mainly by macrophages and NK cells as well as infiltrating T lymphocytes. IL-1, also a proinflammatory stimulus, is also produced in response to injury or infection primarily by macrophages and epithelial cells. One potent inducer of these cytokines is lipopolysaccharide (LPS) or endotoxin, a component of the cell wall of gram-negative bacteria. A common downstream target activated by proinflammatory cytokines as well as LPS is the transcription factor NF-B (11, 29), which is involved in a vast array of intracellular processes in almost all cell types. AP-1 and p38 are other ubiquitous transcription factors stimulated by proinflammatory signals in some cells (36).
The main cell types that respond to injury and inflammation in the brain are part of the glia, a supportive network constituted of astrocytes, microglia, and oligodendroglia. Astrocytes have diverse functions in the brain; they represent the most abundant glial constituent and play a central role in maintaining local tissue homeostasis. The proinflammatory program of astrocytes has been extensively characterized in studies focusing on specific downstream targets as well as more global transcriptional changes by microarray analysis (40, 56, 61). Astrocytes can be activated by proinflammatory stimuli, and one of the outcomes is up-regulation of chemokine production (58, 59, 64). Chemokines are small secretable proteins whose function is to direct immune effector cells to the site of inflammation (21). Astrocytes respond to cytokines such as TNF- and IL-1 but are also highly responsive to LPS (7, 14, 35). Thus, the human astrocytoma cell line U373 provides an excellent model for studying the effects of RID upon stimulation with these ligands (44, 66). In a previous report, we demonstrated that transduction of U373 cells with the entire E3 cassette prevented the expression of TNF--induced chemokines such as monocyte chemoattractant protein (MCP-1) and IL-8 (44). We subsequently showed that this activity maps to RID, and here we demonstrate that RID mediates this activity by down-regulating cell surface levels of the TNFR1. In addition, we wanted to test whether proinflammatory cascades triggered by other factors such as IL-1 and LPS are affected by RID. Both IL-1 and LPS share significant portions of the signaling pathways downstream of the respective receptors, namely, IL-1R and Toll-like receptor 4 (TLR4), including the adaptor molecules required to stimulate signaling (45). However, we find that RID expression has differential inhibitory effects on IL-1R and TLR4 signaling. In both situations, the activation of NFB and AP-1 is inhibited by RID. In contrast, while the up-regulation of chemokines MCP-1 and IL-8 induced by LPS is abolished by RID, the same chemokines up-regulated by IL-1 are only marginally affected. Interestingly, the inhibition of TLR4 signaling by RID does not involve receptor down-regulation on the cell surface, suggesting a novel mechanism of action to achieve a reduction in LPS-mediated inflammation.
(Data in this paper are from a thesis to be submitted by Fernando Delgado-Lopez in partial fulfillment of the requirements for a Ph.D. from the Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.)
MATERIALS AND METHODS
Cells and viruses. The human astrocytoma cell line U373 was grown in alpha minimal essential medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin; the same medium was used for growing the A549 human cell line. HEK293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with FBS and antibiotics. All four cell lines were obtained from the American Type Culture Collection, Rockville, MD. Adenoviral infections were performed in six-well plates with cells at 95% confluence in 1 ml of alpha minimal essential medium supplemented with antibiotics and 10% FBS. Mutant adenoviral vectors expressing, under cytomegalovirus promoter control, RID only, 14.7K protein only, RID plus 14.7K, the entire E3 cassette, or null virus (expressing none of the E3 proteins) were derived from Ad rec700 and kindly provided by W.S. Wold (70). Viruses were added directly to the plate at a concentration of 500 particles/cell (p/cell) (1 PFU equivalent to 50 particles) or at the concentrations indicated. Human recombinant TNF- (R&D Systems) at 10 ng/ml, human recombinant IL-1 (R&D Systems) at 5 ng/ml, or Escherichia coli LPS (Sigma) at 100 ng/ml was added at the times indicated in the corresponding figures. Phorbol myristate acetate (PMA) and okadaic acid were obtained from Biomol.
Virus isolation and purification. Serial dilutions in 300 μl of DMEM were adsorbed to a monolayer of 293 cells growing at about 95% confluence in 60-mm dishes. After an hour, the cells were overlaid with 5 ml of warm 0.9% agar in DMEM and kept in the incubator at 37°C. Fresh medium was added every 2 days, and plaques were isolated after 8 to 15 days postinfection. The plaque-purified virus was expanded in 293 cells. The virus was extracted from approximately 1 x 109 cells. Infected cells were pooled and resuspended in 10 mM TRIS (pH 8.1), lysate was extracted with Freon three times, and the final phase was layered on top of a linear CsCl gradient in 10 mM TRIS (pH 8.1). After 4 h of centrifugation at 23,000 rpm in a SW-28 Beckman rotor, the viral band was isolated and transferred to a fresh CsCl gradient for a second round of centrifugation and isolation. The concentration of the virus was determined by the optical density at 260 nm.
