Rapid co-release of interleukin 1beta and caspase 1 in spinal cord inflammation

Mounting evidence supports the hypothesis that pro-inflammatory cytokines secreted by astrocytes and microglia modulate nociceptive function in the injured CNS and following peripheral nerve damage. Here we examine the involvement of interleukin-1beta (IL-1beta) and microglia activation in nociceptive processing in rat models of spinal cord inflammation. Following application of lipopolysaccharide (LPS) to an ex vivo dorsal horn slice preparation, we observed rapid secretion of IL-1beta which was prevented by inhibition of glial cell metabolism and by inhibitors of either p38 mitogen-activated protein kinase (MAPK) or caspase 1. LPS superfusion also induced rapid secretion of active caspase 1 and apoptosis-associated speck-like protein containing a caspase recruitment domain from the isolated dorsal horn. Extensive microglial cell activation in the dorsal horn, as determined by immunoreactivity for phosphorylated p38 MAPK, was found to correlate with the occurrence of IL-1beta secretion. In behavioural studies, intrathecal injection of LPS in the lumbar spinal cord produced mechanical hyperalgesia in the rat hind-paws which was attenuated by concomitant injections of a p38 MAPK inhibitor, a caspase 1 inhibitor or the rat recombinant interleukin 1 receptor antagonist. These data suggest a critical role for the cytokine IL-1beta and caspase 1 rapidly released by activated microglia in enhancing nociceptive transmission in spinal cord inflammation.     

Emerging inflammasome effector mechanisms

Caspase 1 activation by inflammasome complexes in response to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) induces the maturation and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. Recent reports have begun to identify additional inflammasome effector mechanisms that proceed independently of IL-1β and IL-18. These include the induction of pyroptotic cell death, the restriction of bacterial replication, the activation of lipid metabolic pathways for cell repair and the secretion of DAMPs and leaderless cytokines. These non-canonical functions of caspase 1 illustrate the diverse mechanisms by which inflammasomes might contribute to innate immunity, repair responses and host defence.     

Statin-induced proinflammatory response in mitogen-activated peripheral blood mononuclear cells through the activation of caspase-1 and IL-18 secretion in monocytes

Statins, which inhibit 3-hydroxy-3-methylglutaryl CoA reductase, have been shown recently to promote proinflammatory responses. We show in this study that both atorvastatin and simvastatin induced proinflammatory responses in mitogen-activated PBMCs by increasing the number of T cells secreting IFN-gamma. This is abolished by the presence of mevalonate, suggesting that statins act specifically by blocking the mevalonate pathway for cholesterol synthesis to promote the proinflammatory response. Both statins at low concentrations induced a dose-dependent increase in the number of IFN-gamma-secreting T cells in mitogen-activated PBMCs, whereas at higher concentrations the effect was abolished. The proinflammatory effect of statins was not seen in purified T cells per se activated with mitogen. However, conditioned medium derived from statin-treated PBMCs enhanced the number of IFN-gamma-secreting cells in activated purified T cells. This effect was not blocked by mevalonate, but was abolished by neutralizing Abs to IL-18 and IL-12. Similarly, the up-regulation of IFN-gamma-secreting T cells in PBMCs costimulated with statins and mitogens was blocked by the neutralizing anti-IL-18 and anti-IL-12. We showed that simvastatin stimulates the secretion of IL-18 and IL-1beta in monocytes. Active caspase-1, which is required for the processing and secretion of IL-18 and IL-1beta, was activated in simvastatin-treated monocytes. This was blocked by mevalonate and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone. Taken together, the proinflammatory response mediated by statins in activated PBMCs is mediated mainly via the activation of caspase-1 and IL-18 secretion in the monocytes and to a lesser extent by IL-12.     

The dynamic responses of pro-inflammatory and anti-inflammatory cytokines of human mononuclear cells induced by uromodulin

This study was undertaken to examine the dynamic response of human peripheral blood mononuclear cells (PBMC) in the secretion of proinflammatory and anti-inflammatory cytokines induced by uromodulin (URO). Levels of tumor necrosis factor-alpha (TNFalpha), TNF soluble receptor (sTNFRI and II), interleukin 1-beta (IL-1beta), and IL-1 receptor antagonist (IL-1Ra) in the supernatants of URO-stimulated PBMC were measured by ELISA. URO stimulated the secretion of all these cytokines in a dose dependent manner except sTNFRI. Peak levels of TNFalpha and IL-1beta were reached at 6-12 h, while 5-10 fold higher in sTNFR II and IL-1Ra levels were observed at 24-48 h after URO stimulation. URO-induced secretion of TNFalpha, IL-1beta, sTNFRII and IL-1Ra could be enhanced by human plasma. Specifically, serum proteins including C3, sCD14 and IgG not only bound to URO but also enhanced URO-induced TNFalpha secretion of PBMC. Collectively, our data suggest that URO might have dual immunomodulating effect through regulating the secretion of proinflammatory and anti-inflammatory cytokines, and that serum binding proteins might enhance this activity.     

