Explorer Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll - like receptors exhibit a sustained and additive activation state

Background: Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. Results: RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. Conclusions: These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge. Background Mammalian macrophages respond to a wide range of microbial products with induction of genes required for host defence. Amongst these genes is a suite of inducible Published: 12 October 2001 BMC Immunology 2001, 2:11 Received: 20 August 2001 Accepted: 12 October 2001 This article is available from: http://www.biomedcentral.com/1471-2172/2/11 © 2001 Hume et al; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any non-commercial purpose, provided this notice is preserved along with the article's original URL. For commercial use, contact [email protected] BMC Immunology 2001, 2:11 http://www.biomedcentral.com/1471-2172/2/11 cytokines, including IL-1, TNF-α, IL-6 and IL-12, which are required for protective innate and acquired immunity but also mediate much of the pathology of disseminated infections including toxic shock [1]. The archetypal macrophage activating bacterial product is lipopolysaccharide (LPS) or endotoxin. An understanding of the mechanism of action of LPS was greatly expedited by the identification of the Toll-like receptor [TLR] family and the parallels between recognition of bacterial stimuli in mammals and drosophila. Mice with mutations in the TLR4 gene are hypo-sensitive to LPS [2–4]. Activation of TLR4 on the cell surface which requires coooperation with at least two other surface proteins, CD14 and MD2 [5] leads to recruitment of adaptor proteins (MyD88, IRAK, TRAF6) that ultimately couple the recognition of the microbial product to activation of the transcription factor complex NF-κB [Reviewed in [6]]. Translocation of this transcription factor complex in turn contributes to activation of the promoters of many inducible genes in macrophages [1]. Another member of the TLR family, TLR2, is absolutely required for recognition of a range of surface components of gram-positive organisms including bacterial lipopeptides [7]. Apart from bacterial cell wall components, macrophages are able to recognize and respond to bacterial DNA or CpG-containing immunostimulatory oligonculeotides [8,9]. A recent paper indicates that yet another member of Toll-like receptor family, TLR9, is required for optimal recognition of immunostimulatory DNA [10]. The TLR2, TLR4 and TLR9 pathways apparently converge at the level of MyD88, since macrophages from mice with a mutation in this gene are defective in activation by either gram-negative or gram-positive organisms or bacterial DNA [11,12]. One clinically-important feature of the mammalian response to LPS is the induction of tolerance. An animal exposed to a sub-lethal dose of LPS, or to a non-toxic LPS, enters a refractory period during which it can resist lethal LPS exposure. Several groups have attempted to model the phenomenon of endotoxin tolerance in vitro by studying the ability of macrophages exposed to LPS to recognise a secondary stimulus [5,13–22]. In these models, some responses that are measured (e.g. activation of transcription factor NF-κB, TNF-α mRNA production) occur transiently after LPS addition. Whereas TNF-α mRNA is induced transiently in response to LPS and then rapidly degraded [23] and the protein product is produced and secreted in a bolus from the stimulated cells [24], other mRNAs are elevated for as long as LPS is present, up to 48 hrs after LPS addition [23]. Such genes could be induced by autocrine stimuli produced in the initial phase of activation. Alternatively, LPS may act to cause a change of "steady state", and TNF-α induction and other early response genes could be a feature of a transition state that cannot be reactivated until the fully active state is allowed to completely decay. In such a model, the new steady state could have an intrinsic halflife or it could be maintained by continued stimulation. Explanations for tolerance in macrophages in vitro generally involve selective modulation or repression of some component of the signalling cascade, ranging from the putative LPS receptor TLR4, through IRAK and various regulators of NF-κB [[5,17] and references therein]. Many of the proposed mechanisms of tolerance would imply cross-desensitisation of responses to other bacterial products presumed to share the same pathway, a phenomenon that has been reported between TLR2 and TLR4 [25,26]. It is self-evident that if genes such as TNF-α and c-fos are induced transiently by LPS, there is a state that might be called "tolerance" or "repression" in that the mRNA and protein declines despite the continued presence of the stimulus. The transitory nature of TNF-α induction is probably a consequence of induction of nucleases that specifically degrade the mRNA [27]. Such a feedback mechanism would clearly interfere with subsequent induction by any stimulus for as long as the nuclease activity was retained. By contrast, where mRNAs continue to increase and there is no evidence of feedback control, there is no reason to expect any restriction on additive signaling by different TLR agonists unless there is an intrinsic limit to the amount of mRNA that can be made or common pathway