Western blotting. Cells were lysed in 250 μl of electrophoresis loading buffer (50 mM Tris-HCl, 100 mM dithiothreitol, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol), boiled at 100°C for 3 min, and sonicated. Samples of the extracts were separated using SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to a nitrocellulose membrane for 1.5 h in 20 mM Tris (pH 8)-150 mM glycine at room temperature (RT). The membrane was blocked for 30 min at RT in blocking buffer (1x phosphate-buffered saline[PBS], 5% nonfat milk), washed several times with PBS, and incubated overnight at 4°C with the appropriate antibodies diluted in blocking buffer. After several washes the blot was incubated for 1.5 h at RT with horseradish peroxidase-conjugated antibody diluted 1:3,000 in 2%milk:PBS. Proteins were detected with ECL chemoluminescence (PerkinElmer). Polyclonal antibody for RID was raised against the C terminal of RID (CEISYFNLTGGDD) and produced by Genemed Synthesis Inc. Polyclonal antibodies against TNFR1, TLR4, FAS, IB, phospho-c-Jun, and -tubulin were obtained from Santa Cruz Biotechnology. Anti-phosphoserine-536-p65 and anti-phospho-p38 were purchased from Cell Signaling Technologies. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G was purchased from Amersham Biosciences. Biotinylated mouse anti-TNFR1 and its biotinylated isotype control were purchased from BD Bioscience.
RNase protection assays (RPAs). Total RNA was extracted from U373 cells by use of TRIzol reagent (Life Technologies) per the suggested protocol. In brief, the cells were lysed with 1 ml TRIzol reagent on the plates and then mixed with 0.2 ml of chloroform in Eppendorf tubes, shaken by hand, allowed to stand at room temperature for 5 min, and then centrifuged at 10,000 rpm at 4°C for 15 min. The supernatants were collected and precipitated with isopropanol at –80°C for 15 min and centrifuged at 14,000 rpm at 4°C for 25 min. The pellet was washed with 70% ethanol at room temperature, dried, and resuspended in 10 μl of Ambion nuclease-free water. The yield of the RNA was measured by optical density at 260 nm.
RPAs were performed using an RPA III kit (Ambion) and probes labeled using a Maxiscript T7 kit (Ambion). The multiprobe template set hCK-5 (Pharmingen) was transcribed using T7 RNA polymerase with [32P]UTP (Amersham-Pharmacia), phenol chloroform extracted, precipitated with ethanol and ammonium acetate, and resuspended in 70 μl hybridization buffer. Five micrograms of sample RNA was hybridized with 1.5 μl of probe overnight at 44°C. Samples were then digested with RNase A/T1 for 30 min at 37°C. RNase was inactivated, and the samples were precipitated with proprietary buffer and ethanol. Protected samples were resuspended in 1x loading buffer and separated by 5% acrylamide-8 M urea gel electrophoresis. After drying, the gel was exposed to Kodak film (mass spectrometry).
FACS analysis. Cells were placed on ice for 10 min before washing twice with cold PBS and then incubated with 1% BSA for 10 min, followed by a 10 min incubation with FB (2% FBS in PBS), always on ice. Biotin-anti-TNFR antibody was used at 0.5 μg/ml for 30 min, cells were washed three times, and then phycoerythrin-streptavidin (Molecular Probes) was added at 1 μg/ml in FB for 30 min on ice. After three washes with FB, cells were fixed with 2% paraformaldehyde in PBS. Fluorescence-activated cell sorting (FACS) analysis was done in a FACSCalibur machine.
Cell surface biotinylation. U373 cells growing in 10-cm dishes at 90% confluence were infected with 500 p/cell of various adenoviral vectors and incubated for 20 h before labeling. We followed the instructions for use of a cell surface biotinylation kit from Pierce. Essentially, the monolayer was transferred to a bed of ice for 10 min and remained there during the length of the labeling. Cells were washed twice with ice-cold PBS, and 10 ml of EZ-Link Sulfo-NHS-SS-Biotin in PBS was added for 30 min. The reaction was stopped with quenching solution and the reaction mixture was washed three times; cells were then scraped and lysed for 30 min on ice with the provided lysis buffer. The lysate was clarified by centrifugation, and the supernatant was incubated for 1 h at RT with NeutrAvidin gel in a column. The beads were washed several times and finally eluted under reducing conditions with loading buffer. The eluate was concentrated 5x by ultrafiltration, and 25% of the eluate was loaded on an SDS gel.
