Processing Of Interleukin Research Articles

Inflammation and altered metabolism impede efficacy of functional electrical stimulation in critically ill patients

BackgroundCritically ill patients suffer from acute muscle wasting, which is associated with significant physical functional impairment. We describe data from nested muscle biopsy studies from two trials of functional electrical stimulation (FES) that did not shown improvements in physical function.MethodsPrimary cohort: single-centre randomized controlled trial. Additional healthy volunteer data from patients undergoing elective hip arthroplasty. Validation cohort: Four-centre randomized controlled trial. Intervention: FES cycling for 60-90min/day. Analyses: Skeletal muscle mRNA expression of 223 genes underwent hierarchal clustering for targeted analysis and validation.ResultsPositively enriched pathways between healthy volunteers and ICU participants were “stress response”, “response to stimuli” and “protein metabolism”, in keeping with published data. Positively enriched pathways between admission and day 7 ICU participants were “FOXO-mediated transcription” (admission = 0.48 ± 0.94, day 7 = − 0.47 ± 1.04 mean log2 fold change; P = 0.042), “Fatty acid metabolism” (admission = 0.50 ± 0.67, day 7 = 0.07 ± 1.65 mean log2 fold change; P = 0.042) and “Interleukin-1 processing” (admission = 0.88 ± 0.50, day 7 = 0.97 ± 0.76 mean log2 fold change; P = 0.054). Muscle mRNA expression of UCP3 (P = 0.030) and DGKD (P = 0.040) decreased in both cohorts with no between group differences. Changes in IL-18 were not observed in the validation cohort (P = 0.268). Targeted analyses related to intramuscular mitochondrial substrate oxidation, fatty acid oxidation and intramuscular inflammation showed PPARγ-C1α; (P < 0.001), SLC25A20 (P = 0.017) and UCP3 (P < 0.001) decreased between admission and day 7 in both arms. LPIN-1 (P < 0.001) and SPT1 (P = 0.044) decreased between admission and day 7. IL-18 (P = 0.011) and TNFRSF12A (P = 0.009) increased in both arms between admission and day 7. IL-1β (P = 0.007), its receptor IL-1R1 (P = 0.005) and IL-6R (P = 0.001) decreased in both arms between admission and day 7. No between group differences were seen in any of these (all p > 0.05).ConclusionsIntramuscular inflammation and altered substrate utilization are persistent in skeletal muscle during first week of critical illness and are not improved by the application of Functional Electrical Stimulation-assisted exercise. Future trials of exercise to prevent muscle wasting and physical impairment are unlikely to be successful unless these processes are addressed by other means than exercise alone.

Inhibition of the NLRP3 inflammasome by OLT1177 induces functional protection and myelin preservation after spinal cord injury

Spinal cord injury (SCI) leads to irreversible functional deficits due to the disruption of axons and the death of neurons and glial cells. The inflammatory response that occurs in the injured spinal cord results in tissue degeneration; thus, targeting inflammation after acute SCI is expected to ameliorate histopathological evidence indicative of damage and, consequently, reduce functional disabilities. Interleukin 1 beta (IL-1β) and interleukin 18 (IL-18) are pro-inflammatory cytokines members of the IL-1 family that initiate and propagate inflammation. Here, we report that protein levels of IL-1β and IL-18 were increased in spinal cord parenchyma after SCI, but with different expression profiles. Whereas levels of IL-1β were rapidly increased reaching peak levels at 12 h after the injury, levels of IL-18 did not increase until 7 days after the injury. Since activation of the NLRP3 inflammasome is required for the processing and release of IL-1β and IL-18, we intraperitoneally administered OLT1177, a selective inhibitor of the NLRP3 inflammasome, to reduce the contribution of these cytokines to SCI. At a dose of 200 mg/kg, OLT1177 protected against neurological deficits and histological evidence of damage. OLT1177 also reduced the levels of IL-1β in the spinal cord after contusion injury and diminished the accumulation of neutrophils and macrophages at later time points. These data suggest that targeting the NLRP3 inflammasome with OLT1177 could be a novel therapeutic strategy to arrest neuroinflammation and reduce functional impairments after acute SCI in humans.

