The release of IL-1β is a tightly controlled process that requires induced synthesis of the precursor pro-IL-1β and a second stimulus that initiates cleavage and secretion of mature IL-1β. Although ATP as a second stimulus potently promotes IL-1β maturation and release via P2X7 receptor activation, millimolar ATP concentrations are needed. The human cathelicidin-derived peptide LL37 is a potent antimicrobial peptide produced predominantly by neutrophils and epithelial cells. In this study, we report that LL37 stimulation of LPS-primed monocytes leads to maturation and release of IL-1β via the P2X7 receptor. LL37 induces a transient release of ATP, membrane permeability, caspase-1 activation, and IL-1β release without cell cytotoxicity. IL-1β release and cell permeability are suppressed by pretreatment with the P2X7 inhibitors oxidized ATP, KN04, and KN62. In the presence of apyrase, which hydrolyzes ATP to AMP, the effect of LL37 was not altered, indicating that LL37 rather than autocrine ATP is responsible for the activation of the P2X7 receptor. We conclude that endogenous LL37 may promote IL-1β processing and release via direct activation of P2X7 receptors.
Macrophage responses to Francisella infection have been characterized previously by subdued proinflammatory responses; however, these studies have generally focused on macrophage cell lines or monocyte-derived macrophages. Therefore, we studied the ability of fresh human blood monocytes to engulf and respond to Francisella by using the live vaccine strain variant and Francisella novicida. Because Francisella organisms have been reported to escape from the phagolysosome into the cytosol, we hypothesized that this escape may trigger the activation of caspase-1. Francisella tularensis variants were readily taken up by fresh human CD14 ؉ monocytes, inducing the release of IL-1, as well as IL-8, in a timeand dose-dependent fashion. Importantly, whereas live and dead Escherichia coli, F. novicida, and live vaccine strain, as well as the LPS of E. coli, were able to induce abundant IL-1 mRNA synthesis and intracellular pro-IL-1 production, only live Francisella induced enhanced IL-1 processing and release (51 ؎ 10 vs. 7.1 ؎ 2.1 ng͞ml, for F. novicida vs. E. coli LPS; P ؍ 0.0032). Cytochalasin D blocked the Francisella internalization and the Francisella-induced monocyte IL-1 processing and release but not that induced by the exogenous stimulus E. coli LPS. Also, killing bacteria did not block uptake but significantly diminished the IL-1 processing and release that was induced by Francisella. Blocking bacterial escape from the phagosome into the cytosol also decreased IL-1 but not IL-8 release. These findings demonstrate that Francisella organisms efficiently induce IL-1 processing and release in fresh monocytes by means of a sensing system that requires the uptake of live bacteria capable of phagosome escape.bacteria ͉ caspase-1 ͉ cytokine ͉ phagocytosis M onocytes and macrophages, which are the first-line guards of the innate host-defense system, recognize and phagocytose pathogens. The innate immune system senses the invasion of pathogenic microorganisms through the recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs), of which there are 10 that are known in humans, as well as non-TLRs (1-3). Pathogen recognition induces the release of many proinflammatory and antiinflammatory cytokines, which provide a balanced inflammatory response. At the same time, prototypical intracellular pathogens like Francisella tularensis depend on the intracellular milieu of mononuclear cells for growth and replication (4, 5). In this context, a class of intracellular innate host-defense molecules, which recognize and respond to internalized pathogens, has been identified (3, 6). These molecules [historically called CATERPILLERs (3) and nucleotide-binding oligomerization domain-leucine-rich repeats (NOD-LRRs) (7), and, most recently, NACHT leucine-rich repeats (NLRs) (8)] are homologues of plant-disease-resistance genes and contain leucine-rich repeats, much like TLRs. The human family of NLR consists of 22 (8) or 23 (7) proteins. These NLRs, similar to TLRs, function to regulate pathogen recogniti...