RESULTS
Proinflammatory stimuli activate NFB and AP-1 pathways and induce the expression of MCP-1 and IL-8 in U373 cells. Four human cell lines were evaluated for responsiveness to four different potent proinflammatory stimuli. Three of these stimuli, TNF-, IL-1, and LPS, mediate their action through cell surface receptors, whereas PMA represents a cell-permeative stimulus with an intracellular site of action. All four factors are known to stimulate NF-B and AP-1 signal transduction. The assay used for testing NF-B activation examined degradation of IB, which normally binds the p50/p65 NF-B heterodimer and prevents its translocation to the nucleus. AP-1 activation was assayed by detecting phosphorylation of c-Jun. The four human cell lines tested were U373, 293, A549, and HeLa, which differ significantly in terms of their response to proinflammatory stimuli as well as in the extent to which AP-1 is activated (Fig. 1A). HeLa cells responded weakly and only to TNF- stimulation, as evidenced by minimal IB degradation and c-Jun phosphorylation. In 293 cells, TNF and PMA elicited a strong response in both NF-B and AP-1 activation. In A549 cells, TNF- and IL-1 stimulation degraded IB efficiently, while c-Jun was only marginally phosphorylated. The astrocytoma cell line U373 is the only cell line—among the four tested—which showed consistent activation of the NF-B as well as AP-1 pathways in response to all four stimuli.
The transcription of many chemokines is regulated by NF-B- and AP-1-responsive elements in the promoters of their genes (31, 60). We used RPA, a quantitative method of measuring steady-state levels of specific mRNAs, to analyze various chemokine transcripts. U373 cells were stimulated with TNF-, IL-1, LPS, and PMA for 4 h. Exposure to all four factors up-regulates IL-8 and MCP-1 messages to similar levels (Fig. 1B).
Expression of RID correlate with efficient suppression of TNF- signaling via down-regulation of TNFR1 in U373 cells. Adenoviruses infect cells at various efficiencies; some cells require very high multiplicities of infection (MOI) before effective transduction is achieved, while others are very permissive. We analyzed the kinetics of RID expression in U373 cells as a function of MOI and time. The kinetics of RID expression after infection with 500 p/cell of AdRID showed that the extent of NF-B and AP-1 inhibition correlated with expression of RID; this inhibition was detected as early as 8 h postinfection (Fig. 2A).
As shown previously in a study using human 293 cells (26), FACS analysis confirmed that RID down-regulates the cell surface expression of TNFR1 in U373 cells and that the levels of cell surface TNFR1 decreased in a manner that is dose dependent on RID expression levels (Fig. 2B). Furthermore, this down-regulation is saturable, since infection at MOIs above 1,000 particles per cell makes RID activity less efficient. A dose-response experiment showed that infection for 20 h with as few as 50 p/cell is sufficient for detection of RID and inhibition of TNF- signaling (Fig. 2C). Infection with AdNull (lacking the E3 domain) had no inhibitory effect. In contrast to cell surface expression results, the total cellular levels of TNFR1 remained the same over the course of the infection.
LPS activation of the NF-B and AP-1 pathways is slower than the rapid activation induced by IL-1 and TNF-. In addition to IB degradation, we utilized p65 phosphorylation to assess NF-B activation (p65 conveys the transactivation activity in the p50/p65 heterodimer complex of NF-B). To measure AP-1 and p38 activation, we analyzed c-Jun phosphorylation and p38 phosphorylation, respectively. Both TNF- and IL-1 showed rapid kinetics of NF-B, AP-1, and p38 activation, reaching a maximum level at 10 min postinitiation of stimulation (Fig. 3A) consistent with previous reports (6). The downstream signaling events mediated by LPS were significantly slower, reaching complete activation only after 60 min of stimulation (Fig. 3B). Infection with AdRID (500 p/cell) for 20 h prior to LPS stimulation completely inhibited the LPS-induced IB degradation as well as c-Jun, p65, and p38 phosphorylation, while infection with AdNull (at the same MOI) had no effect on LPS-stimulated cells. Furthermore, experiments conducted to determine the induction rates of IL-8 and MCP-1 mRNA showed that TNF- and IL-1 up-regulated the expression of chemokines with faster kinetics than did LPS (Fig. 3C).
RID inhibits IL-1-mediated signaling. Although the receptors and upstream adaptors of the TNF- and IL-1 pathways are different, they both lead to the activation of the canonical NF-B and AP-1 pathways. While AdNull or Ad14.7K-expressing viruses did not have any effect on either prevention of IB degradation or phosphorylation of c-Jun, p65, or p38 induced by either TNF- or IL-1, AdRID infection prevented IB degradation and c-Jun phosphorylation induced by the same cytokines. Although RID efficiently inhibited p65 and p38 phosphorylation induced by TNF-, it only partially inhibited the phosphorylation of p65 or p38 induced by IL-1 (Fig. 4).
Non-receptor-mediated activation of NF-B and AP-1 by PMA or okadaic acid is not affected by RID. The activation of NFB by phorbol esters and okadaic acid has been previously described (43, 63). These small cell-permeative molecules activate signaling without the mediation of a cell surface receptor. PMA activates protein kinase C (20), while okadaic acid inhibits the PP2 family members of serine/threonine phosphatases (23). We found that RID does not have an impact on PMA- or okadaic acid-mediated activation of the NFB and AP-1 pathways, as judged by the fact that IB degradation and c-Jun phosphorylation are unaffected (Fig. 5). This supports the notion that the target for RID action is a receptor proximal to PMA and okadaic acid action.