Genetic analysis of NLRP3 in recurrent pericarditis

Abstract Background The mechanisms underlying recurrent pericarditis are poorly understood, although the efficacy of colchicine and targeted inhibitors of the interleukin-1 pathway implicate auto-inflammation in its pathogenesis. NLRP3 (NACHT, leucine-rich repeat, and pyrin domain containing protein 3) forms part of the NLRP3 inflammasome that mediates post-translational processing of interleukin-1, and colchicine has also been demonstrated to inhibit this pathway. Pathogenic mutations in exon 3 of NLRP3 cause the auto-inflammatory disease cryopyrin associated periodic syndrome, and the management of this disease and other auto-inflammatory diseases has been transformed over the last two decades by the use of targeted anti-interleukin-1 agents. More recently, NLRP3 inflammasome activity has been demonstrated to be upregulated in the pericardia of patients with recurrent pericarditis. Purpose Given that mutations in exon 3 of NLRP3 are known to cause auto-inflammatory disease and NLRP3 activity is implicated in recurrent pericarditis, we undertook a genetic analysis of exon 3 of NLRP3 in 99 patients with recurrent pericarditis to determine whether pathogenic mutations in this gene are associated with the disease. Methods Exon 3 of NLRP3 was analysed either by Sanger (n=30) or amplicon-based next generation (n=69) sequencing. Variants identified by next generation sequencing were validated by Sanger sequencing. Minor allele frequencies were compared to those in the healthy reference population in the Genome Aggregation database (gnomAD) using Fisher's exact test. p values &amp;lt;0.05 were considered statistically significant. Results No known pathogenic mutations in exon 3 of NLRP3 were identified in 99 patients with recurrent pericarditis. Three patients harboured variants of unknown significance (V198M, T543M, Q703K) in NLRP3. A burden association test comparing the combined frequency of all variants in exon 3 of NLRP3 in this cohort against that observed in healthy controls in gnomAD found no significant difference in allele frequency. Conclusions This is the first reported genetic analysis of NLRP3 in a cohort of patients with recurrent pericarditis and found no evidence to implicate mutations in this gene in the disease. Whilst access to effective therapies for pericarditis is widening, decisions on treatment duration and identification of patients at risk of recurrent or treatment refractory disease remain significant clinical challenges. Further work exploring the mechanisms by which NLRP3 activity is upregulated in recurrent pericarditis is therefore indicated to identify biomarkers of disease activity and to stratify patients at risk of recurrence. Given the recent successes of agents targeting this pathway in coronary artery disease in clinical trials, such studies may also provide important insights that are of wider interest to the field of cardiovascular medicine. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Clinical Research Training Fellowship

Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ stimulated cells guanylate binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, whereas GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-Caspase-4 pathway in anti-bacterial defense.

Abstract 005: A Small-molecule Inhibitor of NLRP3 Inflammasome Activity, MCC950, Reduces Blood Pressure and Restores Renal Function in Hypertensive Mice

Inflammasomes are a family of interleukin-1 processing complexes and master regulators of inflammation. We recently showed that both one-kidney/deoxycorticosterone acetate/salt (1K/DOCA/salt)- and angiotensin II-dependent hypertension in mice are associated with elevated expression of the NLRP3 inflammasome in the kidneys. Moreover, genetic deficiency of a key subunit critical for NLRP3 inflammasome activity protected mice against renal inflammation and chronic pressor responses associated with these models. As a step towards translation of these findings into new therapies, here we investigated whether a highly specific small-molecule inhibitor of NLRP3 inflammasome activity, MCC950, similarly reduces the deleterious effects of 1K/DOCA/salt on blood pressure (BP) and renal function. Male C57BL6/J mice were implanted with telemetry probes for continuous recording of BP (mean arterial (MAP), systolic, diastolic) and heart rate (HR), or placed in metabolic cages for 24 h urine collections to assess renal function. Once baseline parameters were established, mice were uninephrectomized, received a DOCA pellet (2.4 mg/kg/d, s.c. ) and were given 0.9% saline to drink. Following establishment of hypertension (10 d), mice were implanted with osmotic pumps containing either MCC950 (10 mg/kg/d, s.c. ) or vehicle (saline) and followed for 28 d. MAP increased from 102 ± 2 to 133 ± 3 mmHg over the 10 d following 1K/DOCA/salt surgery. In vehicle-treated mice, MAP remained at this elevated level until the end of the treatment period. By contrast, MAP of mice treated with MCC950 gradually declined such that at day 38 it was 15 mmHg lower than that of vehicle-treated mice. Systolic and diastolic BP response was similar to MAP, whereas HR was unaffected by MCC950. Urine, Na + and albumin excretion, and osmolality were all markedly increased after 10 d of 1K/DOCA/salt treatment. Consistent with its effect on BP, MCC950 decreased each of these parameters by 30-40%, whereas vehicle had no effects. In conclusion, we have shown that an inhibitor of NLRP3 inflammasome activation reduces BP and restores renal function in mice with established hypertension, highlighting MCC950 as a promising candidate for future therapies.

