Cytokine secretion requires phosphatidylcholine synthesis

Choline cytidylyltransferase (CCT) is the rate-limiting enzyme in the phosphatidylcholine biosynthetic pathway. Here, we demonstrate that CCT alpha-mediated phosphatidylcholine synthesis is required to maintain normal Golgi structure and function as well as cytokine secretion from the Golgi complex. CCT alpha is localized to the trans-Golgi region and its expression is increased in lipopolysaccharide (LPS)-stimulated wild-type macrophages. Although LPS triggers transient reorganization of Golgi morphology in wild-type macrophages, similar structural alterations persist in CCT alpha-deficient cells. Pro-tumor necrosis factor alpha and interleukin-6 remain lodged in the secretory compartment of CCT alpha-deficient macrophages after LPS stimulation. However, the lysosomal-mediated secretion pathways for interleukin-1 beta secretion and constitutive apolipoprotein E secretion are unaltered. Exogenous lysophosphatidylcholine restores LPS-stimulated secretion from CCT alpha-deficient cells, and elevated diacylglycerol levels alone do not impede secretion of pro-tumor necrosis factor alpha or interleukin-6. These results identify CCT alpha as a key component in membrane biogenesis during LPS-stimulated cytokine secretion from the Golgi complex.     

Uropathogenic Escherichia coli dominantly suppress the innate immune response of bladder epithelial cells by a lipopolysaccharide- and Toll-like receptor 4-independent pathway

Urinary tract infections are a major source of morbidity among women, with the majority caused by uropathogenic Escherichia coli. Our objective was to test if uropathogenic E. coli suppress the innate immune response of bladder epithelial cells. We found that bladder epithelial cells secrete interleukin-6 and interleukin-8 in response to non-pathogenic E. coli, whereas they failed to do so in response to uropathogenic E. coli. Uropathogenic E. coli prevented interleukin-6 secretion in response to non-pathogenic E. coli and a panel of Toll-like receptor agonists, as well as to interleukin-1beta, but not to tumor necrosis factor alpha. These results indicate that receptors with a Toll/interleukin-1 receptor domain are specifically targeted, and that suppression is not a consequence of toxicity. One candidate for mediating immune suppression is bacterial lipopolysaccharide. However, lipopolysaccharide isolated from either uropathogenic or non-pathogenic E. coli stimulated interleukin-6 secretion to similar levels. In addition, uropathogenic E. coli did not stimulate interleukin-6 secretion from cells expressing a dominant negative Toll-like receptor 4, and prevented cells lacking Toll-like receptor 4 from secreting interleukin-6 in response to synthetic lipoprotein. We conclude that uropathogenic E. coli suppress the innate immune response through a pathway partially independent of lipopolysaccharide and Toll-like receptor 4.     

The estrogen-responsive B box protein: a novel enhancer of interleukin-1beta secretion

The estrogen-responsive B box protein (EBBP) and Pyrin belong to a family of structurally related proteins. While mutations in the pyrin gene cause an autoinflammatory disease, the biological function of EBBP is unknown. In this study, we identified the proinflammatory cytokine interleukin-1beta (IL-1beta) as an EBBP-binding partner. Furthermore, caspase-1 and NACHT, LRR and Pyrin domain containing protein (NALP) 1, two components of the recently identified inflammasome, a platform for the activation of caspase-1, also interact with EBBP. These proteins bind to the RFP domain of EBBP, suggesting that this domain of so far unknown function is an important protein-binding domain. EBBP was secreted in a caspase-1-dependent manner from cultured cells, and its secretion was enhanced by IL-1beta. Vice versa, endogenous and overerexpressed EBBP increased IL-1beta secretion. These results provide evidence for a role of EBBP in innate immunity by enhancing the alternative secretion pathway of IL-1beta.     

TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-β1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-β1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-β1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-β1-stimulated invasion. Our results also indicate that signaling events involved in TGF-β1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.     

The nuclear protein HMGB1 is secreted by monocytes via a non-classical,vesicle-mediated secretory pathway

HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1beta is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1beta and HMGB1 respond at different times to different stimuli: IL-1beta secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.     

