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Summary: Interleukin 36 (IL‐36) constitutes a group of cytokines that belong to the IL‐1 superfamily. Emerging evidence has suggested a role of IL‐36 in the pathogenesis of many inflammatory disorders. Intriguingly, in the gastrointestinal tract, IL‐36 has a rather complex function. IL‐36 receptor ligands are overexpressed in both animal colitis models and human IBD patients and may play both pathogenic and protective roles, depending on the context. IL‐36 cytokines comprise three receptor agonists: IL‐36α, IL‐36β and IL‐36γ, and two receptor antagonists: IL‐36Ra and IL‐38. All IL‐36 receptor agonists bind to the IL‐36R complex and exert pleiotropic effects during inflammatory settings. Here, we first briefly review the processing and secretion of IL‐36 cytokines. We then focus on the current understanding of the immunology effects of IL‐36 in gut immunity. In addition, we also discuss the ongoing trials that aim to blockage IL‐36R signalling for treating chronic intestinal inflammation and present some unexplored questions regarding IL‐36 research. [ABSTRACT FROM AUTHOR]

Summary: Excessive and dysregulated inflammation is known to contribute to disease progression. HSP90 is an intracellular chaperone known to regulate inflammatory processes including the NLRP3 inflammasome and secretion of the pro‐inflammatory cytokine interleukin(IL)‐1β. Here, primarily using an in vitro inflammasome ASC speck assay, and an in vivo model of murine peritonitis, we tested the utility of HSP90 inhibitors as anti‐inflammatory molecules. We report that the HSP90 inhibitor EC144 effectively inhibited inflammatory processes including priming and activation of NLRP3 in vitro and in vivo. A specific inhibitor of the β HSP90 isoform was ineffective suggesting the importance of the α isoform in inflammatory signalling. EC144 inhibited IL‐1β and IL‐6 in vivo when administered orally, and was brain‐penetrant. These data suggest that HSP90 inhibitors may be useful for targeting inflammation in diverse diseases that are worsened by the presence of inflammation. [ABSTRACT FROM AUTHOR]

Summary: Interleukin‐1β (IL‐1β) is a potent mediator of innate immunity commonly up‐regulated in a broad spectrum of inflammatory diseases. When bound to its cell surface receptor, IL‐1β initiates a signalling cascade that cooperatively induces the expression of canonical IL‐1 target genes such as IL‐8 and IL‐6. Here, we present galectin‐3 as a novel regulator of IL‐1β responses in corneal keratinocytes. Using the SNAP‐tag system and digitonin semi‐permeabilization, we show that recombinant exogenous galectin‐3 binds to the plasma membrane of keratinocytes and is internalized into cytoplasmic compartments. We find that exogenous galectin‐3, but not a dominant negative inhibitor of galectin‐3 polymerization lacking the N‐terminal domain, exacerbates the response to IL‐1β by stimulating the secretion of inflammatory cytokines. The activity of galectin‐3 could be reduced by a novel d‐galactopyranoside derivative targeting the conserved galactoside‐binding site of galectins and did not involve interaction with IL‐1 receptor 1 or the induction of endogenous IL‐1β. Consistent with these observations, we demonstrate that small interfering RNA‐mediated suppression of endogenous galectin‐3 expression is sufficient to impair the IL‐1β‐induced secretion of IL‐8 and IL‐6 in a p38 mitogen‐activated protein kinase‐independent manner. Collectively, our findings provide a novel role for galectin‐3 as an amplifier of IL‐1β responses during epithelial inflammation through an as yet unidentified mechanism. [ABSTRACT FROM AUTHOR]

Langerhans' cells ( LC) play pivotal roles in skin immune responses, linking innate and adaptive immunity. In aged skin there are fewer LC and migration is impaired compared with young skin. These changes may contribute to declining skin immunity in the elderly, including increased skin infections and skin cancer. Interleukin-1 β ( IL-1 β) and tumour necrosis factor- α ( TNF- α) are mandatory signals for LC migration and previous studies suggest that IL-1 β signalling may be dysregulated in aged skin. Therefore, we sought to explore the mechanisms underlying these phenomena. In skin biopsies of photoprotected young (< 30 years) and aged (> 70 years) human skin ex vivo, we assessed the impact of trauma, and mandatory LC mobilizing signals on LC migration and gene expression. Biopsy-related trauma induced LC migration from young epidermis, whereas in aged skin, migration was greatly reduced. Interleukin-1 β treatment restored LC migration in aged epidermis whereas TNF- α was without effect. In uncultured, aged skin IL-1 β gene expression was lower compared with young skin; following culture, IL-1 β mRNA remained lower in aged skin under control and TNF- α conditions but was elevated after culture with IL-1 β. Interleukin-1 receptor type 2 ( IL1R2) gene expression was significantly increased in aged, but not young skin, after cytokine treatment. Keratinocyte-derived factors secreted from young and aged primary cells did not restore or inhibit LC migration from aged and young epidermis, respectively. These data suggest that in aged skin, IL-1 β signalling is diminished due to altered expression of IL1B and decoy receptor gene IL1R2. [ABSTRACT FROM AUTHOR]

Ikaros is a transcription factor that regulates lymphocyte development from the level of the haematopoietic stem cell. Lack of Ikaros reduces the ability of progenitor cells to commit to the T-cell lineage, resulting in reduced numbers of early thymic T-cell progenitors and mature T cells. Mature CD4 T cells that lack Ikaros have defects in proliferation, T helper cell differentiation, cytokine expression and the ability to become anergic. A role for Ikaros in the naive T cell has not yet been identified. The receptors interleukin-7 receptor α (IL-7R α) and l-selectin are important for ensuring survival and proper homing of naive T cells, respectively. Here we show that lack of Ikaros leads to reduced expression of these receptors in naive T cells, which impacts their ability to home and survive in response to IL-7. We define the mechanism underlying this phenotype as a requirement for Ikaros in maintenance of expression of Foxo1, a transcriptional regulator that is required for their expression. We also demonstrate that CD4 T cells lacking Ikaros are significantly crippled in their ability to become induced regulatory T cells, a phenotype also linked to reduced Foxo1 expression. Finally, we show that restoring Ikaros function to Ikaros-deficient CD4 T cells increases levels of Foxo1 message. Together, these studies define, for the first time, a role for Ikaros in naive T cells and establish it as the first transcriptional regulator required for maintaining levels of Foxo1 gene expression in these cells. [ABSTRACT FROM AUTHOR]

Human ficolin-2 ( FCN-2) and mouse ficolin-A ( FCN-A, a ficolin-2-like molecule in mouse) are activators of the lectin complement pathway, present in normal plasma and usually associated with infectious diseases, but little is known about the role of FCN-A/2 in inflammatory bowel disease ( IBD). In our present study, we found that patients with IBD exhibited much higher serum FCN-2 levels than healthy controls. In the dextran sulphate sodium-induced acute colitis mouse model, FCN-A knockout mice showed much milder disease symptoms with less histological damage, lower expression levels of pro-inflammatory cytokines [interleukin-6 ( IL-6), IL-1 β and tumour necrosis factor- α (TNF- α)], chemokines ( CXCL1/2/10 and CCL4) and higher levels of the anti-inflammatory cytokine IL-10 compared with wild-type mice. We demonstrated that FCN-A/2 exacerbated the inflammatory pathogenesis of IBD by stimulating M1 polarization through the TLR4/MyD88/ MAPK/ NF- κB signalling pathway in macrophages. Hence, our data suggest that FCN-A/2 may be used as a novel therapeutic target for IBD. [ABSTRACT FROM AUTHOR]