126: Inflammasome activation due to polymerized actin triggers an autoinflammatory disease that is dependent on IL-18, not IL-1β

Here, we have characterized the first known auto-inflammatory disease caused by interleukin-18 (IL-18). This is due to a point mutation in the gene Wdr1 , which is required for disassembly of actin filaments in conjunction with ADF/cofilin family proteins. Mice homozygous for this mutation ( Wdr1 rd / rd ) develop severe autoinflammation in the ears and tail due to an intracellular build up of polymerized actin. The serum IL-18 level was significantly elevated in Wdr1 rd / rd mice, compared to littermate controls. Processing and secretion of IL-18 and IL-1β is induced by caspase-1 via activation of the inflammasome complex. Importantly, a delayed onset of the disease was seen in Wdr1 rd / rd mice lacking caspase-1, the inflammasome adaptor ASC or IL-18 itself. In contrast, the time of onset and severity of the inflammatory disease was not altered by a deficiency of IL-1R, G-CSF, interferon γ (IFNγ) or tumor necrosis factor α (TNFα), suggesting that the inflammatory disease was driven by an IL-18 specific inflammasome. Furthermore, we are able to show ex vivo and in vivo, that this IL-18 production specifically comes from monocytes, but not mature macrophages. Overall, our data reveal the mechanism and biological basis of a mouse model of spontaneous sterile inflammatory disease driven by IL-18 but not IL-1.

Expression of the NLRP3 Inflammasome in Cerebral Cortex After Traumatic Brain Injury in a Rat Model

Inflammatory response plays an important role in the pathogenesis of secondary damage after traumatic brain injury (TBI). The inflammasome is a multiprotein complex involved in innate immunity and a number of studies have suggested that the inflammasome plays a critical role in a host inflammatory signaling. Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing 3 (NLRP3) is a key component of the NLRP3-inflammasome, which also includes apoptotic speck-containing protein (ASC) with a cysteine protease (caspase)-activating recruitment domain and pro-caspase1. Activation of the NLRP3-inflammasome causes the processing and release of the interleukin 1 beta (IL-1β) and interleukin 18 (IL-18). Based on this, we hypothesized that the NLRP3-inflammasome could participate in the inflammatory response following TBI. However, the expression of NLRP3-inflammasome in cerebral cortex after TBI is not well known. Rats were randomly divided into control, sham and TBI groups (including 6 h, 1 day, 3 day and 7 day sub-group). TBI model was induced, and animals were sacrificed at each time point respectively. The expression of NLRP3-inflammasome was measured by quantitative real-time polymerase chain reaction, western blot and immunohistochemistry respectively. Immunofluorescent double labeling was performed to identify the cell types of NLRP3-inflammasome's expression. Moreover, enzyme linked immunosorbent assay was used to detect the alterations of IL-1β and IL-18 at each time point post-injury. The results showed that, TBI could induce assembly of NLRP3-inflammasome complex, increased expression of ASC, activation of caspase1, and processing of IL-1β and IL-18. These results suggested that NLRP3-inflammasome might play an important role in the inflammation induced by TBI and could be a target for TBI therapy.

