This study simultaneously examines the secretion of 6 factors (G-CSF, IL-6, IL-10, MIP-1α, RANTES, and TNF-α) by RAW 264.7 cells in response to simultaneous application of multiple stimuli. The multi-cytokine responses were used to examine two issues. As a platform to identify instances of significant nonadditivity, the multi-factorial experiments addressed the general question of complexity and crosstalk in signal transduction. This same profiling process was used to identify previously uncharacterized and specific higher-order interactions that regulated cytokine secretion. With regard to the first point, does greater information complexity, in this case larger numbers of stimuli encountered by a cell, equate to greater complexity in output? In other words, if a cell is capable of responding to a number of individual stimuli that result in a unique set of behaviors, does presenting the total ensemble or various subsets of inputs result in unique sets of responses? Moreover, does such behavior scale with an increase in the number of inputs? The current literature presents contrasting possibilities. High-throughput measurements of protein-protein interactions, such as those obtained through two-hybrid experiments, and the large number of components now identified in cellular signaling systems, lead to speculation that the networks of signal transduction machinery are inordinately complex17, 18
. However, such observations often reflect connections that are possible in an in vitro context and are not necessarily of physiological consequence in vivo. Other studies indicate that cells may have evolved elaborate mechanisms to limit interaction complexity, and may have invested heavily in the macromolecular organization of signaling machinery to limit both the spatial and temporal spreading of signaling events in cells19, 20
. The mechanisms that limit interactions are more difficult to define than those that encourage synergy.
Our study examined this issue in a system in which a number of different factors are known to interact in simple combinations of inputs, and thus allowed us to ask in a meaningful context whether novel signaling regimes arise from complex higher-order combinations of elements. Although our own work5
and other studies21–25
have profiled the extent and context-dependence of nonadditive cellular responses to pairs of stimulators, this is the first study that systematically examines whether complex signaling responses can be mediated in response to stimulation by higher-order combinations of ligands. Importantly, the results show that such higher-order combinations of ligands can induce unique signaling regimes, but that such instances are limited when compared to the total number of combinations possible (~33%, with the majority in the 3-way combinations). This limitation is considered important because all of the ligands used in this study were known to be interactive through their production of nonadditive responses in two-way combinations.
Although we cannot generalize signaling architectures from one set of results, the following conclusions can be drawn in this instance. First, complexity of output responses is “encoded” or “hard-wired” into the system by the presence of interactions between pathways (as evinced by the nonadditivity of the two-way combinations). Increasing the number of stimuli to engage more of these interactions between pathways did not, in general, result in a concomitant, iterative increase in output complexity. Second, the interactions that are realized can uniquely shape overall cellular behavior in response to complex stimuli. Almost every higher-order combination of ligands, (that is, looking at each row in for all combinations of ligands 3- or higher, 14/15 rows) did elicit a significant, nonadditive response in at least one cytokine; however, nonadditive changes among individual cytokines are often not observed even though certain pairs of ligands involved were highly interactive [see responses for MIP-1α and TNF-α ()]. Because such unrealized interactions to ligand combinations varied with individual cytokines, correlations in nonadditive responses among various cytokines observed with dual ligands5
(, top panel) were not maintained with higher-order combinations of the same ligands. Thus, higher-order combinations of ligands gave rise to limited, unique behaviors based on restricted interactions among the signaling pathways.
The estimate of nonadditivity does not inform us about the mechanism by which it happens. Although some cytokines appear to trend together in their responses to the two-ligand perturbations, this does not imply a similarity in how this nonadditivity is mediated for these cytokines. This becomes especially evident when the correlation is lost with higher-order combinations of ligands. Indeed, this suggests that the mechanisms for the correlated behavior with dual ligands are different. By comparing and contrasting responses across combinations of ligands, we can gain some insight into the requirements for the nonadditivity observed, and this will help shape future studies to elucidate the molecular mechanisms involved.
