Mature Interleukin-18 Research Articles

Monoclonal Antibodies Against Mature Interleukin-18 Ameliorate Colitis and Repair Goblet Cell Function

BackgroundNumerous biological interventions and small molecules are used to treat Crohn’s disease; however, the effectiveness of these treatments varies largely. Non-responsiveness to biological therapies is associated with interleukin (IL)-18 gene polymorphisms and high IL-18 expression has been implicated in the pathogenesis of Crohn’s disease.AimsThe aim of this study was to elucidate the expression of precursor and mature IL-18 in patients with Crohn’s disease who exhibited varied responses to cytokine-targeted treatments and determine whether selective inhibition of mature IL-18 offers a novel therapeutic avenue.MethodsWe generated a monoclonal antibody that specifically recognizes the neoepitope of caspase-cleaved mature IL-18. Expression of precursor and mature IL-18 was analyzed in patients with Crohn’s disease. Anti-mature IL-18 monoclonal antibodies were intraperitoneally administered in an acute colitis mouse model, and the disease activity index, body weight loss, tissue pathology, proinflammatory cytokine expression, goblet cell function, and microbiota composition were assessed.ResultsPrecursor and mature IL-18 expression was upregulated and goblet cell function was impaired in patients with Crohn’s disease who were unresponsive to biological therapies. Administration of anti-mature IL-18 antibodies ameliorated induced colitis by repairing goblet cell function and restoring the mucus layer.ConclusionsThe newly developed monoclonal antibody holds promise as a therapeutic alternative for Crohn’s disease.

YinChen WuLing powder attenuates non-alcoholic steatohepatitis through the inhibition of the SHP2/PI3K/NLRP3 pathway.

YinChen WuLing Powder (YCWLP) has been recommended by consensus for the treatment of non-alcoholic steatohepatitis (NASH); nevertheless, its specific pharmacological mechanisms remain to be elucidated. This study aims to dissect the mechanisms underlying the therapeutic effects of YCWLP on NASH using a hybrid approach that encompasses network pharmacology, molecular docking, and in vitro experimental validation. We compiled the chemical constituents of YCWLP from the Traditional Chinese Medicine System Pharmacological Database and Analysis Platform (TCMSP), while potential targets were predicted using the SwissTargetPrediction database. To identify NASH-related candidate targets, comprehensive retrieval was carried out using five authoritative databases. Protein-Protein Interaction (PPI) networks of direct targets of YCWLP in NASH treatment were then constructed using the String database, and functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, were conducted through the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. Core targets were discerned using the Molecular Complex Detection (MCODE) and cytoHubba algorithms. Subsequently, molecular docking of key compounds to core targets was conducted using AutoDock software. Moreover, we established a free fatty acid-induced HepG2 cell model to simulate NASH in vitro, with YCWLP medicated serum intervention employed to corroborate the network pharmacology-derived hypotheses. Furthermore, a combination of enzyme-linked immunosorbent assay (ELISA), and Western blotting analyses was employed to investigate the lipid, hepatic enzyme, SHP2/PI3K/NLRP3 signaling pathway and associated cytokine levels. The network pharmacology analysis furnished a list of 54 compounds from YCWLP and 167 intersecting targets associated with NASH. Through analytic integration with multiple algorithms, PTPN11 (also known as SHP2) emerged as a core target of YCWLP in mitigating NASH. The in vitro experiments validated that 10% YCWLP medicated serum could remarkably attenuate levels of total cholesterol (TC, 1.25 vs. 3.32) and triglyceride (TG, 0.23 vs. 0.57) while ameliorating alanine aminotransferase (ALT, 7.79 vs. 14.78) and aspartate aminotransferase (AST, 4.64 vs. 8.68) leakage in NASH-afflicted cells. In addition, YCWLP significantly enhanced the phosphorylation of SHP2 (0.55 vs. 0.20) and downregulated the expression of molecules within the SHP2/PI3K/NLRP3 signaling axis, including p-PI3K (0.42 vs. 1.02), NLRP3 (0.47 vs. 0.93), along with downstream effectors-cleaved Caspase-1 (0.21 vs. 0.49), GSDMD-NT (0.24 vs. 0.71), mature interleukin-1β (IL-1β, 0.17 vs. 0.48), pro-IL-1β (0.49 vs. 0.89), mature interleukin-18 (IL-18, 0.15 vs. 0.36), and pro-IL-18 (0.48 vs. 0.95). Our research reveals that YCWLP exerts therapeutic effects against NASH by inhibiting lipid accumulation and inflammation, which involves the attenuation of pyroptosis via the SHP2/PI3K/NLRP3 pathway.