Muckle-Wells Syndrome and Male Hypofertility: A Case Series

Muckle-Wells syndrome (MWS) is a rare autoinflammatory disorder associated with NLRP3 gene mutations, which cause excessive caspase-1 activation and processing of interleukin (IL)-1β and IL-18. Here we investigated whether MWS disease may be associated with impaired fertility in male patients. Medical records of all male MWS patients with NLRP3 mutations followed in our tertiary center for inherited autoinflammatory diseases were reviewed retrospectively for data indicating fertility problems. Six of 9 patients were unable to have children despite regular sexual activity during at least 2 years; 3 succeeded in having children through in vitro fertilization. Infertility was the main reason for divorce in 1 patient. Spermiogram analyses were available in 8 of the 9 patients. Oligozoospermia was observed in 5 patients and azoospermia in 3 patients. In 2 patients, treatment with IL-1-targeting drugs for 6 and 12 months, respectively, had a moderate or no effect on spermatozoa counts. In 2 patients testosterone levels were low and testosterone treatment significantly increased spermatozoa counts in 1 of them. MWS may be associated with subfertility and infertility in male patients. Consequently, sexual health and fertility should be assessed systematically in adolescent and adult male patients. Additional studies are required to establish the frequency of subfertility in male MWS patients, to understand when subfertility occurs in the disease natural history, and, finally, to investigate whether early management with IL-1-targeting drugs, or testosterone treatment or early sperm cryo-conservation may help to allow procreation.

Expression and regulation of the NALP3 inflammasome complex in periodontal diseases

Periodontitis is an infectious process characterized by inflammation affecting the supporting structures of the teeth. Porphyromonas gingivalis is a major oral bacterial species implicated in the pathogenesis of periodontitis. Processing of interleukin (IL)-1 family cytokines is regulated by an intracellular innate immune response system, known as the NALP3 [nacht domain-, leucine-rich repeat-, and pyrin domain (PYD)-containing protein 3] inflammasome complex. The aim of the present study was to investigate by quantitative real-time polymerase chain reaction (PCR) the mRNA expression of NALP3, its effector molecule apoptosis associated speck-like protein (ASC), its putative antagonist NLRP2 (NLR family, PYD-containing protein 2), IL-1beta and IL-18 (i) in gingival tissues from patients with gingivitis (n = 10), chronic periodontitis (n = 18), generalized aggressive periodontitis (n = 20), as well as in healthy subjects (n = 20), (ii) in vitro in a human monocytic cell line (Mono-Mac-6), in response to P. gingivalis challenge for 6 h. The clinical data indicate that NALP3 and NLRP2, but not ASC, are expressed at significantly higher levels in the three forms of inflammatory periodontal disease compared to health. Furthermore, a positive correlation was revealed between NALP3 and IL-1beta or IL-18 expression levels in these tissues. The in vitro data demonstrate that P. gingivalis deregulates the NALP3 inflammasome complex in Mono-Mac-6 cells by enhancing NALP3 and down-regulating NLRP2 and ASC expression. In conclusion, this study reveals a role for the NALP3 inflammasome complex in inflammatory periodontal disease, and provides a mechanistic insight to the host immune responses involved in the pathogenesis of the disease by demonstrating the modulation of this cytokine-signalling pathway by bacterial challenge.

Asc and Ipaf Inflammasomes Direct Distinct Pathways for Caspase-1 Activation in Response to Legionella pneumophila

Caspase-1 activation is a key feature of the innate immune response of macrophages elicited by pathogens and a variety of toxins. Here, we determined the requirement for different adapter proteins involved in regulating host processes mediated by caspase-1 after macrophage infection by Legionella pneumophila. The adapter protein Asc was found to be important for caspase-1 activation during L. pneumophila infection. Activation of caspase-1 through Asc did not require the flagellin-sensing pathway involving the host nucleotide-binding domain and leucine-rich repeat-containing protein Ipaf (NLRC4). Asc-dependent caspase-1 activation was inhibited by high extracellular potassium levels, whereas Ipaf-dependent activation was unaffected by potassium treatment. Activation of caspase-1 in macrophages occurred independently of Nalp3 and proteasome activity, suggesting that a previously uncharacterized mechanism for caspase-1 activation through Asc may be triggered by L. pneumophila. Rapid pore formation and pyroptosis induced by L. pneumophila required caspase-1, Ipaf, and bacterial flagellin but occurred independently of Asc. Equivalent levels of active interleukin-18 (IL-18) were detected in the lungs of mice infected with a flagellin-deficient strain of L. pneumophila and Asc-deficient mice infected with wild-type L. pneumophila. Active IL-18 was undetectable in the lungs of Asc-deficient mice infected with an L. pneumophila flagellin mutant, indicating independent roles for Ipaf and Asc in caspase-1-mediated processing and release of IL-18 in vivo. Ipaf-dependent activation of caspase-1 restricted bacterial replication in vivo, whereas Asc was dispensable for restriction of L. pneumophila replication in mice. Thus, L. pneumophila-mediated caspase-1 activation involves the coordinate activities of inflammasomes differentially regulated by Ipaf and Asc.