PYPAF3, a PYRIN-containing APAF-1-like protein, is a feedback regulator of caspase-1-dependent interleukin-1beta secretion

PYPAF3 is a member of the PYRIN-containing apoptotic protease-activating factor-1-like proteins (PYPAFs, also called NALPs). Among the members of this family, PYPAF1, PYPAF5, PYPAF7, and NALP1 have been shown to induce caspase-1-dependent interleukin-1beta secretion and NF-kappaB activation in the presence of the adaptor molecule ASC. On the other hand, we recently discovered that PYNOD, another member of this family, is a suppressor of these responses. Here, we show that PYPAF3 is the second member that inhibits caspase-1-dependent interleukin-1beta secretion. In contrast, PYPAF2/NALP2 does not inhibit this response but rather inhibits the NF-kappaB activation that is induced by the combined expression of PYPAF1 and ASC. Both PYPAF2 and PYPAF3 mRNAs are broadly expressed in a variety of tissues; however, neither is expressed in skeletal muscle, and only PYPAF2 mRNA is expressed in heart and brain. They are also expressed in many cell lines of both hematopoietic and non-hematopoietic lineages. Stimulation of monocytic THP-1 cells with lipopolysaccharide or interleukin-1beta induced PYPAF3 mRNA expression. Furthermore, the stable expression of PYPAF3 in THP-1 cells abrogated the ability of the cells to produce interleukin-1beta in response to lipopolysaccharide. These results suggest that PYPAF3 is a feedback regulator of interleukin-1beta secretion. Thus, PYPAF2 and PYPAF3, together with PYNOD, constitute an anti-inflammatory subgroup of PYPAFs.     

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.     

Inhibition of insulin secretion from rat islets of Langerhans by interleukin-6. An effect distinct from that of interleukin-1.

Glucose-induced insulin secretion from islets cultured in the presence of interleukin-6 (IL-6) for 12-24 h was inhibited to a similar extent as when islets were treated with interleukin-1 beta (IL-1 beta). However, unlike IL-1 beta, IL-6 did not potentiate insulin secretion during an acute (30 min) exposure of islets to the cytokine, nor did it inhibit DNA synthesis during a 24 h culture period. A 12 h pretreatment of islets with tumour necrosis factor-alpha (TNF-alpha) combined with IL-1 beta potentiated the inhibitory effect of IL-1 beta on secretion, such that 20 mM-glucose-induced insulin secretion was abolished. No synergistic inhibition of secretion was observed with TNF-alpha and IL-6. However, IL-1 beta and IL-6 were found to inhibit insulin secretion in an additive manner. These results suggest that IL-6 inhibits insulin secretion in a manner distinct from that of IL-1 beta, and that IL-6 is unlikely to mediate the inhibitory effects of IL-1 beta or TNF-alpha on rat islets of Langerhans.     

Further aspects of IL-1 beta secretion revealed by transfected monkey kidney cells.

Because the cytokine interleukin-1 beta (IL-1 beta) lacks a classical hydrophobic signal sequence, it has been unclear how it is released from cells, and whether release proceeds via a novel mechanism or through non-specific leakage. To address this issue, we have examined the secretion of the recombinant forms of human IL-1 beta from COS monkey kidney cells, which express low levels of endogenous IL-1 beta. Four proteins were expressed: precursor and mature IL-1 beta and precursor and mature IL-1 beta fused to an amino terminal hydrophobic signal sequence from human tissue plasminogen activator. By monitoring the appearance of a known cytosolic protein (ATP citrate lyase) in the medium, we find that the unmodified IL-1 beta s are non-specifically released in very small quantities from the cytosol. On the other hand, the signal sequence-modified IL-1 beta s are glycosylated and efficiently secreted by the ER/Golgi pathway. The secreted, modified-mature protein is also biologically active, suggesting that this pathway has been bypassed for reasons other than maintaining the structural integrity of IL-1 beta. More likely the alternative pathway is a critical aspect of IL-1 biology. The differences in kinetics and quantity of IL-1 beta release from monocytic and COS cells suggest that COS cells lack critical components for the rapid release seen in monocytes.     

Essential role for Ca2+ in regulation of IL-1beta secretion by P2X7 nucleotide receptor in monocytes,macrophages, and HEK-293 cells

Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released.     

Interleukin-1 beta- and forskolin-induced synthesis and secretion of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by transforming growth factor-beta 2.

Interleukin-1 beta and forskolin induce prostaglandin E2 release as well as 14-kDa group II phospholipase A2 gene expression and secretion of the enzyme from rat glomerular mesangial cells. We now report that pretreatment of mesangial cells with transforming growth factor-beta 2 prior to stimulation with interleukin-1 beta or forskolin inhibits the induced release of prostaglandin E2. At the same time the secretion of group II phospholipase A2, measured both as enzyme activity with sn-2-labeled phosphatidylethanolamine as substrate and as enzyme protein in immunoblot experiments, is dose-dependently inhibited by pretreatment of the cells with transforming growth factor-beta 2. Analyses of enzyme activity and enzyme protein levels in the cells indicated that this is not due to inhibition of enzyme secretion with a concomitant increase in cellular levels of the enzyme. Rather, pretreatment of the cells with transforming growth factor-beta 2 largely prevented both the interleukin-1 beta- and the forskolin-induced synthesis of phospholipase A2. This is the first report indicating an inhibition of group II phospholipase A2 gene expression by transforming growth factor-beta 2. In line with those results, transforming growth factor-beta 2 did not induce the synthesis and secretion of group II phospholipase A2. However, under conditions where the interleukin-1 beta-induced expression of group II phospholipase A2 is fully suppressed by transforming growth factor-beta 2, the growth factor itself stimulated prostaglandin E2 synthesis by a mechanism apparently not involving group II phospholipase A2. The immunochemical identification of the inducible and secretable phospholipase A2 from rat mesangial cells as a group II enzyme was confirmed by purification and N-terminal amino acid sequence determination.     