components are limiting. There is no obvious reason why it would be desirable for macrophages to restrict their ability to recognise more than one TLR-related challenge simultaneously, especially in mixed infections. Based upon these considerations, we hypothesised that genes that are induced in a sustained manner by TLR agonists would not display any kind of "tolerance" and that there would be significant advantage to macrophages in being able to integrate multiple signals that induce such genes. To address this hypothesis, we have developed novel reporter gene systems. The firefly luciferase gene product is very unstable in the macrophage cell line RAW264, and stably-transfected cells have provided a sensitive indicator of transient activation of κB-dependent transcription in these cells [28]. We examined the regulation of promoters of the IL-1β, IL-6 and IL-12p40 genes as well as the widely-used κB-responsive gene, ELAM (Eselectin) in stably-transfected RAW264 cells. We demonstrate that sustained activation of the promoters of these genes requires the continued presence of microbial agonist and that signaling by one such agonist does not preclude additional activation through a distinct Tolllike receptor pathway. In the second part of the study, we use constitutively active forms of TLRs to generate the primary activation of reporter gene expression, and BMC Immunology 2001, 2:11 http://www.biomedcentral.com/1471-2172/2/11 again show that their actions are additive with those of agents that act through other TLRs. Finally, we show that primary macrophages can, indeed, respond to multiple TLR stimuli with increased cytokine secretion. In overview, we conclude that macrophages sense microbial challenges continuously and can respond to more than one stimulus simultaneously. Results Generation and characterisation of stably transfected RAW264 cell lines with two integrated luciferase reporters Previous studies examined a stable transfectant of the cell line RAW264, in which the firefly luciferase gene directed by the NF-κB-dependent HIV-1-LTR was integrated into the genome. This line provided a sensitive indicator of response to LPS and to CpG DNA [8,28]. Induction of luciferase activity was transient, reaching a peak after around 2 hrs and then declining rapidly to control levels. The time course of luciferase activation was consistent with transient induction of nuclear NFκB activity [8,28] and TNF-α mRNA [8,28] demonstrated previously using RAW264 cells cultured in similar conditions. Although NF-κB is strongly implicated in regulated gene expression in macrophages, numerous other transcription factors (i.e. PU.1, Ets-2, Sp1, Stat-1, C/EBPβ, γ or δ, IRF-1 etc [1,29]) are regulated or induced in LPS-stimulated cells, so we decided to examine more complex promoters that are not solely dependent upon NF-κB. To examine genes that are induced at a transcriptional level in RAW264 cells, we made a series of stable RAW264 cell transfectants with cytokine promoters driving luciferase. Stable transfection avoids the complication that derives from the ability of macrophages to recognise and respond to plasmid DNA [8]. The interleukin-1β gene is of particular interest, because previous reports indicate that "LPS tolerance" does not prevent re-induction of this gene or of the interleukin 6 gene [30,31]. We cloned the human interleukin-1 β promoter into a renilla luciferase plasmid, which allowed us to produce lines in which the IL-1β induction response could be measured simultaneously with other promoters that may, or may not, exhibit tolerance. RAW264 cells were contransfected by electroporation with two separate reporters and after 2 weeks of selection in G418, several hundred foci of stable transfectants were pooled for further study. Because of the known variation between RAW264 subclones in terms of LPS-inducible gene expression [23] we chose not to study single cell clones. In a separate study, we have confirmed that all clones derived from the pools used in this study express both the firefly and renilla luciferase genes at low levels, but vary considerably in whether that activity is inducible by LPS or other agonists [32]. The expression of luciferase and responsiveness to microbial challenge has been stable in the pools of transfectants for at least 2 months in continuous culture. To our knowledge, this is the first example of the use of multiple reporter genes in a stably-transfected macrophage line. Time and dose response curves for activation of luciferase expression Comparative time and dose response curve analysis were performed for each of the pooled transfectant lines with different combinations of promoters. Each promoter controlling firefly luciferase behaved in a unique manner, regardless of the presence of the IL-1 promoter-renilla luciferase gene in all of the lines, whereas the IL-1 promoter regulation was remarkably consistent in independent lines with different combinations of firefly luciferase reporters. The ELAM-1 promoter, used commonly as an indicator of κB-dependent transcription [33]. displayed the highest basal activity and inducibility. In our studies of the HIV-1-LTR, we found that adherence to tissue culture plastic was stimulatory [unpublished], so the transfected RAW264 cells were plated in the evening and stimulated the next morning after the cells were fully adherent. We