Monoclonal Antibody Against Mature Interleukin-18 Ameliorates Colitis in Mice and Improves Epithelial Barrier Function.

Antitumor necrosis factor (TNF)-α antibodies have improved the outcome of inflammatory bowel disease (IBD); but half of patients remain unresponsive to treatment. Interleukin-18 (IL-18) gene polymorphism is associated with resistance to anti-TNF-α antibodies, but therapies targeting IL-18 have not been clinically applied. Only the mature protein is biologically active, and we aimed to investigate whether specific inhibition of mature IL-18 using a monoclonal antibody (mAb) against a neoepitope of caspase-cleaved mature IL-18 could be an innovative treatment for IBD. The expression of precursor and mature IL-18 in patients with UC was examined. Colitis was induced in C57/BL6 mice by administering dextran sulfate sodium (DSS), followed by injection with anti-IL-18 neoepitope mAb. Colon tissues were collected and subjected to histological analysis, immunohistochemistry, immunoblotting, and quantitative polymerase chain reaction. Colon epithelial permeability and microbiota composition were analyzed. Mature IL-18 expression was elevated in colon tissues of patients with active ulcerative colitis. Administration of anti-IL-18 neoepitope mAb ameliorated acute and chronic DSS-induced colitis; reduced interferon-γ, TNF-α, and chemokine (CXC motif) ligand-2 production and epithelial cell permeability; promoted goblet cell function; and altered the intestinal microbiome composition. The suppressive effect of anti-IL-18 neoepitope mAb was superior to that of anti-whole IL-18 mAb. Furthermore, combination therapy with anti-TNF-α Ab suppressed acute and chronic colitis additively by suppressing cytokine expressions and reducing cell permeability by upregulating claudin1 and occludin expression. Anti-IL-18 neoepitope mAb ameliorates acute and chronic colitis, suggesting that this mAb will be an innovative therapeutic option for IBD.

Generation of antagonistic monoclonal antibodies against the neoepitope of active mouse interleukin (IL)-18 cleaved by inflammatory caspases

Interleukin 18 (IL-18) is a member of the IL-1 family and plays an important role in both the innate and acquired immune systems. It is constitutively expressed as an inactive precursor (24 kDa) in various cell types, and the mature IL-18 (18 kDa) cleaved by inflammatory caspase-1/4 binds to the interleukin-18 receptor, thereby activating downstream signaling pathways. We previously generated anti-human IL-18 antibodies that specifically recognize the human IL-18 neoepitope cleaved by inflammatory caspase-1/4. Because the N-terminal amino acid sequences of the neoepitopes are different between human IL-18 and mouse IL-18, the anti-human IL-18 neoepitope antibodies do not recognize mouse mature IL-18. We have now generated novel anti-mouse IL-18 neoepitope antibodies. We also confirmed CXCL2 secretion from P-815 mouse cells by mouse IL-18 stimulation, and established a simple assay to evaluate the activity of mouse IL-18. Using this evaluation system, we confirmed that the anti-mouse IL-18 neoepitope antibodies could inhibit mouse IL-18. By demonstrating the therapeutic efficacy of the anti-mouse IL-18 neoepitope and function-blocking mAbs established in the present study in mouse models, corresponding to human inflammatory diseases in which IL-18 may be involved, such as inflammatory bowel diseases, we can provide the proof-of-concept that the previously established anti-human IL-18 neoepitope and function-blocking mAbs work in human inflammatory disorders corresponding to mouse models.

Rapid Inflammasome Activation Is Attenuated in Post-Myocardial Infarction Monocytes.