alpha3beta1 integrin induced suppression of the Caco-2 epithelial cell IL-1 signaling pathway leading to NF-(kappa)B activation

Intestinal epithelial cells (IECs) produce several potent cytokines in response to interleukin-1 (IL-1) and may play a role in the inflammatory response. Previously, we determined that treatment of the Caco-2 cells with a cross-linking anti-alpha3 integrin antibody resulted in a suppression of IL-1 induced cytokine secretion and mRNA levels, suggesting that the alpha3beta1 integrin may play a role in the regulation of IEC cytokine responses to IL-1. In this report, treatment of the Caco-2 cells with the anti-alpha3 integrin antibody resulted in a suppression of IL-1 induced levels of NF-kappaB binding activity in nuclear extracts, as determined by EMSA, as well as phosphorylation and degradation of the inhibitor, I(kappa)B(alpha). The anti-integrin antibody treatment was also found to suppress I(kappa)B kinase (IKK) activity and IKK(beta) phosphorylation. Culture of the Caco-2 cells on purified laminin-5, the ligand for the alpha3beta1 integrin, also resulted in suppression of IL-1 induced phosphorylation of I(kappa)B(alpha) and IKK(beta). Together with our previous findings, these results suggest that alpha3beta1 integrin binding results in a suppression of the IL-1 signaling pathway leading to the activation of NF-(kappa)B and ultimately IEC cytokine responses. These studies define a novel regulatory mechanism which may be important in the control of IEC cytokine responses during inflammation.     

Polyene macrolide antifungal drugs trigger interleukin-1β secretion by activating the NLRP3 inflammasome

The use of antimycotic drugs in fungal infections is based on the concept that they suppress fungal growth by a direct killing effect. However, amphotericin and nystatin have been reported to also trigger interleukin-1β (IL-1β) secretion in monocytes but the molecular mechanism is unknown. Here we report that only the polyene macrolides amphotericin B, nystatin, and natamycin but none of the tested azole antimycotic drugs induce significant IL-1β secretion in-vitro in dendritic cells isolated from C57BL/6 mouse bone marrow. IL-1β release depended on Toll-like receptor-mediated induction of pro-IL-1β as well as the NLRP3 inflammasome, its adaptor ASC, and caspase-1 for enzymatic cleavage of pro-IL-1β into its mature form. All three drugs induced potassium efflux from the cells as a known mechanism for NLRP3 activation but the P2X7 receptor was not required for this process. Natamycin-induced IL-1β secretion also involved phagocytosis, as cathepsin activation as described for crystal-induced IL-1β release. Together, the polyene macrolides amphotericin B, nystatin, and natamycin trigger IL-1β secretion by causing potassium efflux from which activates the NLRP3-ASC-caspase-1. We conclude that beyond their effects on fungal growth, these antifungal drugs directly activate the host's innate immunity.     

Gingival and dermal fibroblasts produce interleukin-1 beta converting enzyme and interleukin-1 beta but not interleukin-18 even after stimulation with lipopolysaccharide

Epithelial cells play a critical role in periodontal disease through the secretion of pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18). However, the role played by fibroblasts is still unclear. The rationale of this study was to throw light on the role of gingival fibroblasts in periodontal disease. We thus investigated the expression of IL-1 beta, IL-18, and ICE mRNA and the secretion of the corresponding proteins by human normal gingival fibroblasts before and after stimulation with lipopolysaccharide (LPS) from Porphyromonas gingivalis and Escherichia coli. IL-1 beta, IL-18, and ICE mRNA expression was evaluated by RT-PCR. Proteins were analyzed by Western blot and ELISA. We demonstrated that gingival fibroblasts expressed ICE mRNA. Basal expression of ICE was modulated following cell stimulation with lipopolysaccharide (5 mug/ml). However, gingival fibroblasts expressed low levels of IL-1 beta mRNA. The expression was potentiated by LPS. The expression of IL-1 beta mRNA was followed by the secretion of IL-1 beta but not IL-18 protein. Our study suggests that fibroblasts may be involved in the defense against infections via an IL-1 beta-mediated but not an IL-18-mediated mechanism.     