considered the possibility that overnight culture could lead to accumulation of endogenous stimulatory or inhibitory cytokines, so we examined the effect of replacing the medium. The outcome depended upon the reporter gene. When cells were exposed to fresh medium, ELAMluciferase activity decreased transiently, then increased continuously over a 12 hr incubation (this is not obvious in Fig 1A, because of the much larger effect of added LPS). Following addition of 100 ng/ml LPS to unwashed cells, ELAM-luciferase was detectably elevated 2-fold after 30 mins, and continued to increase relative to unstimulated expression for up to 6–8 hrs, after which it declined (Fig 1A). In cells provided with fresh medium, the tail-off at 6–8 hrs did not occur, and luciferase activity was still increasing at 12 hrs. Hence, endogenous regulators appear to constrain activation of the NF-κBdependent promoter. In the same stably-transfected cells, the IL-1β renilla luciferase did not show either the rapid decline upon medium replacement, or the increase in basal activity thereafter (Fig 1C). However, fresh medium did accelerate the response to LPS, and permit the response to continue rising up to 12 hrs. Fig 1B shows a comparable time course for the IL-12 promoter. The data for the IL-1β promoter in this pool of stable transfectants line were indistinguishable from those obtained with the ELAM/IL-1 transfectants, and have been averaged in Fig 1C. Unlike the IL-1 promoter in the same cells, the response of the IL-12 promoter to LPS BMC Immunology 2001, 2:11 http://www.biomedcentral.com/1471-2172/2/11 was also increased marginally by washing and continued to increase when the control activation had peaked. Similar results were obtained with a population of cells transfected with an IL-6 firefly luciferase reporter together with the IL-1 renilla luciferase reporter (data not shown). These findings suggest that the RAW264 cells produce a feedback suppressor of LPS response that has some selectivity for the target promoter being studied. Because the introduction of fresh medium, or use of freshly plated-cells creates a rising baseline which is difficult to interpret, we chose a standard assay procedure in which the cells were plated late in the afternoon and stimulated the next day for 8 hrs without changing the medium. The sensitivity of the assay allowed us to obviate the effect of accumulated inhibitors following overnight culture via the use of relatively low starting cell density (2 × 105/ml). Reporter gene lines respond to a wide range of microbial agonists with distinct dose response curves The different indicator cells lines each responded to a wide diversity of different agonists of bacterial origin in addition to LPS, including the synthetic bacterial lipopeptide, PAM3-CSK4 [34], peptidoglycan and bacterial DNA (bDNA) or CpG-containing oligonucleotides. Figure 2 shows an example using the IL-6 promoter in combination with the IL-1β promoter in the same cells. This study makes several additional points: 1) Activation of expression of both reporter genes was detectable in response to LPS at Ing/ml. 2) The response of both promoters to low doses (1 or 10 ng/ml) of LPS was transient and declined after 10–15 hrs, where the response to higher LPS concentration (100 ng/ml) was sustained for longer and was still almost maximal after 24 hrs. Hence, the duration, rather than the peak magnitude, of the response was most sensitive to LPS concentration. 3) The different agonists have different relative activities on the two promoters. In keeping with the previous observation that IL-1β is weakly induced by bDNA [8], bDNA was as effective as LPS at inducing IL-6 promoter but only half as effective on the IL-1 promoter. Figure 3 shows dose response curves for LPS, lipopeptide and bacterial DNA for the IL-12, IL-1 and ELAM-1 promoters. Activity of both the IL-1 and IL-12 promoters continued to increase up to 500 ng/ml LPS whereas the activation of ELAM luciferase was detectable at 0.1 ng/ ml and maximally at 10 ng/ml. Given that the ELAM-1 promoter is apparently more sensitive to LPS than other promoters tested, the rising baseline seen upon addition of fresh medium (Fig 1) is likely to be due to endotoxin and/or endotoxin-like activity in serum, which cannot be completed avoided and which the cells themselves degrade with time. The effects of pretreatment The main purpose of creating these lines of RAW264 cells is to use them as convenient indicators of interactions amongst signals generated by different microbe-associated stimuli. We focused upon the IL-12/IL-1 and ELAM-IL-1 lines, which appear to display the spectrum Figure 1 Time course of activation of integrated reporter genes in RAW264 cells. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL-12 promoter driving firefly luciferase, cotransfected with the IL-1β promoter driving renilla luciferase, were cultured overnight as described in Materials and Methods. Where the cells were washed, the medium was aspirated, and replaced immediately with warm medium at time zero. A typical experiment of three is shown. Results for ELAM and IL12 promoters are the average of duplicates that differ by less than 10% from the mean. In the case of the IL-1β data, the results are the average of 4 datapoints obtained with the two separate pooled transfectant lines in the same experiment. 0 50000