Inflammasomes are crucial gatekeepers of the immune response, but their maladaptive activation associates with inflammatory pathologies. Besides canonical activation, monocytes can trigger non-transcriptional or rapid inflammasome activation that has not been well defined in the context of acute myocardial infarction (AMI). Rapid transcription-independent inflammasome activation induced by simultaneous TLR priming and triggering stimulus was measured by caspase-1 (CASP1) activity and interleukin release. Both classical and intermediate monocytes from healthy donors exhibited robust CASP1 activation, but only classical monocytes produced high mature interleukin-18 (IL18) release. We also recruited a limited number of coronary artery disease (CAD, n=31) and AMI (n=29) patients to evaluate their inflammasome function and expression profiles. Surprisingly, monocyte subpopulations isolated from blood collected during percutaneous coronary intervention (PCI) from AMI patients presented diminished CASP1 activity and abrogated IL18 release despite increased NLRP3 gene expression. This unexpected attenuated rapid inflammasome activation was accompanied by a significant increase of TNFAIP3 and IRAKM expression. Moreover, TNFAIP3 protein levels of circulating monocytes showed positive correlation with high sensitive troponin T (hsTnT), implying an association between TNFAIP3 upregulation and the severity of tissue injury. We suggest this monocyte attenuation to be a protective phenotype aftermath following a very early inflammatory wave in the ischemic area. Damage-associated molecular patterns (DAMPs) or other signals trigger a transitory negative feedback loop within newly recruited circulating monocytes as a mechanism to reduce post-injury tissue damage.

A pilot study evaluating GSK1070806 inhibition of interleukin-18 in renal transplant delayed graft function

IntroductionDelayed graft function (DGF) following renal transplantation is a manifestation of acute kidney injury (AKI) leading to poor long-term outcome. Current treatments have limited effectiveness in preventing DGF. Interleukin-18 (IL18), a biomarker of AKI, induces interferon-γ expression and immune activation. GSK1070806, an anti-IL18 monoclonal antibody, neutralizes activated (mature) IL18 released from damaged cells following inflammasome activation. This phase IIa, single-arm trial assessed the effect of a single dose of GSK1070806 on DGF occurrence post donation after circulatory death (DCD) kidney transplantation.MethodsThe 3 mg/kg intravenous dose was selected based on prior studies and physiologically based pharmacokinetic (PBPK) modeling, indicating the high likelihood of a rapid and high level of IL18 target engagement when administered prior to kidney allograft reperfusion. Utilization of a Bayesian sequential design with a background standard-of-care DGF rate of 50% based on literature, and confirmed via extensive registry data analyses, enabled a statistical efficacy assessment with a minimal sample size. The primary endpoint was DGF frequency, defined as dialysis requirement ≤7 days post transplantation (except for hyperkalemia). Secondary endpoints included safety, pharmacokinetics and pharmacodynamic biomarkers.ResultsGSK1070806 administration was associated with IL18-GSK1070806 complex detection and increased total serum IL18 levels due to IL18 half-life prolongation induced by GSK1070806 binding. Interferon-γ−induced chemokine levels declined or remained unchanged in most patients. Although the study was concluded prior to the Bayesian-defined stopping point, 4/7 enrolled patients (57%) had DGF, exceeding the 50% standard-of-care rate, and an additional two patients, although not reaching the protocol-defined DGF definition, demonstrated poor graft function. Six of seven patients experienced serious adverse events (SAEs), including two treatment-related SAEs.ConclusionOverall, using a Bayesian design and extensive PBPK dose modeling with only a small sample size, it was deemed unlikely that GSK1070806 would be efficacious in preventing DGF in the enrolled DCD transplant population.Trial registrationNCT02723786.