IL-1beta-mediated proinflammatory responses are inhibited by estradiol via down-regulation of IL-1 receptor type I in uterine epithelial cells

The objective of this study was to examine the effects of sex hormones on IL-1beta-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1beta in the presence of estradiol or progesterone. Estradiol inhibited the IL-1beta-mediated mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1beta-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10(-7) to 10(-10) M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1beta-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell's ability to respond to IL-1beta. These results suggest that the inhibitory effect of estradiol on IL-1beta-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.     

Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1beta activation by this protease in THP-1 monocytes. ASC enhances IL-1beta secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1beta secretion but that coexpression of ASC with these proteins results in enhanced IL-1beta secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1beta secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.     

Staphylococcal alpha-toxin synergistically enhances inflammation caused by bacterial components

This study was performed to investigate the in vivo effects of staphylococcal alpha-toxin on phagocytosis and the secretion of proinflammatory cytokines at local sites of intraperitoneal toxin-challenged mice. A dosage of 45 hemolytic units (HU) of alpha-toxin induced a marked increase in the peritoneal neutrophil count. The toxin caused a 52% decrease in phagocytosis by peritoneal macrophages, compared with that of control mice receiving Staphylococcus aureus particles alone. However, no effect on phagocytosis in neutrophils was observed. A dosage of 45 HU toxin and the synergistic activity of S. aureus particles strongly induced interleukin (IL) 6 secretion but only mildly induced IL-1alpha secretion. The toxin did not induce the secretion of tumor necrosis factor-alpha (TNF-alpha). Interestingly, S. aureus culture supernatant induced the secretion of TNF-alpha in cultured macrophages. These results suggest that alpha-toxin damages the primary host defense system by inducing the oversecretion of IL-1alpha and IL-6, but not TNF-alpha, via a mechanism that requires the synergistic action of bacterial components.     

Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus

Astrocytes play an important role in initiating and regulating CNS immune responses through the release of proinflammatory cytokines and chemokines. Here we demonstrate that primary astrocytes are capable of recognizing the Gram-positive bacterium Staphylococcus aureus and its cell wall product peptidoglycan (PGN) and respond by producing numerous proinflammatory mediators including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemoattractant protein (MCP-1). Astrocytes have recently been shown to express Toll-like receptor 2 (TLR2), a pattern recognition receptor important for recognizing structural components of various Gram-positive bacteria, fungi, and protozoa. However, the functional significance of TLR2 in mediating astrocyte activation remains unknown. Primary astrocytes from TLR2 knockout mice were used to evaluate the role of TLR2 in astrocyte responses to S. aureus and PGN. The results demonstrate that TLR2 is essential for maximal proinflammatory cytokine and chemokine production, but not phagocytosis, in primary astrocytes following S. aureus and PGN exposure. In addition, both stimuli led to a significant increase in TLR2 mRNA expression in wild-type astrocytes as assessed by real-time quantitative RT-PCR. These findings suggest that astrocytes may play a key role in the initial antibacterial immune response in the CNS through engagement of TLR2.     

Cop, a caspase recruitment domain-containing protein and inhibitor of caspase-1 activation processing.

The production of bio-active interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves binding of the caspase-1 prodomain to a caspase recruitment domain (CARD)-containing serine/threonine kinase known as RIP2/CARDIAK/RICK. We have identified a novel protein, COP (CARD only protein), which has a high degree of sequence identity to the caspase-1 prodomain. COP binds to both RIP2 and the caspase-1 prodomain and inhibits RIP2-induced caspase-1 oligomerization. COP inhibits caspase- 1-induced IL-1beta secretion as well as lipopolysaccharide-induced IL-1beta secretion in transfected cells. Our data indicate that COP can regulate IL-1beta secretion, implying that COP may play a role in down-regulating inflammatory responses analogous to the CARD protein ICEBERG.     

Gingipains from Porphyromonas gingivalis synergistically induce the production of proinflammatory cytokines through protease-activated receptors with Toll-like receptor and NOD1/2 ligands in human monocytic cells