Recent developments in crystal-induced inflammation pathogenesis and management

Crystal-induced inflammation pathogenesis is undergoing a transition with respect to monosodium urate, calcium pyrophosphate dihydrate, and even basic calcium phosphate crystals. It is now recognized that innate immunity could be involved in the earlier pathogenic events and that the inflammasome, along with other signaling pathways, is activated and results in interleukin-1 processing and secretion, ultimately activating cells as a paracrine or autocrine cytokine. Management of acute and chronic monosodium urate crystal-induced inflammation, namely gout, has been critically reviewed by a dedicated European working group, and on the behalf of the European League against Rheumatism, 12 evidence-based recommendations have been reported. Calcium pyrophosphate dihydrate chronic inflammation could benefit from colchicine and from methotrexate as an anti-inflammatory agent.

A novel isoform of pro-interleukin-18 expressed in ovarian tumors is resistant to caspase-1 and -4 processing.

Interleukin-18 (IL-18) is a proinflammatory cytokine synthesized as a 24 kDa inactive precursor (pro-IL-18) by several cell types, and is processed to a bioactive molecule of 18 kDa by the proteinases caspase-1 or caspase-4. All ovarian carcinoma cell lines express pro-IL-18, only in some instances coexpress caspase-1, and always express caspase-4; in any case, they display a defective processing of IL-18. We analysed whether pro-IL-18, present in two ovarian carcinoma cell lysates, could be processed 'in vitro' by recombinant active caspase-1. While most of pro-IL-18 could be cleaved by caspase-1, a residual of pro-IL-18 appeared to be resistant. Cloning and sequence analysis of the whole pro-IL-18 open reading frame demonstrated the existence of an alternatively spliced mRNA variant, which lacked exon-3 (Delta3pro-IL-18). The 12 bp exon-3 encodes for the AEDD amino-acid sequence, which is N-terminal with respect to the cleavage site of caspase-1. Both pro-IL-18 and Delta3pro-IL-18 mRNA isoforms were detected in all ovarian cancer cell lines analysed, while Delta3pro-IL-18 mRNA was undetectable in normal ovarian epithelial cells. The Delta3pro-IL-18 cDNA induced synthesis of an alternative Delta3pro-IL-18 protein upon transfection into a murine cell line. The Delta3pro-IL-18 protein was resistant to proteolytic activation by caspase-1 and -4, although it was capable to bind caspase-1. Aternative splicing of pro-IL-18 exon-3 may represent a novel mechanism of regulation of bioactive IL-18 production in human ovarian tumors.

Correlation of renal tubular epithelial cell-derived interleukin-18 up-regulation with disease activity in MRL-Faslpr mice with autoimmune lupus nephritis.

MRL-Fas(lpr) mice spontaneously develop an autoimmune disease that mimics systemic lupus erythematosus in humans. Infiltrating T cells expressing interferon-gamma (IFNgamma) are responsible for the autoimmune kidney destruction in MRL-Fas(lpr) mice, and interleukin-18 (IL-18) released by mononuclear phagocytes stimulates T cells to produce the IFNgamma. Since MRL-Fas(lpr) T cells are characterized by an overexpression of the IL-18 receptor accessory chain, we sought to determine the impact of IL-18 on the progression of lupus nephritis in MRL-Fas(lpr) mice. IL-18 expression in sera and kidney tissues from MRL-Fas(lpr) mice was determined by enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blotting. IL-18 production by primary cultured tubular epithelial cells (TECs) from MRL-Fas(lpr) and BALB/c mice were examined by RT-PCR, ELISA, and Western blotting. The interactions of TEC-derived IL-18 and MRL-Fas(lpr) T cells were studied in coculture assays. IL-18-related effects on TEC viability and adhesion molecule expression were determined by fluorescence-activated cell sorting and cell proliferation assays. Up-regulation of mature IL-18 was restricted to nephritic MRL-Fas(lpr) kidneys and increased in parallel with the severity of lupus nephritis. IL-18 expression was not confined to infiltrating monocytes but was primarily detected in TECs. Similarly, interleukin-1beta-converting enzyme expression, which is required for the processing of precursor IL-18, was localized in TECs. De novo synthesis of IL-18 by MRL-Fas(lpr) TECs was confirmed by RT-PCR and Western blotting. Functional assays revealed that activated TECs induced IFNgamma production in MRL-Fas(lpr) T cells through IL-18. IL-18, in turn, increased apoptotic TEC death and up-regulation of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. Taken together, our findings suggest that IL-18-producing TECs may directly be involved in the pathogenesis of lupus nephritis.