Only IL‐1β release is inflammasome‐dependent upon ultraviolet B irradiation although IL‐18 is also secreted

DNA damage accumulates in aged postmitotic retinal pigment epithelium (RPE) cells, a phenomenon associated with the development of age-related macular degeneration. In this study, we have experimentally induced DNA damage by ultraviolet B (UVB) irradiation in interleukin-1α (IL-1α)-primed ARPE-19 cells and examined inflammasome-mediated signaling. To reveal the mechanisms of inflammasome activation, cells were additionally exposed to high levels of extracellular potassium chloride, n-acetyl-cysteine, or mitochondria-targeted antioxidant MitoTEMPO, prior to UVB irradiation. Levels of interleukin-18 (IL-18) and IL-1β mRNAs were detected with qRT-PCR and secreted amounts of IL-1β, IL-18, and caspase-1 were measured with ELISA. The role of nucleotide-binding domain and leucine-rich repeat pyrin containing protein 3 (NLRP3) in UVB-induced inflammasome activation was verified by using the NLRP3-specific siRNA. Reactive oxygen species (ROS) levels were measured immediately after UVB exposure using the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA) indicator, the levels of cyclobutane pyrimidine dimers were assayed by cell-based ELISA, and the extracellular levels of adenosine triphosphate (ATP) determined using a commercial bioluminescence assay. We found that pro-IL-18 was constitutively expressed by ARPE-19 cells, whereas the expression of pro-IL-1β was inducible by IL-1α priming. UVB induced the release of mature IL-18 and IL-1β but NLRP3 contributed only to the secretion of IL-1β. At the mechanistic level, the release of IL-1β was regulated by K+ efflux, whereas the secretion of IL-18 was dependent on ROS production. As well as K+ efflux, the cells released ATP following UVB exposure. Collectively, our data suggest that UVB clearly stimulates the secretion of mature IL-18 as a result of ROS induction, and this response is associated with DNA damage. Moreover, in human RPE cells, K+ efflux mediates the UVB-activated NLRP3 inflammasome signaling, leading to the processing of IL-1β.

Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome

AbstractHemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Although profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of interleukin-18 (IL-18). We tested this association in hyperferritinemic and autoinflammatory patients and found a dramatic correlation of MAS risk with chronic (sometimes lifelong) elevation of mature IL-18, particularly with IL-18 unbound by IL-18 binding protein, or free IL-18. In a mouse engineered to carry a disease-causing germ line NLRC4T337S mutation, we observed inflammasome-dependent, chronic IL-18 elevation. Surprisingly, this NLRC4T337S-induced systemic IL-18 elevation derived entirely from intestinal epithelia. NLRC4T337S intestines were histologically normal but showed increased epithelial turnover and upregulation of interferon-γ–induced genes. Assessing cellular and tissue expression, classical inflammasome components such as Il1b, Nlrp3, and Mefv predominated in neutrophils, whereas Nlrc4 and Il18 were distinctly epithelial. Demonstrating the importance of free IL-18, Il18 transgenic mice exhibited free IL-18 elevation and more severe experimental MAS. NLRC4T337S mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18–driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.

Effects of tumor vaccine expressing Granulocyte–Macrophage Colony Stimulating Factor and interleukin-18 fusion on cancer cells and its possible application for cancer immunotherapy

To access antitumor effects of a combined Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and interleukin-18 (IL-18), cDNA fusion of murine GM-CSF and mature IL-18 (GMIL-18) was constructed and transfected in mammalian cells. GMIL-18 fusion protein was highly secreted and displayed bifunctional activities, possessing immune response initiation and cytokine roles, including IFN-γ induction in mouse splenocytes and increased proliferation of GM-CSF-dependent cells, M-NSF-60. The GMIL-18 secreting tumor vaccine was generated and it strongly stimulated differentiation of dendrite cells (DCs) and effusive CD8+ and CD4+ cell infiltration into tumor mice. Moreover, growth of CT26 mouse colon cancer cells was significantly retarded by GMIL-18 (CT26GMIL-18), but not by CT26GM-CSF- or CT26IL-18. The efficiency of prophylactic vaccination was greater than that of therapeutic vaccination in terms of tumor size and its inhibitory role in proliferation. In micrometastasis analysis of tumor models, γ-ray irradiated GMIL-18 tumor vaccine showed a smaller number of liver-meta tumor nodules in mouse liver cells. We concluded that bifunctional GMIL-18 fusion protein could be applied as an immune therapy for cancer treatments.

Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

ABSTRACTInterleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

Expression, purification and monoclonal antibodies preparation of recombinant equine mature interleukin-18

IL-18 is a cytokine originally discovered as an important modulator of immune responses and subsequently shown to be pleiotropic. In this report, we expressed the recombinant equine mature interleukin-18 (rEMIL-18) in E. coli and purified it by nickel affinity gel column chromatography. Purified rEMIL-18 had biological activity commensurate with recombinant human IL-18, as determined by its synergistic effect with recombinant human IL-12 (rhIL-12) on the induction of IFN-γ gene expression in equine peripheral blood mononuclear cells (PBMC). Following intraperitoneal (i.p.) immunization of BALB/c mice with rEMIL-18, nine monoclonal antibodies (mAbs) against equine interleukin-18 (EIL-18) were obtained and characterized. These mAbs recognized different epitopes on equine mature interleukin-18 (EMIL-18) protein based on their reactivity with two peptides containing different amino acid sequences and one of these mAbs has neutralization activity against EIL-18 in an IFN-γ-induction assay.

Adjuvant effects of chicken interleukin-18 in avian Newcastle disease vaccine

Interleukin-18 (IL-18) can induce interferon-γ (IFN-γ) production and promote Th1 immunity, and hence, it modulates immune functions. In the present study, the in vitro and in vivo immunomodulatory activities of full length or mature chicken IL-18 expressed in a prokaryotic expression system (pCHIL18-F and pCHIL18-M, respectively) and chicken IL-18 expressed in a eukaryotic expression system (euCHIL18) were examined. Results showed that pCHIL18-F, pCHIL18-M and euCHIL18 significantly enhanced IFN-γ mRNA expression in chicken splenocytes, which successfully increased IFN-γ-induced nitric oxide (NO) synthesis by macrophages. Vaccination with cell-cultured Newcastle disease vaccine (NDTC) co-administrated with pCHIL18-F, pCHIL18-M or euCHIL18 resulted in significant increments of hemagglutination inhibition (HI) titers, cell proliferation of peripheral blood mononuclear cells (PBMC), and ratios of CD8 + to CD4 + in chickens compared with inoculation of PBS or NDTC alone. Thus, full length and mature chicken IL-18 expressed using a prokaryotic system and using a eukaryotic system showed equivalent in vitro and in vivo biological activities, and all forms effectively enhanced cell-mediated and humoral immunity, suggesting possible future use as a potential adjuvant in chicken NDTC vaccine production.

Involvement of interleukin 18 in indomethacin-induced lesions of the gastric mucosa in adjuvant-induced arthritis rat

It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs) have significant side effects, such as gastroenteropathy, and rheumatoid arthritis patients taking NSAIDs are more susceptible to NSAIDs-induced gastric lesions in comparison with other patients. The pathogenic mechanism of these lesions is not fully understood. We demonstrate whether interleukin 18 (IL-18) expression relate the aggravation of gastric lesion in adjuvant-induced arthritis (AA) rats following the oral administration of indomethacin. Arthritis was induced by injecting 50 μl of a suspension of 10 mg/ml heat-killed butyricum ( Mycobacterium butyricum) in Bayol F oil into the plantar region of the right hind foot and tail of Dark Agouti rats resulting in an arthritis incidence of 100%. Two weeks after injection, the rats were administered indomethacin (40 mg/kg) orally, and were killed under deep ether anesthesia 6 h later. The gastric mucosa was then examined. Oral administration of indomethacin caused hemorrhagic lesions in the gastric mucosa of AA rats, and the lesion score for AA rats following indomethacin treatment was significantly higher than for normal rats administered indomethacin. The expression of the IL-18 mRNA and mature IL-18 protein in the gastric mucosa of AA rats administered indomethacin were also higher in comparison with normal rats receiving indomethacin. In addition, interferon-γ and nitric oxide levels in the gastric mucosa of AA rats were increased by the oral administration of indomethacin. It is possible that IL-18 expression in AA rats is more sensitive to indomethacin, and the IL-18 may play a role in the aggravation of gastric lesions in AA rats treated with indomethacin.