Age affects the immune response to vaccination, with individuals at the extremes of age responding poorly. The initial inflammatory response to antigenic materials shapes the subsequent adaptive response and so understanding is required about the effect of age on the profile of acute inflammatory mediators. In this study we measured the local and systemic inflammatory response after influenza vaccination or infection in neonatal, young adult and aged mice. Mice were immunized intramuscularly with inactivated influenza vaccine with and without the adjuvant MF59 and then challenged with H1N1 influenza. Age was the major factor affecting the inflammatory profile after vaccination: neonatal mice had more interleukin-1 α ( IL-1 α), C-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GMCSF), young adults more tumour necrosis factor- α (TNF), and elderly mice more interleukin-1 receptor antagonist ( IL-1RA), IL-2RA and interferon- γ-induced protein 10 (IP10). Notably the addition of MF59 induced IL-5, granulocyte colony-stimulating factor (G-CSF), Keratinocyte Chemotractant (KC) and monocyte chemoattractant protein 1 (MCP1) in all ages of animals and levels of these cytokines correlated with antibody responses. Age also had an impact on the efficacy of vaccination: neonatal and young adult mice were protected against challenge, but aged mice were not. There were striking differences in the localization of the cytokine response depending on the route of exposure: vaccination led to a high serum response whereas intranasal infection led to a low serum response but a high lung response. In conclusion, we demonstrate that age affects the inflammatory response to both influenza vaccination and infection. These age-induced differences need to be considered when developing vaccination strategies for different age groups. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is a multi-drug resistant, Gram-negative bacteria and infection with this organism is one of the major causes of mortality in intensive care units. Inflammasomes are multiprotein oligomers that include caspase-1, and their activation is required for maturation of interleukin-1 β ( IL-1 β). Inflammasome signalling is involved in host defences against various microbial infections, but the precise mechanism by which A. baumannii activates inflammasomes and the roles of relevant signals in host defence against pulmonary A. baumannii infection are unknown. Our results showed that NLRP3, ASC and caspase-1, but not NLRC4, are required for A. baumannii-induced production of IL-1 β in macrophages. An inhibitor assay revealed that various pathways, including P2X7R, K+ efflux, reactive oxygen species production and release of cathepsins, are involved in IL-1 β production in macrophages in response to A. baumannii. Interleukin-1 β production in bronchoalveolar lavage ( BAL) fluid was impaired in NLRP3-deficient and caspase-1/11-deficient mice infected with A. baumannii, compared with that in wild-type ( WT) mice. However, the bacterial loads in BAL fluid and lungs were comparable between WT and NLRP3-deficient or caspase-1/11-deficient mice. The severity of lung pathology was reduced in NLRP3- deficient, caspase-1/11- deficient and IL-1-receptor-deficient mice, although the recruitment of immune cells and production of inflammatory cytokines and chemokines were not altered in these mice. These findings indicate that A. baumannii leads to the activation of NLRP3 inflammasome, which mediates IL-1 β production and lung pathology. [ABSTRACT FROM AUTHOR]

Interleukin-1 β ( IL-1 β), a potent pro-inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL-1 β is controlled by a multi-protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals ( CC) may promote atherosclerosis development by activation of the inflammasome. High-density lipoprotein ( HDL) has consistently been shown to be anti-atherogenic and to have anti-inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL-1 β secretion in response to cholesterol crystals in THP-1 cells and in human-monocyte-derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro- IL-1 β and NLRP3, and reducing caspase-1 activation. In summary, we have described a novel anti-inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome. [ABSTRACT FROM AUTHOR]

Preterm birth occurs in 10% of pregnancies and is a major cause of neonatal morbidity and mortality. The majority of cases of early preterm labour are associated with infection/inflammation, which places the fetal central nervous system at risk. Targeting immune activation is therefore an appealing therapeutic strategy for the prevention of preterm labour and neonatal brain injury. The expression of many labour-associated and inflammatory-response genes is controlled by the transcription factors nuclear factor-jB (NF-κB) and activator protein-1 (AP-1), which makes them therapeutic targets of interest. Sulfasalazine (SASP) has been shown to inhibit NF-κB and reduce lipopolysaccharide-induced cytokine concentrations in fetal membrane explants and reduce the rate of Escherichia coli-induced preterm labour in mice. Its effects upon AP-1 in the context of pregnancy are unknown. In this study the effect of SASP on interleukin- 1β (IL-1β) -induced NF-κB and AP-1 activity, cytokine production and cyclo-oxygenase-2 (COX-2) expression was examined in amniocytes and myocytes. A supra-therapeutic concentration (5 mM) was required to inhibit IL-1b-induced NF-κB (P < 0<0001) in amniocytes and IL-1β-induced NF-κB (P < 0.01), AP-1 (P < 0.01) and COX-2 (P < 0.05) in myocytes. Despite inhibiting IL-1β-induced cytokines, a basal increase in IL-6 (P < 0.01), IL-8 (P < 0.0001) and tumour necrosis factor-α (TNF-α) (P < 0.001) was seen with 5 mM SASP in amniocytes, and significant cytotoxic effects were seen in myocytes. The therapeutic concentration of 0.015 mM had no inhibitory effects on pro-inflammatory mediators, but led to an augmented response to IL-1β-induced IL-6 (P < 0.01), IL-8 (P < 0.05) and TNF-α (P < 0.05) in amniocytes and IL-8 (P < 0.05) in myocytes. SASP is therefore an unlikely therapeutic candidate for the prevention of inflammation-induced preterm labour. [ABSTRACT FROM AUTHOR]

Zinc is an essential trace element that plays pivotal roles in multiple facets of the immune system. Besides its catalytic and structural roles, zinc alsofunctions as an intracellular signalling molecule, and changes in zinc levels can cause both direct and indirect modulation of immune responses. Further, cytoplasmic levels of bioavailable zinc in immune cells are largely influenced by many extracellular stimuli. Here we provide evidence that zinc represses memory T helper type 17 responses in humans by inhibiting interleukin-1β (IL-1β)-mediated signal. In vitro zinc treatment of CD4+ T cells in the presence of activated monocytes inhibited interferon-c-producing cells and IL-17-producing cells, but not IL-4- producing cells. Of note, production of IL-17+ cells from memory CD4+ T cells, which is significantly up-regulated by lipopolysaccharide-stimulated monocytes, was preferentially repressed by zinc. Increased cytoplasmic zinc in T cells suppressed IL-1β signalling through repression of phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4), so leading to an inhibitory effect on T helper type 17 responses facilitated by monocyte- derived IL-1β in humans. These findings suggest that extracellular zinc bioavailability may affect memory CD4+ T-cell responses by modulating the zinc-mediated signalling pathway. [ABSTRACT FROM AUTHOR]

Over one million newborns die annually from sepsis with the highest mortality in premature and low-birthweight infants. The inflammasome plays a central role in the regulation of innate immunity and inflammation, and is presumed to be involved in protective immunity, in large part through the caspase-1-dependent activation of interleukin-1 β (IL-1 β) and IL-18. Studies in endotoxic shock, however, suggest that endogenous caspase-1 activity and the inflammasome contribute to mortality primarily by promoting excessive systemic inflammatory responses. We examined whether caspase-1 and the inflammasome also regulate neonatal inflammation, host protective immunity and myelopoiesis during polymicrobial sepsis. Neonatal (5-7 days) C57BL/6 and caspase-1/11−/− mice underwent a low-lethality caecal slurry model of intra-abdominal sepsis (LD25-45). Ablation of caspase-1/11, but not apoptosis-associated speck-like protein containing a CARD domain or nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), improved neonatal survival following septic challenge compared with wild-type mice ( P < 0·001), with decreased concentrations of inflammatory cytokines in the serum and peritoneum. Surprisingly, caspase-1/11−/− neonates also exhibited increased bone marrow and splenic haematopoietic stem cell expansion ( P < 0·001), and increased concentrations of granulocyte and macrophage colony-stimulating factors in the peritoneum ( P < 0·001) after sepsis. Ablation of caspase-1/11 signalling was also associated with increased recruitment of peritoneal macrophages and neutrophils ( P < 0·001), increased phagocytosis by neutrophils ( P = 0·003), and decreased bacterial colonization ( P = 0·02) in the peritoneum. These findings suggest that endogenous caspase-1/11 activity, independent of the NLRP3 inflammasome, not only promotes the magnitude of the inflammatory response, but also suppresses protective immunity in the neonate, so contributing to innate immune dysfunction and poor survival in neonatal sepsis. [ABSTRACT FROM AUTHOR]