Thus, even in a multifactorial system such as the cytokine network in which multiple crosstalk and feedback loops are present, information complexity is encoded in sparse and discrete instances. This suggests that context-dependence, or the ability of the system to recognize two stimuli in conjunction and to respond differently from either stimulus taken individually, is encoded through select interactions or pathways that are not extensively or promiscuously activated. Several other studies have argued that such limitation of interaction complexity is evolved in architectures that enhance “robustness” or dependability of signal transduction19, 20
. If this is true, it stands to reason that the few higher-order interactions observed are very likely to indicate biologically important phenotypes.
A second objective of this study was to identify any novel higher-order interactions between the signaling mechanisms downstream of the receptor for LPS (a major antigenic factor of gram-negative bacteria) and the 4 families of receptors used. Although the molecular mechanisms by which these pathways exert their effects cannot be definitively determined by this approach, our data are highly consistent with published mechanisms that regulate cytokine secretion (as outlined below) and allow for tractable generation of hypotheses for future experimentation.
We focused specifically on the novel observation that the downstream pathways from IFN-β and cAMP interacted to simultaneously both amplify and inhibit G-CSF and MIP-1α produced in response to KDO. Furthermore, secretion of IL-6 was greatly potentiated when all three ligands were combined. These data demonstrate the multiple regulatory nature of each pathway to simultaneously and differentially modulate multiple cytokines as discussed below.
The secretion of many cytokines is regulated through de novo synthesis of their nascent transcript. Stimulation of the secretion of G-CSF by KDO was inhibited by IFN-β, but was potentiated by cAMP-dependent pathways. When all three ligands were combined, a less-than-additive, intermediate amount of G-CSF was secreted. The relative pattern of secreted G-CSF obtained with single, double, and triple combinations of ligands mirrored the pattern observed in the expression of its mRNA (), which suggests that these interacting pathways regulated secretion of this cytokine by regulating the expression of its mRNA.
Regulation of MIP-1α by the same ligands showed opposing effects and suggests the existence of additional mechanisms. Unlike its effect on secretion of G-CSF, cAMP-dependent pathways suppressed secretion of MIP-1α induced by KDO and this was paralleled by attenuation of the KDO-induced expression of MIP-1α mRNA (). This modulation of mRNA expression for both cytokines indicates that the intersection between the TLR4 and cAMP pathways for regulation of secretion either resides upstream of transcription or at the regulation of transcript production or stability. One putative node of intersection could be in the regulation of nuclear factor κB (NF-κB), a key mediator of the TLR-induced secretion of many cytokines; however, the opposing action of cAMP on G-CSF and MIP-1α indicates that this cannot uniquely account for both effects.
In contrast to G-CSF, the greater-than-additive amounts of MIP-1α secreted in the presence of KDO and IFN-β was accomplished while the abundance of MIP-1α mRNA either did not change or showed a less-than-additive response. This result has also been observed in purified human monocytes cultured with IFN-α and LPS26
. Thus, regulation by IFN-β appears to occur through two pathways. Stimulation of MIP-1α secretion by IFN-β alone correlated with a modest increase in the abundance of MIP-1α mRNA and predicts a transcriptional mechanism of regulation. However, a lack of increased abundance of mRNA in contrast to the greater-than-additive secretion obtained with the combination of IFN-β and KDO predicts an additional mechanism that is not transcriptional in nature, such as an increase in the rate of translation, trafficking through secretory vesicles, or both. Whereas regulation of the secretory pathway for newly synthesized cytokines, such as MIP-1α, is not well understood, potential mechanisms are suggested by regulation of the secretion of TNF-α. Recent evidence suggests roles for vesicle-associated membrane protein 3 (VAMP3) and Rab11, a small guanosine triphosphatase (GTPase) implicated in membrane trafficking, in the secretion of TNF-α. LPS induces the production of both VAMP3 and Rab11. Macrophages that overexpress VAMP3 or are transfected with a constitutively active form of Rab11 show increased delivery of TNF-α to the cell surface27
. In contrast, the delivery of TNF-α to the cell surface, but not the synthesis of TNF-α, is blocked in macrophages that express dominant-negative forms of VAMP3 or Rab11. Furthermore, production of both VAMP3 and Rab11 is induced more quickly by priming cells with IFN-γ. It is possible that IFN-β, when combined with KDO, induces a similar synergistic increase in VAMP3 and Rab11 or some equivalent pathway.