Anticarcinogenesis pathways activated by bovine lactoferrin in the murine small intestine

Oral administration of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs in rats, and lung metastasis in mice. A likely mechanism by which bLF mediates its anticarcinogenesis effects is by enhanced expression of cytokines and subsequent activation of immune cells. Oral administration of bLF enhances expression of interleukin-18 (IL-18) mRNA in the mucosa of the small intestine of mice. Importantly, the pepsin hydrolysate of bLF (bLFH) also induced expression of IL-18 mRNA in the mouse small intestine and a peptide produced by pepsin digestion of bLF, bovine lactoferricin (bLFcin), induced expression of mature IL-18 in organ culture. In addition to IL-18, bLF and bLFcin both induced significant increases in caspase-1 activity in peritoneal macrophages and in organ cultures. The increase of mature IL-18 by macrophages was inhibited by caspase-1 inhibitor: caspase-1 is known to cleave the proform of IL-18 to produce active mature IL-18. Finally, bLF also induced expression of IFNγ by peritoneal macrophages. Importantly, in IFNγ knockout (GKO) mice, bLF administration resulted in increased expression of caspase-1 protein, but induction of IL-18 mRNA, caspase-1 activity, and mature IL-18 was not observed. These results indicate that orally administered bLF can induce expression of IFNγ and caspase-1 in the small intestine. IFNγ in turn increases expression of target genes, including IL-18. Active caspase-1 then cleaves pro-IL-18 to generate mature IL-18. Thus, bLF activates an effector pathway mediated by IFNγ, caspase-1, and IL-18. We also show that ingested bLF is able to activate more than a single effector pathway. For example, in GKO mice while bLF administration could not activate the IFNγ/caspase-1/IL-18 effector pathway, it was able to inhibit tumor growth and metastasis by activation of an IFNα/IL-7 effector pathway.

Rates of infiltration by macrophages and dendritic cells and expression of interleukin‐18 and interleukin‐12 in the chronic inflammatory lesions of Sjögren's syndrome: Correlation with certain features of immune hyperactivity and factors associated with high risk of lymphoma development

To evaluate the expression profile of infiltrating macrophages and dendritic cells (DCs) as well as of interleukin-18 (IL-18) and IL-12 in the minor salivary gland (MSG) lesions of patients with Sjögren's syndrome (SS), and to assess the relationship of these factors with disease parameters. Macrophages, DCs, T cells, B cells, proIL-18, mature IL-18, and IL-12 were detected by single- and double-labeling immunohistochemistry in MSG specimens from 21 patients with primary SS (13 of 21 tested for IL-12), 7 patients with secondary SS, and 9 disease control patients. Expression profiles were assessed for correlations with various disease parameters, including adverse predictors of lymphoma development. MSGs from patients with SS (but not from disease controls) manifested increased infiltration by macrophages and DCs, strong expression of IL-18 by macrophages (particularly in B cell-rich areas and in germinal center-like structures in primary SS), and expression of IL-12 by mononuclear cell infiltrates. In primary SS, high infiltration by macrophages correlated with SG enlargement (P = 0.01). The DC infiltration rate correlated positively with the macrophage infiltration rate (P = 0.04), occurrence of SG enlargement (P = 0.03), and presence of C4 hypocomplementemia (P = 0.05), and inversely with serum C4 complement levels (P = 0.001). The rate of infiltration by IL-18-expressing cells correlated positively with biopsy focus scores (P < 0.001), larger infiltrates of macrophages (P = 0.01), DCs (P = 0.01), and B cells (P = 0.02), and SG enlargement (P = 0.02), and negatively with serum C4 complement levels (P = 0.02). The rate of infiltration by IL-12-expressing cells correlated inversely with that by IL-18-expressing cells (P = 0.001), biopsy focus scores (P = 0.003), and SG enlargement (P = 0.01), and positively with serum C4 complement levels (P = 0.05). In patients with primary SS, infiltration of the SG by macrophages and DCs and expression of IL-18 and IL-12 appear to play active roles in the expansion and organization of infiltrative injuries and have a correlation with certain predictors of lymphoma development.