Enterohaemorrhagic Escherichia coli ( EHEC) O157:H7 infection in humans can cause acute haemorrhagic colitis and severe haemolytic uraemic syndrome. The role of enterohaemolysin (Ehx) in the pathogenesis of O157:H7-mediated disease in humans remains undefined. Recent studies have revealed the importance of the inflammatory response in O157:H7 pathogenesis in humans. We previously reported that Ehx markedly induced interleukin-1 β ( IL-1 β) production in human macrophages. Here, we investigated the disparity in Ehx-induced IL-1 β production between human and mouse macrophages and explored the underlying mechanism regarding the activation of NOD-like receptor family, pyrin domain containing 3 ( NLRP3) inflammasomes. In contrast to the effects on human differentiated THP-1 cells and peripheral blood mononuclear cells, Ehx exerted no effect on IL-1 β production in mouse macrophages and splenocytes because of a disparity in pro- IL-1 β cleavage into mature IL-1 β upon caspase-1 activation. Additionally, Ehx significantly contributed to O157:H7-induced ATP release from THP-1 cells, which was not detected in mouse macrophages. Confocal microscopy demonstrated that Ehx was a key inducer of cathepsin B release in THP-1 cells but not in mouse IC-21 cells upon O157:H7 challenge. Inhibitor experiments indicated that O157:H7-induced IL-1 β production was largely dependent upon caspase-1 activation and partially dependent upon ATP signalling and cathepsin B release, which were both involved in NLRP3 activation. Moreover, inhibition of K+ efflux drastically diminished O157:H7-induced IL-1 β production and cytotoxicity. The findings in this study may shed light on whether and how the Ehx contributes to the development of haemolytic uraemic syndrome in human O157:H7 infection. [ABSTRACT FROM AUTHOR]

Macrophages play important roles in the innate immune system during infection and systemic inflammation. When bacterial lipopolysaccharide ( LPS) binds to Toll-like receptor 4 on macrophages, several signalling cascades co-operatively up-regulate gene expression of inflammatory molecules. The present study aimed to examine whether salt-inducible kinase [ SIK, a member of the AMP-activated protein kinase ( AMPK) family] could contribute to the regulation of immune signal not only in cultured macrophages, but also in vivo. LPS up-regulated SIK3 expression in murine RAW264.7 macrophages and exogenously over-expressed SIK3 negatively regulated the expression of inflammatory molecules [interleukin-6 ( IL-6), nitric oxide ( NO) and IL-12p40] in RAW264.7 macrophages. Conversely, these inflammatory molecule levels were up-regulated in SIK3-deficient thioglycollate-elicited peritoneal macrophages ( TEPM), despite no impairment of the classical signalling cascades. Forced expression of SIK3 in SIK3-deficient TEPM suppressed the levels of the above-mentioned inflammatory molecules. LPS injection (10 mg/kg) led to the death of all SIK3-knockout ( KO) mice within 48 hr after treatment, whereas only one mouse died in the SIK1- KO ( n = 8), SIK2- KO ( n = 9) and wild-type ( n = 8 or 9) groups. In addition, SIK3- KO bone marrow transplantation increased LPS sensitivity of the recipient wild-type mice, which was accompanied by an increased level of circulating IL-6. These results suggest that SIK3 is a unique negative regulator that suppresses inflammatory molecule gene expression in LPS-stimulated macrophages. [ABSTRACT FROM AUTHOR]

The specific function of human skin-resident dendritic cell (DC) subsets in the regulation of immunity or tolerance is still a matter of debate. Langerhans cells (LC) induce anti-viral immune responses but, conversely to dermal DC, maintain tolerance to bacteria. However, the definite function of epidermal LC and cutaneous DC appears even more complex under inflammatory conditions. Here we investigated the immune responses of human immature monocyte-derived DC (MoDC) and LC-like cells (MoLC) upon stimulation with different Toll-like receptor ligands in the presence or absence of pro-inflammatory cytokines tumour necrosis factor- α (TNF- α) and interleukin-1 β (IL-1 β). In MoDC, bacterial antigens selectively up-regulated CD83 and CD86 expression and induced the release of T helper type 1 (Th1) and Th17 cytokines and led to a higher CCR7-dependent migratory capacity compared with a low responsiveness of MoLC. Importantly, MoLC activation with lipopolysaccharide under inflammatory conditions strongly enhanced a phenotypically mature state, increased IL-12p70, IL-23 and IL-6 production and Th1 cytokine secretion by CD4+ T cells. Treatment with poly(I:C) specifically up-regulated surface expression of co-stimulatory molecules and increased release of IL-12p70 in MoLC and co-stimulation with TNF- α and IL-1 β further elevated Th1 and Th17 cytokine production. Poly(I:C)-induced up-regulation of type I interferon mRNA levels in MoLC and MoDC was Toll-like receptor 3-dependent but not, or only weakly, modulated by pro-inflammatory cytokines. Our results indicate that inflammatory conditions greatly facilitate recognition of bacteria by MoLC. Furthermore, we suggest a critical involvement of both subsets in innate defence against viruses, whereas inflammatory skin environments additionally favour MoLC as potent inducers of Th1 and Th17 cytokines. [ABSTRACT FROM AUTHOR]

Pancreatitis is caused by long-term heavy alcohol consumption, which results in injury and death of pancreatic acinar cells ( PAC). The PAC play a pivotal role in mediating early inflammatory responses but the underlying mechanisms remain poorly understood. Treatment of C57BL/6 mice with ethanol and cerulein resulted in increased staining for acinar interleukin-1 β (IL-1 β), chemokine (C-C motif) ligand 3 ( CCL3), or connective tissue growth factor ( CTGF/ CCN2) by Day 16 and this was associated with increased infiltration of F4/80-positive macrophages and increased expression of pancreatic CTGF/ CCN2 mRNA. Compared with wild-type Swiss Webster mice, ethanol treatment of pan-green fluorescent protein ( GFP)- CTGF/ CCN2 transgenic mice caused enhanced acinar staining for GFP or CTGF/ CCN2 and a significant increase in pancreatic infiltration of F4/80-positive macrophages or NIMP-R14-positive neutrophils. Treatment of primary mouse PAC or the rat AR42J PAC line with ethanol or CTGF/ CCN2 resulted in enhanced expression of IL-1 β or CCL3. Conditioned medium from CTGF/ CCN2-treated AR42J cells induced chemotaxis in NR8383 macrophages and this response was abrogated in a dose-dependent manner by addition of BX471, an inhibitor of chemokine (C-C motif) receptor 1. These results reveal that acinar CTGF/ CCN2 plays a novel role in alcohol-induced inflammatory processes in the pancreas by increasing infiltration of macrophages and neutrophils and increasing acinar production of inflammatory mediators such as IL-1 β or CCL3. The early production of CTGF/ CCN2 by PAC to drive inflammation is distinct from its previously reported production by pancreatic stellate cells to drive fibrosis at later stages of pancreatic injury. [ABSTRACT FROM AUTHOR]