RID strongly inhibits induction of chemokines by LPS and TNF- but not that by IL-1. Transduction of U373 cells with the entire AdE3 cassette inhibits chemokines induced by TNF- stimulation (44). Further mapping studies of the astrocytoma cell line as well as other human cell lines showed that this effect was specific to RID (data not shown). The E3 cassette and the mutant vectors expressing individual E3 proteins were also evaluated in the context of IL-1, LPS, or PMA stimulation. In contrast to the robust inhibition by RID of signaling and chemokine expression triggered by TNF- and LPS and the significant inhibition of IB degradation and c-Jun activation triggered by IL-1, the chemokines up-regulated by IL-1 were not affected to the same extent (Fig. 6). Specifically, while IL-8 and MCP-1 messages were completely suppressed by RID after TNF- or LPS stimulation, the same chemokines, when stimulated by IL-1, were only marginally inhibited. On the other hand, the fact that RID significantly disrupts IL-1 signaling leading to NFB and AP-1 activation but only marginally affects chemokine expression suggests that the cell surface expression of IL-1R is not affected.
The differential effects of RID on three proinflammatory challenges are independent of each other. There are several alternative explanations for the observed differential effects of RID, the most likely being that IL-1 activates pathways in addition to NFB and AP-1 that can lead to chemokine synthesis, especially since the inhibition of IL-1 signaling by RID is not complete (Fig. 4A). However, it is also possible that RID targets only one molecule or protein complex common to the receptors for those three ligands. This component could be more accessible after TNFR1 or TLR4 stimulation compared to its accessibility after IL-1R stimulation. To test this, we simultaneously stimulated U373 cells with various combinations of TNF-, IL-1, and LPS and compared the effects of RID under these circumstances (Fig. 7). In order to appreciate the slight effect of RID on IL-1-induced chemokine expression, gels were underexposed (20 min). As shown in Fig. 7, every time the cells were exposed to IL-1 stimulation, RID was unable to completely inhibit chemokine expression. This indicates that IL-1 has other mechanisms, unaffected by RID, to signal induction of chemokine expression.
Biotinylated human TLR4 is not down-regulated from the cell surface by RID. Despite the availability of a few commercial antibodies against TLR4, we were unable to assess the endogenous levels of this receptor in astrocytoma cells by FACS analysis, probably because of low levels of TLR4 expression on these cells or low affinity of the antibodies (73). Therefore, we used a non-cell-permeative cross-linker to biotinylate and isolate the cell surface proteins by affinity chromatography for Western blotting experiments with specific antibodies. As shown in Fig. 8, cell surface expression of TLR4 is not affected by RID. We measured cell surface expression of FAS, a molecule known to be down-regulated by RID, as a positive control. In addition, RID was also biotinylated and isolated by the affinity protocol (Fig. 8).
DISCUSSION
There is increasing evidence that TLRs play crucial roles in diverse pathogenic scenarios, including sepsis, asthma, transplant rejection, and several inflammatory diseases (3). Therefore, the identification of novel inhibitors of these signaling pathways will have direct clinical implications. Here, we provide evidence that the adenovirus-derived RID can block signaling induced by LPS, the ligand of TLR4, as well as signaling induced by IL-1. Because the intracellular domains of TLR4 and the IL-1R are highly homologous and share many downstream effectors, it was expected that RID would inhibit IL-1 and LPS signaling comparably. However, we found significant variations between the two. While RID is able to inhibit equivalently IB degradation and c-Jun phosphorylation after IL-1 or LPS stimulation, the expression of IL-8 and MCP-1 chemokines induced by LPS is completely abrogated by RID but only marginally inhibited following IL-1 stimulation. The observation that RID does inhibit IB degradation but does not completely inhibit phosphorylation of p65 induced by IL-1 may be explained by the fact that p65 can be phosphorylated by kinases in pathways other than the classical NF-B pathway. Its activity can also be modulated by phosphorylations at other serine residues as well as acetylation of lysine residues (18). Likewise, the versatility of the ways in which p65 activity is modified may help explain the small effect that RID has on the chemokine expression induced by IL-1. Because RID expression down-regulates the cell surface expression of several receptors, it seemed likely that the mechanism for inhibiting LPS-induced signaling and chemokine expression might involve the cell surface down-regulation of TLR4. However, cell surface biotinylation experiments showed that RID does not down-regulate the cell surface expression of TLR4. The fact that IL-1-induced chemokines are only marginally affected by RID provides indirect supporting evidence that the cell surface expression of the IL-1 receptor is also not targeted by RID.
One possibility to be considered is whether U373 cells have mechanisms of response to LPS that do not involve TLR4 signaling. For example, an alternative LPS receptor might be down-regulated by RID. Others have shown that in U373 cells, LPS transduces signaling only through TLR4, specifically through Bruton's tyrosine kinase, as an essential component of the signal transduction machinery (39). However, since there is a report showing that LPS can also signal through TLR2 (54), we did stimulate the cells with zymosan (a known ligand for TLR2) and observed neither NF-B and AP-1 activation nor chemokine up-regulation (data not shown). In addition, we also determined that TLR2 cell surface levels are not affected by RID (data not shown).