Efficient production of biologically active porcine interleukin-18 by coexpression with porcine caspase-1 using a baculovirus expression system.

We previously reported that the precursor form of porcine interleukin-18 (IL-18) expressed by the baculovirus system was able to be secreted efficiently into the supernatant of insect cells, whereas only small amounts of mature IL-18 were secreted from insect cells. As insect cells do not normally have the IL-1beta converting enzyme (caspase-1), which is required for processing of the precursor IL-18 into the mature IL-18, we recently cloned porcine caspase-1 cDNA. In this study, we constructed a recombinant baculovirus containing the cDNA encoding porcine caspase-1 and showed that the coexpression of caspase-1 and the precursor IL-18 enabled insect cells to secrete mature IL-18 into the culture supernatant efficiently. Moreover, inhibition of caspase-1 activity by its specific inhibitor prevented the processing of precursor IL-18 into the mature form. These results indicated that the processing and secretion of precursor IL-18 into the mature form in insect cells were enhanced by the artificial introduction of caspase-1 activity for cleavage.

Processing of interleukin-18 by human vascular smooth muscle cells.

Elena Westphal, Ivar Friedrich*, Rolf-Edgar Silber*, Karl Werdan, and Harald Loppnow Martin-Luther-Universitat Halle-Wittenberg, Klinik und Poliklinik fur Innere Medizin III, Forschungslabor, Magdeburger Str. 21, and *Klinik fur Herzund Thoraxchirurgie, ErnstGrube-Stra e 40, D-06097 Halle (Saale), GERMANY Corresponding author: PD Dr. H. Loppnow, Martin-Luther-Universitat Halle-Wittenberg, Klinik und Poliklinik fur Innere Medizin III, Forschungslabor, Magdeburger Str. 21,D-06097 Halle (Saale), GERMANY (Email: Harald.Loppnow@medizin.uni-halle.de)

Effect of the selective and non-selective cysteine protease inhibitors on the intracellular processing of interleukin 6 by HEPG2 cells.

The effects were measured and compared of three nonselective cysteine cathepsin inhibitors (leupeptin, trans-Epoxy-succinyl-L-Leucylamido(4-guanidino)-butane (E-64), and Z-Phe-Ala-CH2F) and a selective cathepsin B inhibitor, CA074Me, on the intracellular processing of 125I-labeled human recombinant Interleukin 6 (IL-6) by HepG2 cells. The uptake and processing of 125I-IL-6 by cells treated with inhibitors was followed over a 7-h period. All inhibitors caused an increased residence time of IL-6 inside the cell and a corresponding decrease in the output of non-trichloroacetic acid-precipitable fragments of radiolabeled protein. Maximal effect was achieved with leupeptin at 200 microM, with which the rate of IL-6 digestion was reduced to 50% that of control cells. The specific inhibitor CA074Me was the least effective in slowing the intracellular processing of IL-6. The effects of all of the inhibitors on the production of haptoglobin, either stimulated by IL-6 or basal, was negligible over a similar time period, indicating continued cell viability. The data from this model suggest that cathepsin inhibitors would not interfere with lysosomal processing to an extent which would prohibit the development of selective and potent cathepsin inhibitors for the treatment of diseases in which individual cysteine cathepsins play clearly pathophysiological roles.

Interleukin-18 Regulation of Interferon γ Production and Cell Proliferation as Shown in Interleukin-1β–Converting Enzyme (Caspase-1)-Deficient Mice