Ragweed pollen extract ( RWE) possesses intrinsic NADPH oxidase activity that induces oxidative stress by initiating the production of intracellular reactive oxygen species ( ROS). The ROS are important contributors to the manifestation of allergic inflammation; furthermore, concomitant exposure to an allergen and an endotoxin trigger a stronger inflammatory response. One of the main pro-inflammatory cytokines produced in inflammatory responses is interleukin-1β ( IL-1β), and its production is associated with caspase-1-containing inflammasome complexes. Intracellular ROS have been implicated in NLRP3 inflammasome-mediated IL-1β production, therefore, we aimed to study whether RWE influences the function of NLRP3 inflammasome. Here we describe that, in the presence of NADPH, RWE significantly elevates lipopolysaccharide-induced IL-1β production of THP-1 cells as well as human primary macrophages and dendritic cells. We also demonstrate that increased IL-1β production is mediated through NLRP3 inflammasome in THP-1 macrophages. We provide evidence that RWE elevates cytosolic ROS level in these cells, and ROS inhibitors abolish IL-1β production. Furthermore, we show that RWE enhances lipopolysaccharide-induced gene transcription/expression of pro- IL-1β and key components of the inflammasome via a ROS-dependent mechanism. [ABSTRACT FROM AUTHOR]

Summary Oxidized low-density lipoprotein (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to the formation of lipid-laden macrophages (foam cells). Fcγ receptors mediate uptake of oxLDL-IC, whereas scavenger receptors internalize oxLDL. We have previously reported that oxLDL-IC, but not free oxLDL, activate macrophages and prolong their survival. Sphingomyelin is a major constituent of cell membranes and lipoprotein particles and acid sphingomyelinase (ASMase) hydrolyses sphingomyelin to generate the bioactive lipid ceramide. ASMase exists in two forms: lysosomal (L-ASMase) and secretory (S-ASMase). In this study we examined whether oxLDL and oxLDL-IC regulate ASMase differently, and whether ASMase mediates monocyte/macrophage activation and cytokine release. The oxLDL-IC, but not oxLDL, induced early and consistent release of catalytically active S-ASMase. The oxLDL-IC also consistently stimulated L-ASMase activity, whereas oxLDL induced a rapid transient increase in L-ASMase activity before it steadily declined below baseline. Prolonged exposure to oxLDL increased L-ASMase activity; however, activity remained significantly lower than that induced by oxLDL-IC. Further studies were aimed at defining the function of the activated ASMase. In response to oxLDL-IC, heat-shock protein 70B' (HSP70B') was up-regulated and localized with redistributed ASMase in the endosomal compartment outside the lysosome. Treatment with oxLDL-IC induced the formation and release of HSP70-containing and IL-1β-containing exosomes via an ASMase-dependent mechanism. Taken together, the results suggest that oxLDL and oxLDL-IC differentially regulate ASMase activity, and the pro-inflammatory responses to oxLDL-IC are mediated by prolonged activation of ASMase. These findings may contribute to increased understanding of mechanisms mediating macrophage involvement in atherosclerosis. [ABSTRACT FROM AUTHOR]

There is increasing evidence that activation of inflammatory responses in a variety of tissues is mediated co-operatively by the actions of more than one cell type. In particular, the monocyte has been implicated as a potentially important cell in the initiation of inflammatory responses to Toll-like receptor (TLR)-activating signals. To determine the potential for monocyte-regulated activation of tissue cells to underpin inflammatory responses in the vasculature, we established cocultures of primary human endothelial cells and monocytes and dissected the inflammatory responses of these systems following activation with TLR agonists. We observed that effective activation of inflammatory responses required bidirectional signalling between the monocyte and the tissue cell. Activation of cocultures was dependent on interleukin-1 (IL-1). Although monocyte-mediated IL-1β production was crucial to the activation of cocultures, TLR specificity to these responses was also provided by the endothelial cells, which served to regulate the signalling of the monocytes. TLR4-induced IL-1β production by monocytes was increased by TLR4-dependent endothelial activation in coculture, and was associated with increased monocyte CD14 expression. Activation of this inflammatory network also supported the potential for downstream monocyte-dependent T helper type 17 activation. These data define co-operative networks regulating inflammatory responses to TLR agonists, identify points amenable to targeting for the amelioration of vascular inflammation, and offer the potential to modify atherosclerotic plaque instability after a severe infection. [ABSTRACT FROM AUTHOR]

The role of neutrophils in the immune response has long been regarded as mainly phagocytic, but recent publications have indicated the production of several cytokines by polymorphonuclear leucocytes (PMN). The results of the individual reports, however, vary considerably. In this study, we established a cytokine profile of pure human neutrophils and demonstrated that minor contamination of peripheral blood mononuclear cells (PBMCs) in PMN preparations can lead to false-positive results. In our hands, peripheral blood PMN fail to produce the pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumour necrosis factor-α (TNF-α). Instead, they secrete large amounts of the chemokine IL-8 and the anti-inflammatory IL-1 receptor antagonist (IL-1ra). Additionally, PMN preparations of a high purity show production of the chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β and growth-related oncogene-α (GRO-α), as well as macrophage colony-stimulating factor (M-CSF). The neutrophil therefore represents a novelty by producing the antagonist of IL-1β (i.e. IL-1ra) in the absence of IL-1β itself. To support our results, we differentiated stem cells from human cord blood into PMN and monocytes, respectively. These in vitro-differentiated PMN showed the same cytokine profile as peripheral blood PMN lacking IL-1β, while differentiated monocytes produced the expected IL-1β in addition to IL-1ra. The clear anti-inflammatory nature of their cytokine profile enables PMN to antagonize pro-inflammatory signals in experimental conditions. It is therefore possible that PMN play a key role in immune regulation by counteracting a dysregulation of the inflammatory process. Clinical studies, in which administration of recombinant G-CSF had a favourable effect on the outcome of severe infections and even sepsis without worsening inflammation, could thus be explained by our results. [ABSTRACT FROM AUTHOR]

In this study, we investigated how pregnancy influences cytokine production in response to stimulation of the innate and the adaptive immune system, respectively. Peripheral blood mononuclear cells (PBMCs) from allergic ( n = 44) and non-allergic ( n = 36) women were collected at three time-points: during the third trimester, at delivery and at a non-pregnant state 2 years after delivery. The production of interleukin-1β (IL-1β), IL-6, IL-10 and IL-12 was measured by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot assay (ELISPOT). The spontaneous cytokine production, and the response following stimulation with agents that primarily activate the adaptive part of the immune system [phytohaemagglutinin (PHA), allergen extracts from cat and birch], or lipopolysaccharide (LPS) that activate innate immunity was measured in vitro. There was a significantly higher spontaneous in vitro production of IL-1β, IL-6 and IL-10 by PBMCs during pregnancy than 2 years after pregnancy, and this was not affected by the allergic status of the women. Conversely, in PHA-stimulated cell cultures there was a lower production of IL-10 and IL-12 during pregnancy than 2 years after pregnancy. LPS-induced IL-6 levels were significantly lower in PBMCs obtained during pregnancy than at 2 years after pregnancy. In addition, we made the interesting observation that in allergic women total immunoglobulin E (IgE) levels were significantly lower 2 years after pregnancy compared to the levels during pregnancy. Taken together, our results indicate that while atopic allergy in women does not have a substantial effect on cytokine production, pregnancy has an obvious effect on the immune system in terms of cytokine production as well as on the total IgE levels. [ABSTRACT FROM AUTHOR]

The intraperitoneal injection of lipopolysaccharide (LPS) (400 µg/kg body weight) induced the expression of mRNAs of inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α in the submandibular gland (SMG) of C3H/HeN mice but not that of C3H/HeJ mice, a mutant strain for Toll-like receptor-4 (TLR-4– mutant). The mRNA levels of these cytokines in the SMG of the wild-type mice increased as early as 3 hr after injection, peaked at 3–6 hr, and had decreased again by 24 hr. In this study, we particularly focused on IL-1β, and induction by this endotoxin was investigated in detail. Denervation of the superior cervical trunk and chorda tympani nerve did not diminish the LPS-induced elevation of IL-1β mRNA in the SMG, indicating the irrelevance of the central nervous system in this induction. TLR-4 mRNA and protein were shown to be strongly expressed in the SMG, suggesting the direct action of LPS on this gland. IL-1β proteins were localized in the secretory granules of granular convoluted tubular (GCT) cells, and their molecular weights in the gland were 17·5 and 20 kDa. IL-1β of the same size appeared in the saliva 6 hr after LPS injection in C3H/HeN but not in C3H/HeJ mice. The present study thus suggests that IL-1β, an inflammation cytokine, is induced and secreted into the saliva in response to endotoxin injected intraperitoneally. [ABSTRACT FROM AUTHOR]