Adenoviruses have been reported to trigger NF-B activation and chemokine production (12, 13, 53). These observations were made in the context of utilizing adenovirus as a vector for gene therapy, using very high titers of viral vectors that lack RID (most adenoviral vectors have the whole E3 cassette deleted). Under these conditions, structural components of viral capsids may induce the production of proinflammatory molecules. In our experiments, relatively low viral titers were used (in general, 500 p/cell, equivalent to 10 PFU/cell), conditions in which activation of NF-B or chemokine production is not induced by the virus itself. Likewise, we did not observe any change in cell viability or morphology under any of our conditions. In continued use of adenoviruses as vector platforms for gene delivery, it will be important to assess viral input carefully to minimize the intrinsic proinflammatory properties of the vector. It might also be beneficial to include RID (or some component of RID) in the vectors as a means to diminish the intrinsic proinflammatory characteristics of the vectors.
When combinations of the various proinflammatory challenges were tested, the levels of inhibition by RID were not altered in comparisons of its effects after stimulation with one stimulus to those resulting from stimulation with a mixture of them, which implies that RID blocks signaling from the three stimuli by targeting each of the individual pathways in a specific manner. Several strategies have been used to identify the molecular targets of RID. By comparing human astrocytoma cell lines that either overexpress or are deficient in caveolin-1, we determined that RID does not require caveolin-1 for its inhibitory effects (F. Delgado-Lopez and M. Horwitz, unpublished data). In addition, disrupting clathrin-mediated endocytosis by overexpressing a dominant-negative form of the clathrin heavy chain (50) did not affect the ability of RID to down-regulate chemokine expression (Delgado-Lopez and Horwitz, unpublished). However, in 293 cells the μ2 subunit of adaptor protein 2 is involved in clathrin-dependent TNFR1 down-regulation by RID (19), a distinction that could be explained by cell type differences. Here it is pertinent to note that 293 cells do not express significant levels of chemokines regardless of the stimuli, presumably because this cell line is transformed by the adenovirus E1 expression cassette (1). The targets of RID, other than cell surface receptors, are likely to be upstream of the IB kinase (IKK) complex, a nodal point where several kinases—activated by diverse stimuli—converge to signal IKK to phosphorylate IB, a reaction that signals IB for degradation. This notion is supported by the inability of RID to block the signaling at the level of IB in response to PMA and okadaic acid. On the other hand, upstream of IKK, the forced expression of the adaptor molecule MyD88 inhibits IL-1- and LPS-induced NFB activation by preventing IRAK-1 phosphorylation by IRAK-4 (17, 38). RID, therefore, probably does not function at the level of MyD88, since it does not inhibit equivalently IL-1 and LPS signaling. Furthermore, since TAK-1, a mitogen-activated protein kinase kinase kinase, is the molecule in the IL-1 and LPS pathways where the p38 signaling and the AP-1 signaling diverge, it is possible that RID may be working downstream of TAK-1.
In conclusion, the data presented here indicate that RID targets molecules other than cell surface receptors, and this implies there are novel mechanisms for RID function yet to be resolved. Further mapping of pathway components intersecting with the RID complex should provide important information on specificity that could facilitate the use of RID for therapeutic purposes.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grant 1PO1DK52956.
Special gratitude is extended to Todd Evans, Tsoline Kojaoghlanian, and Philipp Scherer for the time they spent reviewing the manuscript.
Dedicated to the memory of Marshall S. Horwitz.
Deceased.
REFERENCES
Aiello, L., R. Guilfoyle, K. Huebner, and R. Weinmann. 1979. Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad-5 or 293). Virology 94:460-469.
Alcami, A. 2003. Viral mimicry of cytokines, chemokines and their receptors. Nat. Rev. Immunol. 3:36-50.
Andreakos, E., B. Foxwell, and M. Feldmann. 2004. Is targeting Toll-like receptors and their signaling pathway a useful therapeutic approach to modulating cytokine-driven inflammation Immunol. Rev. 202:250-265.
Aubert, M., and J. A. Blaho. 2001. Modulation of apoptosis during herpes simplex virus infection in human cells. Microbes Infect. 3:859-866.
Baillie, J., D. A. Sahlender, and J. H. Sinclair. 2003. Human cytomegalovirus infection inhibits tumor necrosis factor alpha (TNF-) signaling by targeting the 55-kilodalton TNF- receptor. J. Virol. 77:7007-7016.
Barchowsky, A., D. Frleta, and M. P. Vincenti. 2000. Integration of the NF-B and mitogen-activated protein kinase/AP-1 pathways at the collagenase-1 promoter: divergence of IL-1 and TNF-dependent signal transduction in rabbit primary synovial fibroblasts. Cytokine 12:1469-1479.
Basu, A., J. K. Krady, and S. W. Levison. 2004. Interleukin-1: a master regulator of neuroinflammation. J. Neurosci. Res. 78:151-156.