Gingipains (HRgpA, RgpB and Kgp) are cysteine proteinases and virulence factors of Porphyromonas gingivalis , the major causative bacterium of periodontal disease. To study synergistic effects of gingipains and signalling via Toll-like receptors (TLRs) and NOD1/2, we investigated effects of a gingipain on the secretion of proinflammatory cytokines from monocytic THP-1 cells in the presence of pathogen-associated molecular patterns (PAMPs). Gingipains stimulated interleukin (IL)-8's secretion from THP-1 cells, which was completely inhibited by proteinase inhibitors of gingipain and increased in the presence of PAMPs. Synergistic effects of gingipains and PAMPs were also seen in the secretion of IL-6 and MCP-1 and reduced to about 50% the secretion of IL-8 from THP-1 cells treated with siRNA targeting either protease-activated receptor (PAR)-1, -2 or -3. PAR agonist peptides mimicked the synergistic effects of gingipains with PAMPs. These results indicate that gingipains stimulate the secretion of cytokines from monocytic cells through the activation of PARs with synergistic effects by PAMPs. This is the first report of synergism of signalling via PARs, and TLRs or NOD1/2. The host defence system against P. gingivalis may be triggered through the activation of PARs by gingipains and augmented by PAMPs from this pathogen via TLRs or NOD1/2.     

Nod1, a CARD protein,enhances pro-interleukin-1beta processing through the interaction with pro-caspase-1

The production of bioactive interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves interaction between the caspase recruit domains (CARDs) of caspase-1 and a serine/threonine kinase RIP2. While the association of Nod1 with both caspase-1 and RIP2 is already known, the consequences of these interactions are poorly understood. Because Nod1 also binds to RIP2, we hypothesized that Nod1 plays a role in pro-caspase-1 activation and IL-1beta processing. We show here that Nod1 binds to both RIP2 and caspase-1 by CARD interactions. Nod1 enhances pro-caspase-1 oligomerization and pro-caspase-1 processing. Nod1 enhances caspase-1-induced IL-1beta secretion, as well as lipopolysaccharide (LPS)-induced IL-1beta secretion in transfected cells. Moreover, HT1080 cells stably transfected with Nod1 showed higher LPS-induced IL-1beta secretion than non-transfected cells, suggesting a role of Nod1 in LPS-induced responses. Our data indicate that Nod1 can regulate IL-1beta secretion, implying that Nod1 may play a role in inflammatory responses to bacterial LPS.     

Interleukin-10 and interleukin-13 inhibit proinflammatory cytokine-induced ceramide production through the activation of phosphatidylinositol 3-kinase

Ceramide produced by hydrolysis of plasma membrane sphingomyelin (SM) in different cells including brain cells in response to proinflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta)] plays an important role in coordinating cellular responses to stress, growth suppression, and apoptosis. The present study underlines the importance of IL-10 and IL-13, cytokines with potent antiinflammatory properties, in inhibiting the proinflammatory cytokine (TNF-alpha and IL-1beta)-mediated degradation of SM to ceramide in rat primary astrocytes. Treatment of rat primary astrocytes with TNF-alpha or IL-1beta led to rapid degradation of SM to ceramide, whereas IL-10 and IL-13 by themselves were unable to induce the degradation of SM to ceramide. Interestingly, both IL-10 and IL-13 prevented proinflammatory cytokine-induced degradation of SM to ceramide. Both IL-10 and IL-13 caused rapid activation of phosphatidylinositol (PI) 3-kinase, and inhibition of that kinase activity by wortmannin and LY294002 potently blocked the inhibitory effect of IL-10 and IL-13 on proinflammatory cytokine-mediated induction of ceramide production. This study suggests that the inhibition of proinflammatory cytokine-mediated degradation of SM to ceramide by IL-10 and IL-13 is mediated through the activation of PI 3-kinase. As ceramide induces apoptosis and IL-10 and IL-13 inhibit the induction of ceramide production, we examined the effect of IL-10 and IL-13 on proinflammatory cytokine-mediated apoptosis. Inhibition of TNF-alpha-induced apoptosis by IL-10 and IL-13 suggests that the antiapoptotic nature of IL-10 and IL-13 is probably due to the inhibition of ceramide production.     

Fas (CD95) induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages

Fas (CD95, APO-1) is regarded as the prototypical cell death receptor of the TNFR superfamily. Fas-induced apoptosis is generally considered to be a noninflammatory process, contributing to the silent resolution of immune and inflammatory responses. However, accumulating evidence indicates that Fas may also induce cellular activation signals. We hypothesized that Fas could activate proinflammatory cytokine responses by normal human monocytes and macrophages. Monocytes were isolated by negative immunoselection from the PBMC fraction of venous blood from healthy volunteers, and monocyte-derived macrophages were cultivated in vitro. Both monocytes and monocyte-derived macrophages released TNF-alpha and IL-8 following Fas ligation, and conditioned medium from Fas-activated monocytes and macrophages induced the directed migration of neutrophils in a chemotaxis assay. Fas-induced monocyte cytokine responses were associated with monocyte apoptosis, nuclear translocation of NF-kappaB, and cytokine gene expression and were blocked by caspase inhibition but not by inhibition of IL-1beta signaling. In contrast, Fas-induced macrophage cytokine responses occurred in the absence of apoptosis and were caspase independent, indicating maturation-dependent differences in the Fas signaling pathways that lead to proinflammatory cytokine induction. Rather than contributing to the resolution of inflammation, Fas ligation on circulating monocytes and tissue macrophages may induce proinflammatory cytokine responses that can initiate acute inflammatory responses and tissue injury.     