We have previously reported that human neutrophils pretreated with tumour necrosis factor-α (TNF-α) and then exposed to a variety of agents such as immune complexes, zymosan, phorbol 12-myristate 13-acetate (PMA), C5a, fMLP, or granulocyte–macrophage colony-stimulating factor (GM-CSF), undergo a dramatic stimulation of apoptosis, suggesting that TNF-α is able to prime an apoptotic death programme which can be rapidly triggered by different stimuli. We report here that this response involves the participation of Mac-1 (CD11b/CD18), is dependent on caspases 3, 8 and 9, and is associated with both a loss of mitochondrial transmembrane potential and a down-regulation in expression of the anti-apoptotic protein, Mcl-1. Interestingly, we also found that the anti-apoptotic cytokine interleukin-1 (IL-1) improves the ability of TNF-α to promote apoptosis, supporting the notion than TNF-α, acting together with IL-1, may favour the depletion of neutrophils from the inflammatory areas during the course of acute inflammation. [ABSTRACT FROM AUTHOR]

The aim of this study was to construct and purify a novel interleukin-1 receptor antagonist (IL-1ra)–interleukin-10 (IL-10) fusion protein and determine its biological function and anti-inflammatory effects. The isolated cDNAs of two inhibitory cytokines (IL-1ra, IL-10) were used to construct a cDNA for the IL-1ra–IL-10 fusion protein. The expressed recombinant cytokines and fusion product were purified and their biological properties analysed. The anti-IL-1 effect was evaluated by using a thymocyte-proliferation assay, and the IL-10 effect was investigated by the inhibition of interferon-γ (IFN-γ) production from splenocytes. The clinical response and histological analyses were studied in an adjuvant arthritic rat model. The fusion protein was 38 000 molecular weight in size. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting demonstrated that the purified protein was recognized by both IL-1ra and IL-10 antibodies. The fusion protein significantly inhibited IL-1-mediated thymocyte proliferation and concanavalin A (ConA)-primed IFN-γ production from splenocytes. The fusion protein also suppressed joint swelling (paw circumference reduced from 5·0 ± 0·2 to 4·1 ± 0·1 cm; paw thickness ≈ 2 mm in difference) and synovial inflammation in adjuvant arthritis of rats. Our investigations indicate that this fusion protein effectively suppresses inflammatory arthritis and may initiate a trend for future clinical application to target multiple molecules at the same time. [ABSTRACT FROM AUTHOR]

Summary Previous studies in mice have reported a decrease in epidermal Langerhans cell (LC) density in aged skin, however, the impact of this reduction on LC function and cutaneous immune responses is unclear. In the present series of experiments, the frequency of major histocompatibility complex class II+ LC in the epidermis of older (6-month-old) mice was found to be reduced significantly compared with that observed for young (6–8-week-old) mice. LC mobilization and the subsequent accumulation of dendritic cells (DC) in regional lymph nodes in response to topical challenge with a chemical allergen were found to be less vigorous in older mice. Flow cytometric analyses of DC derived from the draining lymph nodes of fluorescein isothiocyanate (FITC)-sensitized mice revealed that the frequency of FITC+ -DC arriving in draining lymph nodes was also reduced in older mice but that the fluorescence intensity was comparable. Control and allergen-treated-older mice also displayed decreased total lymph node cellularity. Contact hypersensitivity responses were found not to be compromised in older mice. However, the cytokine regulation of LC migration in the two age groups of mice did differ. LC migration provoked by intradermal injection of tumour necrosis factor-α (TNF-α) was reduced in older animals, whereas, the percentage of LC that migrated in response to exogenous interleukin-1β (IL-1β) was comparable for both young and aged mice. Since both allergen- and TNF-α-induced LC responses are known to require receipt by LC of a signal from IL-1β for effective migration, the suggestion is that impaired LC migration in older mice may be due to a reduced availability of epidermal IL-1β. [ABSTRACT FROM AUTHOR]

Summary Interleukin-1 (IL-1) consists of two molecules, IL-1α and IL-1β, and IL-1 receptor antagonist (IL-1Ra) is a natural inhibitor of these molecules. Although the adjuvant effects of exogenously administered IL-1 in the humoral immune response are well known, the roles of endogenous IL-1 and the functional discrimination between IL-1α and IL-1β have not been elucidated completely. In this report, we investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Both primary and secondary antibody production against T-dependent antigen, sheep red blood cells (SRBC), was significantly reduced in IL-1α/β-/- mice, and was enhanced in IL-1Ra-/- mice. The intrinsic functions of B cells, such as antibody production against type 1 T-independent antigen, trinitrophenyl–lipopolysaccharide and proliferative responses against mitogenic stimuli, were normal in IL-1α/β-/- mice. The proliferative response of T cells and cytokine production upon stimulation with anti-CD3 monoclonal antibody were also normal, as was the phagocytotic ability of antigen-presenting cells (APCs). However, SRBC-specific proliferative response and cytokine production of T cells through the interaction with APCs were markedly impaired in IL-1α/β-/- mice, and enhanced in IL-1Ra-/- mice. Moreover, we show that SRBC-specific antibody production was reduced in IL-1β-/- mice, but not in IL-1α-/- mice. These results show that endogenous IL-1β, but not IL-1α, is involved in T-cell-dependent antibody production, and IL-1 promotes the antigen-specific T-cell helper function through the T-cell–APC interaction. [ABSTRACT FROM AUTHOR]

SummaryWe investigated the changes which occur in gene expression in the human macrophage cell line, THP1, at 1, 6 and 12 hr following infection with Mycobacterium tuberculosis. The analysis was carried out at the transcriptome level, using microarrays consisting of 375 human genes generally thought to be involved in immunoregulation, and at the proteomic level, using two-dimensional gel electrophoresis and mass spectrometry. The analysis of the transcriptome using microarrays revealed that many genes were up-regulated at 6 and 12 hr. Most of these genes encoded proteins involved in cell migration and homing, including the chemokines interleukin (IL)-8, osteopontin, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), regulated on activation, normal, T-cell expressed and secreted (RANTES), MIP-1β, MIP-3α, myeloid progenitor inhibitory factor-1 (MPIF-1), pulmonary and activation regulated chemokine (PARC), growth regulated gene-β (GRO-β), GRO-γ, MCP-2, I-309, and the T helper 2 (Th2) and eosinophil-attracting chemokine, eotaxin. Other genes involved in cell migration which were up-regulated included the matrix metalloproteinase MMP-9, vascular endothelial growth factor (VEGF) and its receptor Flk-1, the chemokine receptor CCR3, and the cell adhesion molecules vesicular cell adhesion molecule-1 (VCAM-1) and integrin a3. In addition to the chemokine response, genes encoding the proinflammatory cytokines IL-1β (showing a 433-fold induction), IL-2 and tumour necrosis factor-α (TNF-α), were also found to be induced at 6 and/or 12 hr. It was more difficult to detect changes using the proteomic approach. Nevertheless, IL-1β was again shown to be strongly up-regulated. The enzyme manganese superoxide dismutase was also found to be strongly up-regulated; this enzyme was found to be macrophage-, rather than M. tuberculosis, derived. The heat-shock protein hsp27 was found to be... [ABSTRACT FROM AUTHOR]