Benedict, C. A. 2003. Viruses and the TNF-related cytokines, an evolving battle. Cytokine Growth Factor Rev. 14:349-357.
Benedict, C. A., T. A. Banks, and C. F. Ware. 2003. Death and survival: viral regulation of TNF signaling pathways. Curr. Opin. Immunol. 15:59-65.
Benedict, C. A., P. S. Norris, T. I. Prigozy, J. L. Bodmer, J. A. Mahr, C. T. Garnett, F. Martinon, J. Tschopp, L. R. Gooding, and C. F. Ware. 2001. Three adenovirus E3 proteins cooperate to evade apoptosis by tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2. J. Biol. Chem. 276:3270-3278.
Bonizzi, G., and M. Karin. 2004. The two NF-B activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25:280-288.
Borgland, S. L., G. P. Bowen, N. C. Wong, T. A. Libermann, and D. A. Muruve. 2000. Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-B. J. Virol. 74:3941-3947.
Bowen, G. P., S. L. Borgland, M. Lam, T. A. Libermann, N. C. Wong, and D. A. Muruve. 2002. Adenovirus vector-induced inflammation: capsid-dependent induction of the C-C chemokine RANTES requires NF-kappa B. Hum. Gene Ther. 13:367-379.
Bowman, C. C., A. Rasley, S. L. Tranguch, and I. Marriott. 2003. Cultured astrocytes express toll-like receptors for bacterial products. Glia 43:281-291.
Boyer, J. L., G. Kobinger, J. M. Wilson, and R. G. Crystal. 2005. Adenovirus-based genetic vaccines for biodefense. Hum. Gene Ther. 16:157-168.
Burgert, H. G., and S. Kvist. 1985. An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell 41:987-997.
Burns, K., S. Janssens, B. Brissoni, N. Olivos, R. Beyaert, and J. Tschopp. 2003. Inhibition of interleukin 1 receptor/Toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4. J. Exp. Med. 197:263-268.
Chen, L. F., and W. C. Greene. 2004. Shaping the nuclear action of NF-B. Nat. Rev. Mol. Cell Biol. 5:392-401.
Chin, Y. R., and M. S. Horwitz. 2005. Mechanism for removal of tumor necrosis factor receptor 1 from the cell surface by the adenovirus RID/ complex. J. Virol. 79:13606-13617.
Crabos, M., R. Imber, T. Woodtli, D. Fabbro, and P. Erne. 1991. Different translocation of three distinct PKC isoforms with tumor-promoting phorbol ester in human platelets. Biochem. Biophys. Res. Commun. 178:878-883.
Cyster, J. G. 2005. Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu. Rev. Immunol. 23:127-159.
DiPerna, G., J. Stack, A. G. Bowie, A. Boyd, G. Kotwal, Z. Zhang, S. Arvikar, E. Latz, K. A. Fitzgerald, and W. L. Marshall. 2004. Poxvirus protein N1L targets the I-B kinase complex, inhibits signaling to NF-B by the tumor necrosis factor superfamily of receptors, and inhibits NF-B and IRF3 signaling by toll-like receptors. J. Biol. Chem. 279:36570-36578.
Dounay, A. B., and C. J. Forsyth. 2002. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor. Curr. Med. Chem. 9:1939-1980.
Efrat, S., G. Fejer, M. Brownlee, and M. S. Horwitz. 1995. Prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes. Proc. Natl. Acad. Sci. USA 92:6947-6951.
Elsing, A., and H. G. Burgert. 1998. The adenovirus E3/10.4K-14.5K proteins down-modulate the apoptosis receptor Fas/Apo-1 by inducing its internalization. Proc. Natl. Acad. Sci. USA 95:10072-10077.
Fessler, S. P., Y. R. Chin, and M. S. Horwitz. 2004. Inhibition of tumor necrosis factor (TNF) signal transduction by the adenovirus group C RID complex involves downregulation of surface levels of TNF receptor 1. J. Virol. 78:13113-13121.
Fessler, S. P., F. Delgado-Lopez, and M. S. Horwitz. 2004. Mechanisms of E3 modulation of immune and inflammatory responses. Curr. Top. Microbiol. Immunol. 273:113-135.
Gooding, L. R., I. O. Sofola, A. E. Tollefson, P. Duerksen-Hughes, and W. S. Wold. 1990. The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis. J. Immunol. 145:3080-3086.
Hayden, M. S., and S. Ghosh. 2004. Signaling to NF-B. Genes Dev. 18:2195-2224.
Hilgendorf, A., J. Lindberg, Z. Ruzsics, S. Honing, A. Elsing, M. Lofqvist, H. Engelmann, and H. G. Burgert. 2003. Two distinct transport motifs in the adenovirus E3/10.4-14.5 proteins act in concert to down-modulate apoptosis receptors and the epidermal growth factor receptor. J. Biol. Chem. 278:51872-51884.
Hoffmann, E., O. Dittrich-Breiholz, H. Holtmann, and M. Kracht. 2002. Multiple control of interleukin-8 gene expression. J. Leukoc. Biol. 72:847-855.