Neutrophil elastase (NE)-deficient mice demonstrate a nonredundant role for NE in neutrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo

Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE(-/-)) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE(-/-) mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1beta and TNF-alpha and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE(-/-) mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1beta, KC, and macrophage inflammatory protein-1alpha in NE(-/-) mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.     

Cutting edge: the "death" adaptor CRADD/RAIDD targets BCL10 and suppresses agonist-induced cytokine expression in T lymphocytes

The expression of proinflammatory cytokines and chemokines in response to TCR agonists is regulated by the caspase-recruitment domain membrane-associated guanylate kinase 1 (CARMA1) signalosome through the coordinated assembly of complexes containing the BCL10 adaptor protein. We describe a novel mechanism to negatively regulate the CARMA1 signalosome by the "death" adaptor protein caspase and receptor interacting protein adaptor with death domain (CRADD)/receptor interacting protein-associated ICH-1/CED-3 homologous protein with a death domain. We show that CRADD interacts with BCL10 through its caspase recruitment domain and suppresses interactions between BCL10 and CARMA1. TCR agonist-induced interaction between CRADD and BCL10 coincides with reduction of its complex formation with CARMA1 in wild-type, as compared with Cradd-deficient, primary cells. Finally, Cradd-deficient spleen cells, CD4(+) T cells, and mice respond to T cell agonists with strikingly higher production of proinflammatory mediators, including IFN-γ, IL-2, TNF-α, and IL-17. These results define a novel role for CRADD as a negative regulator of the CARMA1 signalosome and suppressor of Th1- and Th17-mediated inflammatory responses.     

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide

Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-kappaB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-kappaB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-kappaB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1beta or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1beta- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1beta and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1beta and LPS.     

Impact of cutaneous IL-10 on resident epidermal Langerhans' cells and the development of polarized immune responses

Prolonged topical exposure of BALB/c mice to chemical contact and respiratory allergens stimulates, respectively, preferential Th1- and Th2-type responses with respect to serum Ab isotype and cytokine secretion phenotypes displayed by draining lymph node cells. We now report that differential cytokine secretion patterns are induced rapidly in the skin following first exposure to the contact allergen 2,4-dinitrochlorobenzene (DNCB) and the respiratory sensitizer trimellitic anhydride (TMA). TMA induced early expression of IL-10, a cytokine implicated in the negative regulation of Langerhans cell (LC) migration, whereas exposure to DNCB resulted in production of the proinflammatory cytokine IL-1beta. Associated with this, TMA provoked LC migration with delayed kinetics compared with DNCB, and local neutralization of IL-10 caused enhanced LC mobilization in response to TMA with concomitant up-regulation of cutaneous IL-1beta. We hypothesize that these differential epidermal cytokine profiles contribute to the polarization of immune responses to chemical allergens via effects on the phenotype of activated dendritic cells arriving in the draining lymph node. Thus, TMA-exposed dendritic cells that have been conditioned in vivo with IL-10 (a potent inhibitor of the type 1-polarizing cytokine IL-12) are effective APCs for the development of a Th2-type response.     

Induction of interleukin-1beta by interleukin-4 in lipopolysaccharide-treated mixed glial cultures: microglial-dependent effects

Glial-secreted proinflammatory mediators are dynamically involved in central nervous system responses to exogenous stimuli such as infection, neurotoxins, and nerve injury. The therapeutic use of anti-inflammatory agents may reduce certain central nervous system pathology induced by inflammatory responses. We investigated the role of interleukin (IL)-4 in modulating the production of proinflammatory mediators from lipopolysaccharide-stimulated mixed glia in vitro. Interestingly, IL-4 significantly enhanced IL-1beta secretion and did not affect monocyte chemoattractant protein-1 release, even though IL-4 considerably inhibited IL-6, tumor necrosis factor alpha, and nitric oxide production from rat neonatal mixed glia. Further, IL-4 exhibited inhibitory effects on IL-1beta production in microglial-enriched cultures, while significantly increasing IL-1beta production in microglial-depleted glia. The enhancing effect of IL-4 on IL-1beta production was found to be inversely correlated with the percentage of microglia present in the mixed glial population. In summary, IL-4 did not act as a global anti-inflammatory cytokine and in fact, under certain situations enhanced IL-1beta secretion. We conclude that IL-4 exerts its anti-inflammatory effects in a limited and target-specific manner, which is delicately regulated by the cellular microenvironment. Therefore, precaution should be taken when clinically using IL-4 to treat diseases manifested by overt inflammatory responses.