Summary Multinucleated giant cells (MGC) are a common feature of granulomas. The mechanism of their formation has been studied extensively, but their function has not been completely characterized. A new method for the in vivo production of MGC was developed involving subcutaneous injection of microscopic nitrocellulose particles with adsorbed mycobacterial antigens into the footpads of sensitized BALB/c mice (immune [I]-MGC), or by nitrocellulose administration to non-sensitized mice (foreign body [FB]-MGC). The development of granulomas with a highly enriched MGC population was observed 2 weeks after the nitrocellulose injection. MGC were larger with a greater number of nuclei in I-MGC than in FB-MGC. From days 7–28 after nitrocellulose administration, the production of interleukin-1α (IL-1α) and tumour necrosis factor-α (TNF-α) was demonstrated in both MGC types by in situ reverse transcription–polymerase chain reaction (RT–PCR) and immunohistochemistry. After 2 months, the MGC had ceased production of IL-1α and TNF-α, but the expression of transforming growth factor-β (TGF-β) was very high, occurring together with extensive fibrosis. These results suggest that MGC are an active source of inflammatory cytokines, which can contribute to the initiation, maintenance and down-regulation of granulomatous inflammation induced by immunological and inert substances. [ABSTRACT FROM AUTHOR]

Extracellular matrices (ECMs) can stimulate human monocytic cells to express interleukin-1β (IL-1β), a proinflammatory cytokine implicated in the regulation of tissue inflammation. In this study, we explored the intracellular mechanisms responsible for ECM induction of IL-1β using human promonocytic U937 cells transfected with the full-length human IL-1β gene promoter connected to a reporter gene. Using this system, we demonstrated that the ECM molecules fibronectin (FN), type I collagen (Coll), fibrin (Fb) and laminin (Lm) induced transcription of the IL-1β gene (which was associated with a modest increase in IL-1β protein secretion) in suspended cells, when used in their soluble monomeric form. This effect was mimicked or blocked by anti-integrin monoclonal antibodies (mAbs) and was dependent on the activation of protein kinase C (PKC). Three of the ECMs tested (FN, Coll and Fb) induced the activation of mitogen-activated protein kinases (MAPKs), whereas Lm had no effect. FN, Coll and Fb (but not Lm) also induced DNA binding of the transcription factor activator protein-1 (AP-1), but not that of nuclear factor-κB. Co-transfection of U937 cells with a competing AP-1 oligomer blocked the IL-1β response induced by FN, but not that induced by the other ECMs. By inhibiting AP-1 translocation, glucocorticoids also blocked the FN-induced response, but not that of the other ECMs. These studies suggest that the signalling pathways which mediate ECM induction of IL-1β expression in human monocytic cells converge at the level of PKC activation. However, they diverge in other aspects, as demonstrated by the differential activation of MAPKs and the need for diverse transcription factors. [ABSTRACT FROM AUTHOR]

Bacterial endotoxin increased the expression of mRNA (maximal after 4 hr) for interleukin-1β (IL-1β) and the release of mature protein from intact human fetal membranes. In contrast, the change in expression of mRNA for type 2 cyclo-oxygenase (COX-2) was biphasic, with peaks after 0·5–1 hr and after 8 hr of culture. An antibody to IL-1β was without effect after 4 hr of culture, inhibited endotoxin-stimulated prostaglandin E2 (PGE2) production after 8 hr of culture, and caused a parallel decrease in the expression of mRNA for COX-2. We conclude that endotoxin induced the expression of COX-2 through IL-1β-independent and IL-1β-dependent mechanisms, and these differences are time dependent. Corticotrophin-releasing hormone (CRH) or platelet-activating factor (PAF) also increased the expression of mRNA for IL-1β and the release of IL-1β from some, but not all, fetal membranes. The antibody to IL-1β did not affect CRH-stimulated or PAF-stimulated PGE2 production or COX-2 expression. We conclude that CRH and PAF can induce the expression of IL-1β, but this is not obligatory for increased PGE2 release, and the effect of these stimuli on COX-2 expression is a direct, IL-1β-independent effect. [ABSTRACT FROM AUTHOR]

Purified, monocyte-derived interleukin-l (IL-l) was found to provide growth support for Epstein- Barr virus (EBV) transformed B-lymphocytes seeded at densities below which their own autostimulatory factors were limiting. By contrast, highly purified resting B cells triggered via their receptors for antigen failed to respond to identical preparations of IL-l by DNA synthesis. That successful priming of the B cells had occurred was evidenced by a transient rise in RNA synthesis and the ability of the cells to respond to T-cell supernatants by DNA synthesis. The findings indicate that while IL- l might perform an autostimulatory function in B lymphocyte proliferation it is not by itself sufficient to provide growth support for resting B cells activated through their receptors for antigen. The implications of these observations for autocrine models of B-cell growth are discussed. [ABSTRACT FROM AUTHOR]

This study investigates the effect of interferons on the induction of human monocyte secretion of interleukin-1 (IL-1) activity by lipopolysaccharides (LPS). Monocytes do not spontaneously produce IL-1 and the addition of interferons to the culture does not lead to detectable secretion. Addition of LPS alone induces the release of measurable amounts of IL-1 activity. The addition of low doses (1-10 units/mI) of alpha, beta, or gamma interferon to the LPS-stimulated cultures further increases this secretion by 50%. The addition of 1000 units/ml of alpha or beta interferon leads to inhibition of IL-1 release. By contrast, gamma interferon is a dose dependent enhancer of IL-1 release. The effect of gamma interferon is on the production of IL-1 and is not an enhancement of IL-1 activity in the biological assay. Results demonstrate that addition of gamma interferon to monocytes increases the rate of secretion of IL-1 by these cells. Gamma interferon also appears to abrogate the loss in the ability of monocytes to produce IL-1 activity after overnight culture. This last result parallels the maintenance of the expression of the HLA-DR surface marker on monocytes by gamma interferon. These results may help define a mechanism involving IL-1 generation which could have bearing on the in vivo pyrogenic effects of purified gamma interferon. [ABSTRACT FROM AUTHOR]

The injection of BCG vaccine in C57BL/6J mice results in the suppression of the generation of cytotoxic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC) and of mitogenic rections to concanavalin A (Con A). Suppression is mediated by macrophage-like suppressor cells. Since previous work had indicated that suppression involved the inhibition of the production of interlcukin-2 (IL-2), the effects of BCO on interleukin-l (IL-1), a monokine required for IL-2 production, were investigated. It was found that the release of IL-l-like activity in spleen cell cultures stimulated with LPS or Con A was increased by previous BCG treatment of the cell donors. In MLC, the release of IL-l-like activity was also increased by BCG. However, the detection of IL- l -like activity in MLC supernatants was prevented by the presence of a suppressor factor. In this case, the IL-l- like activity could be separated with gel filtration from the suppressor factor which had higher molecular weight. The production of IL-I-like activity by CBA/J spleen cells, which are not suppressed by 8CC, was not significantly different from that of C57BL/63 cells, which are markedly suppressed. Moreover, the addition of IL-I to the BCG-suppressed cultures not only did not restore normal reactivity, but actually further suppressed CTL formation. It was concluded that BCG- induced suppression cannot be attributed to decreased IL-i activity. The suppressor factor discovered during these investigations may have a role in this type of suppression. [ABSTRACT FROM AUTHOR]

The faster degradation of antigen by macrophages in Biozzi low (L) responder mice, compared to Biozzi high (H) responder mice, is thought to be responsible for their lower antibody response. We have measured four functions associated with macro- phages to see whether macrophages from L mice were generally more active than those from H mice. Peritoneal macrophages obtained from normal mice were compared with those from groups of mice given Mycobacterium boris BCG or Propionibacteriumacnes. Cells from normal H mice gave a stronger oxidative burst when triggered with phorbol myristate acetate, and were more cytotoxic for tumour cells than cells from L mice. Cells from all mice injected with 8CC or P. acnes gave a stronger oxidative burst, and were more cytotoxic for tumour cells; again, both responses were higher in H mice than in L mice. By contrast, when groups of mice that had received P. aenes were given endotoxin and bled, higher titres of tumour necrosis factor were found in the sera of L mice. Spleen cells from both lines of mice released similar levels of interleukin-1, both spontaneously and in response to lipopolysaccharide. Our results suggest that these various macrophage responses are expressed independently in H and L mice. [ABSTRACT FROM AUTHOR]