The potentiation of secretion of IL-6 that resulted from the simultaneous stimulation of cells with IFN-β, KDO, and ISO or 8Br indicates that these three pathways converge at least at one site (). A similar potentiation in the secretion of IL-10 was not observed under the same conditions (). This seems surprising because IL-6 induces the secretion of IL-10 in these cells5
(). In the same experiments, potentiation of IL-10 was observed with the combination of KDO and IL-6 and correlated with dual combinations of ligands that potentiate secretion of IL-6, such as KDO paired with either IFN-β or 8Br ( and ). The lack of synergy at the first time point might be expected because the concentration of secreted IL-6 (about 100 pM) was insufficient to induce production of IL-10. In this case, the additive nature of the triple response would suggest that the synergy in IL-10 production produced by the combinations of KDO-IFN-β and the KDO-cAMP pathway stems from separate parallel pathways. However, the amount of IL-6 secreted by the three combined ligands at longer times of incubation exceeded the threshold for stimulating the secretion of IL-10. A potential explanation for a lack of synergy could be that the IL-6 pathway is simply saturated by the other ligands. Alternatively, the absence of potentiated amounts of IL-10 by the increased quantities of extracellular IL-6 reflects the existence of an underlying inhibitory mechanism in which IL-6 can also mediate attenuation of IL-10 secretion, which is synergized by other ligand pairs (). Such established mechanisms of cytokine regulation may include suppressors of cytokine signaling (SOCS) proteins to inhibit signal transduction from the IL-6 receptor, the downregulation of functional IL-6 receptor itself, or the induction of a decoy receptor antagonist in analogy to mechanisms that regulate IL-1 and IL-1028
TGF-β inhibited the potentiation of IL-6 by KDO in combination with IFN-β, ISO or 8Br. When combined with KDO and IFN-β, more prominent inhibition at later time points suggests that TGF-β may act through a secondary mechanism, which potentially involves secretion of an additional factor to mediate its suppressive effects. In general, the data presented here demonstrate that TGF-β in this experimental paradigm inhibits cytokine secretion and has the potential to reduce powerful stimulatory paradigms. The ability of TGF-β to inhibit is consistent with its known immunosuppressive properties29, 30
. However, IL-6 is also a pleiotropic cytokine whose activities have been characterized as anti- and pro-inflammatory31
and the ability of TGF-β to markedly modify its secretion in different venues may produce the divergent effects.
The profiles of cytokine secretion reported here can be applied to well-studied paradigms as well as novel areas where the roles of the cytokines are less defined. For example, the interactions of IFN-β with signaling by TLR4 potentially play a role in defense against viruses. The TLR family of receptors is one of two classes of receptors that can detect viruses within a host organism11, 13, 14, 32
, and type I IFNs such as IFN-β are necessary for viral clearance; thus, mice devoid of the type I IFN receptors are highly susceptible to various viral pathogens33–35
. Pathways dependent on cAMP have been proposed to mediate aspects of inflammation and fever through their mediation of TLR-dependent cytokines by prostaglandin E2
) receptors36, 37
receptors, which couple to Gs
, the stimulatory heterotrimeric guanine nucleotide–binding protein (G protein) that activates adenylyl cyclase and effects increases in the concentration of cAMP.
We have provided a systematic experimental paradigm to investigate the cytokine secretion response by cells in response to stimulation by multiple ligands, a scenario likely to occur in vivo. The higher-order responses uncovered in this study demonstrate both a rich diversity in the interactions that are used to coordinate responses to multiplex stimuli as well as a high degree of constraint in the number of pathway interactions that are realized. Thus, the context in which various stimuli are detected by the cell leads to limited, but unique behaviors. The limitation of unique responses suggests the biological importance of maintained crosstalk and the interactions identified with this limited analysis of TLR4-dependent cytokine secretion in macrophages. This systematic approach should be applicable to other systems and help identify key interactions for regulating other cellular paradigms.