Interleukin-18 (IL-18) is a costimulatory factor for interferonγ (IFNγ) production. Processing of pro–IL-18 by IL-1β–converting enzyme (ICE) leads to the release of bioactive IL-18. Compared with wild-type (WT) mice, splenocytes from ICE-deficient mice produced low IFNγ after lipopolysaccharide (LPS) or zymosan (50% and 80% reduction). In contrast, IFNγ production was unimpaired in ICE-deficient mice using Concanavalin A (Con A). Comparable results were obtained when endogenous IL-18 was blocked with a neutralizing antibody. LPS-induced IFNγ was also reduced by an ICE inhibitor. Exogenous IL-18 augmented zymosan-induced IFNγ production in WT mice. In ICE-deficient cells, IFNγ production was only partially restored by IL-18. The reduced levels of IFNγ in ICE-deficient mice were not due to a lack of IL-12, because zymosan induced IL-12 equally in WT and in ICE-deficient mice. IFNγ is an important regulator of cell proliferation. In accordance, splenocytes from ICE-deficient mice proliferated more when stimulated with LPS, but not with Con A. Furthermore, in ovalbumin-sensitized ICE-deficient mice, proliferation of lymph node cells in response to the specific antigen was not altered. Exogenous IFNγ inhibited, whereas blockade of endogenous IFNγ or IL-18 increased, LPS induced splenocyte proliferation both in WT and in ICE-deficient mice. Our results show that IL-18 is an IL-12–independent regulator of IFNγ production and of cell proliferation induced by microbial stimuli. However, ICE-dependent processing of IL-18 is not needed for response to mitogens or antigens.

Interleukin-18: A Novel Cytokine That Augments Both Innate and Acquired Immunity

Publisher Summary Interleukin-18 (IL-18) is a multifunctional cytokine, the roles of which extend over both acquired and innate immunity, and its major role has been thought to be the strong ability to induce IFN-γ production from T or NK cells. This chapter describes the IL-18/IL-l8R system and its pathophysiological roles in immune response. Although IL-18-producing cells were thought to be activated macrophages at first, recent investigations reveal that various types of cells, including keratinocytes, osteoclasts, and adrenal cortex cells, express IL-18, suggesting that IL-18 plays important physiological roles in fields other than the immune system. IL-18 shares many similarities to IL-1, such as in its amino acid sequence. Preliminary experiments indicate that IL-18 is involved in the defense mechanisms against infection and in tumor rejection, and it is also involved in the suppressions of allergy via suppression of IgE production. Thus, it can be considered that IL-18 is involved in the various diseases in diverse manners; however, more investigation is needed to answer several questions about the regulation of production and processing of IL-18 along with the role that it plays in defense against pathogens.

Processing of interleukin-1 in cells of monocytic lineage is differentiation-dependent.

The interleukin-1 (IL-1) alpha and beta precursor proteins are processed and released from several cell types in the absence of a canonical signal peptide. To gain some insight into the mechanisms that allow the production of IL-1 alpha and beta, we have investigated by immunoprecipitation the synthesis, their release and processing in a promyeloblastic cell line of tumoral origin, U937, and in peripheral blood monocytes. We show that U937 monocytic cells, on induction with a tumor-promoting agent, synthesize and release into the culture medium proIL-1 beta but do not process it. Similarly, peripheral blood monocytes left in adherence for 24 h or longer, prior to addition of lipopolysaccharide, synthesize and release proIL-1 alpha and beta without detectable processing of either cytokine. Processing and release of IL-1 alpha and beta by peripheral blood monocytes can be observed when monocytes are left to adhere for periods less than 15 h before lipopolysaccharide addition. IL-1 alpha and beta show similar kinetics of release from the cells, suggesting the existence of a common mechanism regulating their secretion. Since peripheral blood monocytes left in adherence in the presence of lipopolysaccharide differentiate into macrophages, we conclude that release and processing of IL-1 can occur independently and that processing depends on the stage of differentiation of monocytes, i.e. only the monocytes at an early stage of differentiation produce 17-kDa IL-1 alpha and beta.

Human interleukin 1 beta is not secreted from hamster fibroblasts when expressed constitutively from a transfected cDNA.

To understand the secretion and processing of interleukin-1 (IL-1), a Chinese hamster fibroblast cell line (R1610) was transfected with a human IL-1 beta cDNA under the control of the SV40 early promoter and linked to the gene for neomycin resistance. After selecting for transfected cells resistant to G418, two clones were found to constitutively express the IL-1 beta 31-kD precursor which was almost exclusively located in the cytosol. Pulse-chase experiments failed to show any secretion of IL-1 and very little IL-1 activity was detectable in cell supernatants. Furthermore, surface membrane IL-1 activity could not be detected, although low levels of activity could be released upon brief trypsin treatment. Therefore, unlike monocytes, these fibroblast cells lack the mechanism for secreting and processing of IL-1 beta.