The decrease in T-cell proliferation with age is due, in part, to the decline in the production of IL-2. Since IL-1 is needed to trigger IL-2 production, we determined the IL-I producing capacity of peritoneal macrophages of young (2-4 months) and old (24-26 months) BALB/c and C57BL/6 mice. Mice were stimulated with LPS, and their peritoneal macro-phages were obtained 3 days later, purified, and assessed for IL-1 production by coculturing them with splenic I cells at a ratio of 1 :5 in the presence of LPS. Supernatants were obtained 4 days later when the PGE2 and IL-2 activities were minimal and IL-1 activity maximal. IL-1 activity was assessed for their ability to augment the proliferative activity of indicator thymocytes in their response to PHA stimulation. The results revealed that (i) IL-1 production by cells of old BALB/c and C57BL/6 mice is reduced to about 40% and 30% that of young mice, respectively; (ii) indomethacin enhances IL-1 production by cells of both young and old mice to the same extent; and (iii) reduction in the IL-1 producing capacity by cells of old mice results from altered activities of both the IL-1 producing peritoneal macrophages and the augmenting T cells. [ABSTRACT FROM AUTHOR]

The present report examines the behaviour of a slowly proliferating pure population of thymus-derived macrophages in long-term culture, regarding their ability to secrete interleukin-l (IL-I) and prostaglandin E2 (PGE2), to stimulate a mixed leucocyte reaction (MLR) and to lyse tumour cells in vitro. Following stimulation with LPS, high levels of IL-I were released to the medium. IL- I release was significantly augmented by the addition of indomethacin during stimulation. Thymus-derived macrophages constitutively secreted significant levels of PGE2. These cells served as excellent stimulators in a one-way MLR, substantiating the claim that pure populations of macrophages effectively stimulate an allogeneic response in vitro. Thym us-derived macrophages showed tumoricidal activity following activation with either high concentrations of LPS or suboptimal concentrations of LPS and 1-cell lymphokine. These findings portray a close interrelationship and reciprocal regulation between thymus-derived macro- phages and T lymphocytes. [ABSTRACT FROM AUTHOR]

Human T lymphocyte colonies were grown in methylcellulose semi-solid cultures in the presence of phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA). Surface marker analysis showed lower percentages of OKT3- and OKT4-positive cells in PMA-induced colonies than those in PHA-indoced colonies. The percentage of OKIa1-positive cells in PMA-induced colonies was approximately twice that in PHA-induced colonies The percentage of OKT9- positive cells in PMA-induced colonies was significantly lower than that in PHA-induced colonies. These data suggest that the subsets of PMA-induced colony cells express a more immature phenotype than that of PHA-induced colony cells and that, among PMA-induced colony cells, there are fewer T cells in the proliferative status at the time tested. When 3 × 105/ml monocyte-depleted T cells, at which concentration of seeded cells neither PHA nor PMA could induce colony growth, were cultured in the presence of both PHA and PMA. T cell colony growth was observed. In T cell colonies induced by a combination of PHA and PMA, the percentages of OKT3-, OKT4- and OKT8-positive cells were different from those in colonies induced by either PHA or PMA alone. These results suggest that PMA acts not only as a substitute for monocytes and/or interleukin-1, but may directly affect lymphocyte proliferation induced by a combination of PHA and PMA. [ABSTRACT FROM AUTHOR]

The specific primary in vitro plaque-forming cell (PFC) response of C57B1/6 nu/nu spleen cells to the Type 2 T-independent (TI-2) antigen DAGG-Ficoll was analysed in the absence of T cell help in a serum-free medium. Lipopolysaccharide (LPS) enhancement of the antigen-specific response was shown to be mediated by soluble factors contained in the supernatants of LPS-induced bone marrow-derived macrophages. The activity of these supernatants was not associated with residual LPS, since the antigenspecific response of B cells from mice genetically deficient in LPS receptors was equally well enhanced. The activity of these supernatants was associated with interleukin-1 (IL-1) and partially purified IL-1 prepared from P388D1 cells also enhanced the primary in vitro response to DAGG-Ficoll. Limiting dilution analysis experiments showed that only in the presence of exogenously added IL-1 could a single cell type, presumably the B cells, be shown to be limiting. [ABSTRACT FROM AUTHOR]

Mice injected with Propionibacterium acnes, when challenged with lipopolysaccharide release a range of soluble mediators into their serum. Included among these is lymphocyte activating factor (LAF, interleukin-l). The release of LAP in vivo was detected only when serum samples were assayed at high dilution because inhibitors of its activity in vitro were also present. The kinetics of release of LAF in viva after injection of P. acnes was dependent on the mouse strain used. LAP was also detected in serum collected from nude mice, implying that the LAF activity measured in vitro was not due to contamination with T cell products, such as interleukin 2. [ABSTRACT FROM AUTHOR]

Reports on the kinetics of the potentiation of phytohemagglutinin-induced DNA synthesis by rat lymph node cells in the presence of non-mitogenic lymphocyte activating factor (LAF) produced by rat peritoneal macrophages in serum-free cultures. Maximal potentiating effect of LAF when added to lymph node cell cultures between zero and 24 hours after their initiation; Finding that 2-mercaptoethanol did not substitute for the biological activity of rat LAF.

Culture fluids of murine peritoneal cells contain two distinct, non-dialysable principles (LAF) with thymocyte proliferation-stimulating properties. One of them is elaborated from phagocytic cells (presumably macrophages), the other is released by non-phagocytic cells (lymphocytes). cGMP added exogenously stimulates the production and/or release of LAF from phagocytic cells, but not from non-phagocytic cells. The phagocytic cells (but not the lymphocytes) require intact RNA and protein synthesis for LAF release. The action of LAF differs from those of cGMP itself by being nondialysable, and unable to prevent the inhibitory action of cAMP on mitogen-stimulated lymphocyte proliferation. [ABSTRACT FROM AUTHOR]

Immune mechanisms, including production of pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF), play an important role in early atherogenesis. The study of the mechanisms responsible for the increased cytokine production capacity of hypercholesterolemic hosts is therefore crucial for finding new strategies aimed to stop the development of atherosclerosis. We assessed the lipopolysaccharide (LPS)-induced cytokine production of macrophages from low-density lipoproteins (LDL)-receptor knock-out (LDLR-/-) mice, which have a seven- to ninefold higher plasma LDL concentration. Macrophages of LDLR-/- mice produced approximately twofold more IL-1α and IL-1β in response to LPS when compared with macrophages of control mice (LDLR+/+). TNF-α synthesis was only slightly increased. Removal of CD14 by phospholipase C treatment of cells decreased cytokine production by 50% (IL-1) to 80% (TNF), but the differences between LDLR-/- and LDLR+/+ remained the same. In contrast, treatment of cells with anti-CD11c monoclonal antibody inhibited the IL-1α and IL-1β production in LDLR-/- mice towards normal values, while no effect could be seen on TNF. In conclusion, LDLR-/- macrophages stimulated with LPS synthesize more IL-1α and IL-1β than controls and this phenomenon is mediated by the CD11c/CD18 receptor. [ABSTRACT FROM AUTHOR]