Horton, T. M., T. S. Ranheim, L. Aquino, D. I. Kusher, S. K. Saha, C. F. Ware, W. S. Wold, and L. R. Gooding. 1991. Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis. J. Virol. 65:2629-2639.
Horwitz, M. S. 2001. Adenoviruses, p. 2301-2326. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.
Horwitz, M. S. 2004. Function of adenovirus E3 proteins and their interactions with immunoregulatory cell proteins. J. Gene Med. 6(Suppl. 1):S172-S183.
Hua, L. L., and S. C. Lee. 2000. Distinct patterns of stimulus-inducible chemokine mRNA accumulation in human fetal astrocytes and microglia. Glia 30:74-81.
Huang, Q., J. Yang, Y. Lin, C. Walker, J. Cheng, Z. G. Liu, and B. Su. 2004. Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3. Nat. Immunol. 5:98-103.
Imperiale, M. J., and S. Kochanek. 2004. Adenovirus vectors: biology, design, and production. Curr. Top. Microbiol. Immunol. 273:335-357.
Janssens, S., K. Burns, J. Tschopp, and R. Beyaert. 2002. Regulation of interleukin-1- and lipopolysaccharide-induced NF-B activation by alternative splicing of MyD88. Curr. Biol. 12:467-471.
Jefferies, C. A., S. Doyle, C. Brunner, A. Dunne, E. Brint, C. Wietek, E. Walch, T. Wirth, and L. A. O'Neill. 2003. Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor B activation by Toll-like receptor 4. J. Biol. Chem. 278:26258-26264.
John, G. R., S. C. Lee, X. Song, M. Rivieccio, and C. F. Brosnan. 2005. IL-1-regulated responses in astrocytes: relevance to injury and recovery. Glia 49:161-176.
Kojaoghlanian, T., P. Flomenberg, and M. S. Horwitz. 2003. The impact of adenovirus infection on the immunocompromised host. Rev. Med. Virol. 13:155-171.
Krajcsi, P., T. Dimitrov, T. W. Hermiston, A. E. Tollefson, T. S. Ranheim, S. B. Vande Pol, A. H. Stephenson, and W. S. Wold. 1996. The adenovirus E3-14.7K protein and the E3-10.4K/14.5K complex of proteins, which independently inhibit tumor necrosis factor (TNF)-induced apoptosis, also independently inhibit TNF-induced release of arachidonic acid. J. Virol. 70:4904-4913.
Krappmann, D., A. Patke, V. Heissmeyer, and C. Scheidereit. 2001. B-cell receptor- and phorbol ester-induced NF-B and c-Jun N-terminal kinase activation in B cells requires novel protein kinase C's. Mol. Cell. Biol. 21:6640-6650.
Lesokhin, A. M., F. Delgado-Lopez, and M. S. Horwitz. 2002. Inhibition of chemokine expression by adenovirus early region three (E3) genes. J. Virol. 76:8236-8243.
Li, X., and J. Qin. 2005. Modulation of Toll-interleukin 1 receptor mediated signaling. J. Mol. Med. 83:258-266.
Li, Y., J. Kang, J. Friedman, L. Tarassishin, J. Ye, A. Kovalenko, D. Wallach, and M. S. Horwitz. 1999. Identification of a cell protein (FIP-3) as a modulator of NF-B activity and as a target of an adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis. Proc. Natl. Acad. Sci. USA 96:1042-1047.
Lichtenstein, D. L., P. Krajcsi, D. J. Esteban, A. E. Tollefson, and W. S. Wold. 2002. Adenovirus RID subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis. J. Virol. 76:11329-11342.
Lichtenstein, D. L., K. Toth, K. Doronin, A. E. Tollefson, and W. S. Wold. 2004. Functions and mechanisms of action of the adenovirus E3 proteins. Int. Rev. Immunol. 23:75-111.
Lybarger, L., X. Wang, M. Harris, and T. H. Hansen. 2005. Viral immune evasion molecules attack the ER peptide-loading complex and exploit ER-associated degradation pathways. Curr. Opin. Immunol. 17:71-78.
Molinete, M., S. Dupuis, F. M. Brodsky, and P. A. Halban. 2001. Role of clathrin in the regulated secretory pathway of pancreatic -cells. J. Cell Sci. 114:3059-3066.
Morrison, T. E., A. Mauser, A. Klingelhutz, and S. C. Kenney. 2004. Epstein-Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor alpha-induced signaling and apoptosis by downregulating tumor necrosis factor receptor 1. J. Virol. 78:544-549.
Murphy, P. M. 2001. Viral exploitation and subversion of the immune system through chemokine mimicry. Nat. Immunol. 2:116-122.
Muruve, D. A., M. J. Barnes, I. E. Stillman, and T. A. Libermann. 1999. Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo. Hum. Gene Ther. 10:965-976.