Summary Chemotherapy and radiotherapy, whilst highly effective in the treatment of neoplasia, can also cause damage to healthy tissue. In particular, the alimentary tract may be badly affected. Severe inflammation, lesioning and ulceration can occur. Patients may experience intense pain, nausea and gastro‐enteritis. They are also highly susceptible to infection. The disorder (mucositis) is a dose‐limiting toxicity of therapy and affects around 500 000 patients world‐wide annually. Oral and intestinal mucositis is multi‐factorial in nature. The disruption or loss of rapidly dividing epithelial progenitor cells is a trigger for the onset of the disorder. However, the actual dysfunction that manifests and its severity and duration are greatly influenced by changes in other cell populations, immune responses and the effects of oral/gut flora. This complexity has hampered the development of effective palliative or preventative measures. Recent studies have concentrated on the use of bioactive/growth factors, hormones or interleukins to modify epithelial metabolism and reduce the susceptibility of the tract to mucositis. Some of these treatments appear to have considerable potential and are at present under clinical evaluation. This overview deals with the cellular changes and host responses that may lead to the development of mucositis of the oral cavity and gastrointestinal tract, and the potential of existing and novel palliative measures to limit or prevent the disorder. Presently available treatments do not prevent mucositis, but can limit its severity if used in combination. Poor oral health and existing epithelial damage predispose patients to mucositis. The elimination of dental problems or the minimization of existing damage to the alimentary tract, prior to the commencement of therapy, lowers their susceptibility. Measures that reduce the flora of the tract, before therapy, can also be helpful. Increased production of free radicals and the induction of inflammation are early events in the onset of mucositis. Prophylactic administration of scavengers or anti‐inflammatories can partially counteract or limit some of these therapy‐mediated effects, as can the use of cryotherapy. The regular use of mouthwashes, mouth coatings, antibiotics and analgesics is essential, prior to and during loss and ablation of the epithelial layer. Granulocyte–macrophage colony‐stimulating factor/granulocyte colony‐stimulating factor or the use of laser light therapy may aid restitution and repair. Glutamine supplements may be beneficial in the repair/recovery phase.
Receptor interacting protein-2 (RIP2) is a caspase recruitment domain (CARD)-containing kinase that interacts with caspase-1 and plays an important role in NF-κB activation. Apoptosis-associated speck-like protein containing a CARD (ASC) is a PYRIN and CARD-containing molecule, important in the induction of apoptosis and caspase-1 activation. Although RIP2 has also been linked to caspase-1 activation, RIP2 knockout animals fail to show a defect in caspase-1-mediated processing of proIL-1β to its active form. Therefore, RIP2 function in binding to caspase-1 remains poorly understood. We hypothesized that caspase-1 may serve as a scaffolding molecule that promotes RIP2 interaction with IκB kinase-γ thus inducing NF-κB activation. We further hypothesized that ASC, which also interacts with caspase-1 via its CARD, may interfere with the caspase-1 RIP2 interaction. In HEK293 cells, ASC induced prominent activation of caspase-1 and proIL-1β processing. RIP2 transient transfection induced transcription of an NF-κB reporter gene. This RIP2-induced NF-κB activity and caspase-1 binding was inhibited in a dose-dependent fashion by ASC. Consistent with a role for caspase-1 as a scaffold for RIP2, caspase-1 knockout macrophages were suppressed in their ability to activate NF-κB, and septic caspase-1 knockout animals produced less IL-6, a functional marker of NF-κB activity. Lastly, THP-1 cells treated with small interfering RNA for ASC decreased their caspase-1 activity while enhancing their NF-κB signal. These data suggest that ASC may direct caspase-1 away from RIP2-mediated NF-κB activation, toward caspase-1-mediated processing of proIL-1β by interfering with the RIP2 caspase-1 interaction.
Macrophages and their precursors, monocytes, are key cells involved in the innate immune response. Although both monocytes and macrophages produce caspase-1, the key enzyme responsible for pro-IL-1β processing; macrophages are limited in their ability to activate the enzyme and release functional IL-1β. In this context, because mutations in the pyrin gene (MEFV) cause the inflammatory disorder familial Mediterranean fever, pyrin is believed to regulate IL-1β processing. To determine whether variations in pyrin expression explain the difference between monocytes and macrophages in IL-1β processing and release, pyrin was studied in human monocytes and monocyte-derived macrophages. Although monocytes express pyrin mRNA and protein, which is readily inducible by endotoxin, monocyte-derived macrophages express significantly less pyrin mRNA and protein. Pyrin levels directly correlated with IL-1β processing in monocytes and macrophages; therefore, we asked whether pyrin might promote IL-1β processing and release. HEK293 cells were transfected with pyrin, caspase-1, apoptotic speck protein with a caspase recruitment domain, and IL-1β. Pyrin induced IL-1β processing and release in a dose-dependent manner. Conversely, pyrin small interference RNA suppressed pro-IL-1β processing in both THP-1 cells and fresh human monocytes. In summary, both pyrin expression and IL-1β processing and release are diminished upon the maturation of monocytes to macrophages. When pyrin is ectopically expressed or silenced, IL-1β processing and release parallels the level of pyrin. In conclusion, in the context of endotoxin-induced activation of mononuclear phagocytes, pyrin augments IL-1β processing and release.
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