Beta-endorphin secretion by human peripheral blood mononuclear cells: regulation by glucocorticoids

Corticotropin-releasing factor (CRF), a 41-aminoacid neuropeptide, can induce lymphocytes to production of beta-endorphin (beta E). Furthermore, the neuropeptide Arginine-Vasopressin (AVP) can enhance CRF-induced production of beta E. We have demonstrated that CRF acts by stimulating monocytes to production of the cytokine interleukin-1 (IL-1). IL-1 can in its turn activate the lymphocytes to secretion of beta E. Here we demonstrate that the glucocorticoid analogue dexamethasone is capable of modulating CRF-induced beta E secretion by lymphocytes. It appeared that dexamethasone can inhibit secretion of lymphocyte-derived beta E. The mechanism by which dexamethasone exerts its inhibitory activity is by blocking CRF-induced production of IL-1, thereby preventing induction of beta E secretion by B cells. These results support the concept that peptide hormones and glucocorticoids are mediating a reciprocal modulation of neuroendocrine and immunological activities.     

Defect in mevalonate pathway induces pyroptosis in Raw 264.7 murine monocytes

The inhibition of mevalonate pathway by the aminobisphosphonate alendronate (ALD) has been previously associated with an augmented lipopolysaccharide-induced interleukin-1beta (IL-1β) secretion in monocytes, as demonstrated in an auto-inflammatory disease known as mevalonate kinase deficiency (MKD). In this study we investigated the effect of ALD + LPS on monocyte cell line (Raw 264.7) death. ALD strongly augmented LPS-induced programmed cell death (PCD) as well as IL-1β secretion in Raw murine monocytes, whereas necrosis was rather unaffected. ALD + LPS induced caspase-3 activation. Inhibition of IL-1β stimulation partially restored cell viability. These findings suggest that the inhibition of mevalonate pathway, together with a bacterial stimulus, induce a PCD partly sustained by the caspase-3-related apoptosis and partly by caspase-1-associated pyroptosis. The involvement of pyroptosis is a novel hit in our cell model and opens discussions about its role in inflammatory cells with chemical or genetic inhibition of mevalonate pathway.     

Interleukin-1 beta is more potent than interleukin-1 alpha in suppressing follicle-stimulating hormone-induced differentiation of ovarian granulosa cells

Most studies have shown that the immune and inflammatory actions of interleukin-1 alpha and beta exhibit the identical biological spectrums of activity with similar dose-response curves. We have previously demonstrated that interleukin-1 beta suppresses follicle-stimulating hormone-induced differentiation of ovarian granulosa cells. In these experiments, we show that although the human recombinant preparations of interleukin-1 alpha and beta exhibit a similar directional inhibition of ovarian granulosa cell differentiation, there is a significant difference in the dose-response relationships between the two forms. Interleukin-1 beta was 31 times and 18 times more potent than interleukin-1 alpha in suppressing follicle-stimulating hormone-induced luteinizing hormone receptor development and progesterone secretion, respectively, from rat granulosa cells. However, there was no difference in the dose-dependent activities of interleukin-1 alpha and beta in stimulating murine thymocyte proliferation. These results suggest that interleukin-1 beta is more effective in influencing ovarian granulosa cell function than interleukin-1 alpha.     

A novel thioredoxin h is secreted in Nicotiana alata and reduces S-RNase in vitro

Thioredoxins type h are classified into three subgroups. The subgroup II includes thioredoxins containing an N-terminal extension, the role of which is still unclear. Although thioredoxin secretion has been observed in animal cells, there is no evidence suggesting that any thioredoxin h is secreted in plants. In this study, we report that a thioredoxin h, subgroup II, from Nicotiana alata (NaTrxh) is secreted into the extracellular matrix of the stylar transmitting tract tissue. Fractionation studies showed that NaTrxh is extracted along with well characterized secretion proteins such as S-RNases and NaTTS (N. alata transmitting tissue-specific protein). Moreover, an NaTrxh-green fluorescent fusion protein transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana leaves was also secreted, showing that NaTrxh has the required information for its secretion. We performed reduction assays in vitro to identify potential extracellular targets of NaTrxh. We found that S-RNase is one of the several potential substrates of the NaTrxh in the extracellular matrix. In addition, we proved by affinity chromatography that NaTrxh specifically interacts with S-RNase. Our findings showed that NaTrxh is a new thioredoxin h in Nicotiana that is secreted as well as in animal systems. Because NaTrxh is localized in the extracellular matrix of the stylar transmitting tract and its specific interaction with S-RNase to reduce it in vitro, we suggest that this thioredoxin h may be involved either in general pollen-pistil interaction processes or particularly in S-RNase-based self-incompatibility.