Using a semi-quantitative multiplex reverse transcription–polymerase chain reaction assay, we examined cytokine mRNA expression for interleukin-1α (IL-1α), IL-2, IL-10, IL-12p40, tumour necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in skin samples obtained from C4-deficient (C4D) guinea-pigs inoculated intradermally with virulent Treponema pallidum (VTP). Controls included unmanipulated animals, guinea-pigs injected with T. pallidum-free rabbit inflammatory testicular fluid (ITF) alone, or mixed with heat-killed organisms (HKTP). The expression of IL-1α, IL-12p40, and TNF-α mRNA [T helper type 1 (Th1)] remained within the normal range in both infected and control animals throughout the experimental period. However, a significant increase (P<0·05) in IL-10 mRNA (Th2) was found exclusively in the VTP-inoculated animals from 3 to 30 days post-infection. Another unique characteristic of the inflammatory response in infected guinea-pigs was the appearance, between 11 and 30 days post-inoculation, of a substantial number of eosinophils in addition to infiltrating mononuclear cells. The results showed a local Th2 response which is consistent with an inadequate immune response. This is reflected by the lengthy and incomplete clearance of the pathogen from the local site of entry and the chronic infection of distant organs. [ABSTRACT FROM AUTHOR]

Human monocytic cells express interleukin-1β (IL-1β) when stimulated with the extracellular matrix glycoprotein, fibronectin (FN). Protein kinase C (PKC) activation is considered important for this process; however, the metabolic steps at which PKC acts upon to mediate the FN-induced IL-1β response remain unclear. We performed an analysis of the mechanisms by which two PKC inhibitors, Calphostin C and Staurosporine, prevent the FN-induced IL-1β response. Both inhibitors blocked the secretion of IL-1β protein into the media of peripheral blood mononuclear cells exposed to FN. Immunoprecipitation analysis revealed that under these circumstances, Calphostin C inhibited the production of IL-1β protein, whereas Staurosporine allowed protein production, but inhibited its secretion. To determine the mechanisms responsible for these differences, we turned to human U937 promonocytic cells. U937 cells transfected with the human full-length IL-1β promoter connected to a luciferase reporter gene were submitted to transcription assays, Northern blotting, and DNA electrophoresis mobility gel shift assays. These studies revealed that Calphostin C inhibited the nuclear translocation of the transcription factor activator protein-1 (AP-1) which is considered necessary for FN induction of IL-1β gene transcription, and prevented the transcription of the IL-1β gene. In contrast, Staurosporine alone induced AP-1 translocation and stimulation of the gene. Overall, our data indicate that Calphostin C prevents the transcription of the IL-1β gene thereby inhibiting protein synthesis. Based on the high specificity of this compound for PKC, we conclude that PKC is necessary for FN-induced IL-1β protein production. In contrast, Staurosporine prevented secretion of IL-1β by unknown mechanisms. [ABSTRACT FROM AUTHOR]

The balance between pro- and anti-inflammatory cytokines has been implicated in the pathogenesis of infectious and auto-immune diseases, and its modulation has been proposed as a potential therapeutic target. The results reported in the present study show that modulators of the renin–angiotensin system, such as the angiotensin-converting enzyme (ACE)-inhibitor captopril and the angiotensin II receptor type I antagonist valsartan, have potent inhibitory effects on the lipopolysaccharide (LPS)-stimulated production of pro-inflammatory cytokines tumour necrosis factor (TNF) and interleukin-1 (IL-1) in vitro. The anti-inflammatory cytokine IL-1Ra is increased by captopril, whereas IL-6 production is decreased by valsartan. These effects are exerted mainly at high concentrations of the drugs. Administration of one dose of captopril or valsartan in therapeutic dosages to patients with essential hypertension did not influence LPS-stimulated production of cytokines by whole blood. In conclusion, despite inhibitory effects on pro-inflammatory cytokine production in vitro, it is unlikely that captopril or valsartan could be used in anticytokine therapeutic strategies in vivo. [ABSTRACT FROM AUTHOR]

Phagocytosis by human monocytes is an important primary survival mechanism particularly during bacterial infection. However, the processes that control the events and mediators involved in the activation of monocytes and their impact on the phagocytosis of bacteria are poorly understood. The effect of bacterial endotoxin, interleukin-1β (IL-1β), fatty acids and prostaglandin E2 (PGE2) on the phagocytosis of fluoroscein isothiocyanate (FITC)-labelled Escherichia coli (O157) by human blood monocytes and U937 cells was studied by flow cytometry. Endotoxin increased the phagocytosis of labelled bacteria by both monocytes and U937 cells. IL-1β and the polyunsaturated fatty acids; dihomo-γ-linolenic and arachidonic acids also increased the phagocytic activity of both monocytes and U937 cells. In contrast, PGE2 suppressed phagocytosis in a concentration-dependent manner. The cyclo-oxygenase inhibitor, ketoprofen, further enhanced the increased phagocytic activity in the presence of endotoxin and interleukin-1 (IL-1) indicating suppression by endogenous prostaglandins. This was confirmed by the data which showed that lipopolysaccharide (LPS) and IL-1 increased PGE2 release and ketoprofen inhibited release. Endotoxin and fatty acids increased IL-1β release also, whereas PGE2 inhibited release. The data suggest that phagocytic activity may be linked to changes in IL-1 levels. The data presented in this study also suggest that monocyte phagocytosis in the course of bacterial infection would be altered during pathophysiological events which result in elevation of extracellular fatty acids. [ABSTRACT FROM AUTHOR]

The induction phase of contact sensitization is associated with the movement of epidermal Langerhans cells (LC) from the skin and their migration, via afferent lymphatics, to draining lymph nodes where they accumulate as immunostimulatory dendritic cells (DC). It has been demonstrated previously that tumour necrosis factor-α (TNF-α) provides an important signal for LC migration and that in the absence of this cytokine, movement of LC from the epidermis to regional lymph nodes is inhibited. Recent evidence indicates that interleukin-1β (IL-1β), a cytokine produced in murine epidermis exclusively by LC, may also play a role in LC migration. The purpose of the investigations described here was to clarify, using relevant neutralizing anti- cytokine antibodies, the contributions made by TNF-α and IL-1β to the migration of LC from the epidermis. It was found that like anti-TNF-2, anti-IL-1β administered systemically to mice (by intraperitoneal injection), prior to skin sensitization with the contact allergen oxazolone, resulted in a marked inhibition of DC accumulation in draining lymph nodes. It was shown also that anti-IL- 1β inhibited TNF-α-induced LC migration and DC accumulation and that, in similar fashion, the stimulation of LC migration and DC accumulation induced by IL-1β was compromised by prior treatment with anti-TNF-α. Based upon these data it is proposed that the stimulation of LC migration in response to skin sensitization requires the receipt by LC of two independent signals, one provided by TNF-α and the other by IL-1β. Morphological analyses of LC in epidermal sheets prepared from animals exposed to these cytokines with or without prior systemic treatment with anti-cytokine antibody suggested that the changes induced in LC by TNF-α and IL/1β may include the altered expression of adhesion molecules and acquisition of the ability to interact with and pass through the basement membrane. [ABSTRACT FROM AUTHOR]

Cytokines produced by cells of the immune system, including macrophages, can influence inflammatory responses to viral infection. This has been exploited by viruses, which have developed strategies to direct the immune response towards ineffective responses. African swine fever virus (ASFV) is a double-stranded DNA virus that infects macrophages of domestic swine. In this study, primary cells of monocyte-macrophage lineage were obtained from the lungs, peritoneum or blood of domestic swine and, after infection with ASFV, supernatants were tested for cytokines using biological assays. The cytokine transforming growth factor-β (TGF-β) was detected after infection of macrophage preparations, but tumour necrosis factor (TNF) and interleukin-1 (IL-1) were not detected. ASFV-infected and uninfected macrophage populations were also tested to assess their ability to respond to cytokines by enhancing production of superoxide in the respiratory burst mechanism. Responses to interferon-γ, (IFN-γ) and lipopolysaccharide (LPS) were suppressed in macrophage populations infected with virus, even at low multiplicities of infection. Addition of TGF-β to uninfected macrophages resulted in a similar suppression of response, but antibody to TGF-β did not prevent suppression induced by virus. These results are discussed in relation to the pathology of African swine fever. [ABSTRACT FROM AUTHOR]