Muta, T., and K. Takeshige. 2001. Essential roles of CD14 and lipopolysaccharide-binding protein for activation of toll-like receptor (TLR)2 as well as TLR4 reconstitution of TLR2- and TLR4-activation by distinguishable ligands in LPS preparations. Eur. J. Biochem. 268:4580-4589.
Naniche, D., and M. B. Oldstone. 2000. Generalized immunosuppression: how viruses undermine the immune response. Cell. Mol. Life Sci. 57:1399-1407.
Pang, Y., Z. Cai, and P. G. Rhodes. 2001. Analysis of genes differentially expressed in astrocytes stimulated with lipopolysaccharide using cDNA arrays. Brain Res. 914:15-22.
Park, K. J., S. H. Choi, S. Y. Lee, S. B. Hwang, and M. M. Lai. 2002. Nonstructural 5A protein of hepatitis C virus modulates tumor necrosis factor alpha-stimulated nuclear factor kappa B activation. J. Biol. Chem. 277:13122-13128.
Parri, R., and V. Crunelli. 2003. An astrocyte bridge from synapse to blood flow. Nat. Neurosci. 6:5-6.
Rezaie, P., G. Trillo-Pazos, I. P. Everall, and D. K. Male. 2002. Expression of beta-chemokines and chemokine receptors in human fetal astrocyte and microglial co-cultures: potential role of chemokines in the developing CNS. Glia 37:64-75.
Roebuck, K. A., L. R. Carpenter, V. Lakshminarayanan, S. M. Page, J. N. Moy, and L. L. Thomas. 1999. Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-B. J. Leukoc. Biol. 65:291-298.
Schwamborn, J., A. Lindecke, M. Elvers, V. Horejschi, M. Kerick, M. Rafigh, J. Pfeiffer, M. Prullage, B. Kaltschmidt, and C. Kaltschmidt. 2003. Microarray analysis of tumor necrosis factor alpha induced gene expression in U373 human glioblastoma cells. BMC Genomics 4:46. [Online.] http://www.biomedcentral.com/1471-2164/4/46.
Shisler, J., C. Yang, B. Walter, C. F. Ware, and L. R. Gooding. 1997. The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis. J. Virol. 71:8299-8306.
Sonoda, Y., T. Kasahara, Y. Yamaguchi, K. Kuno, K. Matsushima, and N. Mukaida. 1997. Stimulation of interleukin-8 production by okadaic acid and vanadate in a human promyelocyte cell line, an HL-60 subline. Possible role of mitogen-activated protein kinase on the okadaic acid-induced NF-B activation. J. Biol. Chem. 272:15366-15372.
Speth, C., M. P. Dierich, and S. Sopper. 2005. HIV-infection of the central nervous system: the tightrope walk of innate immunity. Mol. Immunol. 42:213-228.
Stewart, A. R., A. E. Tollefson, P. Krajcsi, S. P. Yei, and W. S. Wold. 1995. The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane. J. Virol. 69:172-181.
Tanaka, C., H. Kamata, H. Takeshita, H. Yagisawa, and H. Hirata. 1997. Redox regulation of lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) gene expression mediated by NF kappa B and AP-1 in human astrocytoma U373 cells. Biochem. Biophys. Res. Commun. 232:568-573.
Tollefson, A. E., A. R. Stewart, S. P. Yei, S. K. Saha, and W. S. Wold. 1991. The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus form a complex and function together to down-regulate the epidermal growth factor receptor. J. Virol. 65:3095-3105.
Tollefson, A. E., K. Toth, K. Doronin, M. Kuppuswamy, O. A. Doronina, D. L. Lichtenstein, T. W. Hermiston, C. A. Smith, and W. S. Wold. 2001. Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins. J. Virol. 75:8875-8887.
Toth, K., K. Doronin, M. Kuppuswamy, P. Ward, A. E. Tollefson, and W. S. Wold. 2005. Adenovirus immunoregulatory E3 proteins prolong transplants of human cells in immunocompetent mice. Virus Res. 108:149-159.
Toth, K., M. Kuppuswamy, K. Doronin, O. Doronina, D. Lichtenstein, A. Tollefson, and W. Wold. 2002. Construction and characterization of E1-minus replication-defective adenovirus vectors that express E3 proteins from the E1 region. Virology 301:99-108.
von Herrath, M. G., S. Efrat, M. B. Oldstone, and M. S. Horwitz. 1997. Expression of adenoviral E3 transgenes in beta cells prevents autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94:9808-9813.
Vossen, M. T., E. M. Westerhout, C. Soderberg-Naucler, and E. J. Wiertz. 2002. Viral immune evasion: a masterpiece of evolution. Immunogenetics 54:527-542.
Wang, R., J. Stephens, and M. J. Lacy. 2003. Characterization of monoclonal antibody HTA125 with specificity for human TLR4. Hyb. Hybridomics 22:357-365.
Windheim, M., A. Hilgendorf, and H. G. Burgert. 2004. Immune evasion by adenovirus E3 proteins: exploitation of intracellular trafficking pathways. Curr. Top. Microbiol. Immunol. 273:29-85.(Fernando Delgado-Lopez an)