Interleukin-18 is increased only in a minority of patients with active Crohn’s disease

It has been suggested that Crohn's Disease (CD) is associated with an elevated T helper 1 response as manifested by increased production of interleukin-18 (IL-18). Local concentrations of neutralizing IL-18 binding proteins (IL-18 bp) may counteract biological functions of mature IL-18 in mucosal inflammation. Therefore, we investigated the IL-18/IL-18 bp system in a large group of patients with active inflammatory bowel disease (IBD) to identify patients that could respond theoretically to IL-18 neutralizing treatment strategies. IL-18 and IL-18 bp messenger RNA (mRNA) expression in colonic mucosa from patients with active CD (n = 72), active ulcerative colitis (UC; n = 32), and non-IBD controls (infectious colitis or diverticulitis; n = 19) and normal, non-diseased controls (n = 20) were measured by reverse-transcribed real-time polymerase chain reaction. Mature IL-18 protein and IL-18 bp expression in inflamed mucosa were assessed by Western blotting. Although IL-18 mRNA was increased in some patients with CD, the increase was not statistically significant. Densitometric evaluation of IL-18/alpha-actin ratio in patients with active CD (n = 20) and patients with UC (n = 10) demonstrated an increased ratio of IL-18 protein in CD when compared to UC (1.04 vs 0.72 [median]). On closer inspections, only 7/20 CD patients had an increased IL-18 protein expression in inflamed areas compared to noninflamed mucosa. IL-18 expression in active CD is heterogeneous, only a minority of patients expresses elevated levels. Further treatment strategies targeting IL-18 expression in active CD should be concentrated on this subgroup of patients.

Engineering human IL-18 with increased bioactivity and bioavailability

Cytokines in plasmid form can act as potent adjuvants when co-administered with DNA vaccines, resulting in an enhanced immune response to the DNA-encoded antigen. This is true of interleukin-18 (IL-18), which has been shown to serve as an adjuvant in conjunction with certain DNA vaccines. To determine if the properties of IL-18 could be optimized for use as a DNA vaccine adjuvant, a model of IL-18/IL-18R binding was developed to identify variants of human IL-18 that were predicted to improve receptor interactions and potentially bioactivity. The linkage of mature IL-18 to a secretion signal sequence provided improved protein expression from mammalian cells and signal peptidase cleavage of this protein produced the authentic N-terminus. The IL-18 variant proteins secreted this way were bioactive, as demonstrated by their ability to induce interferon gamma (IFNγ) expression by human peripheral blood mononuclear cells (PBMCs) and to bind to IL-18R, as demonstrated by BIAcore analysis. The IL-18 variants were inhibited by IL-18 binding protein (IL-18BP), the soluble inhibitor of IL-18, as measured by neutralization of the IFNγ response in PBMCs. One variant, V11I/T63A, demonstrated increases both in bioactivity and mammalian cell expression as compared to native IL-18, indicating that this molecule may be particularly well suited for use as a DNA-encoded vaccine adjuvant.

Expression and alternative processing of IL‐18 inhuman neutrophils

Interleukin-18 (IL-18), a member of the IL-1 cytokine superfamily, is an important regulator of both innate and acquired immune responses. We demonstrate here constitutive expression of IL-18 by human neutrophils. Unexpectedly, we observed that neutrophils from peripheral blood or rheumatoid synovial compartments contained not only pro and mature IL-18, but also several novel smaller-molecular-weight IL-18-derived species. Using specific protease inhibitors, and serine protease gene-targeted mice, we demonstrate that these IL-18-derived products arose through caspase-independent cleavage events mediated by the serine proteases, elastase and cathepsin G. Moreover, we report that the net effect of elastase treatment of mature recombinant IL-18 was to reduce its IFN-gamma-inducing activity. Thus, human neutrophils contain IL-18 and IL-18-derived molecular species that can arise through novel enzymatic processing pathways. Through cytosolic, membrane or secretory expression of such processing enzymes, together with generation of IL-18 itself, neutrophils likely play a critical role in regulating IL-18 activities during early innate immune responses.

Effects of specific interleukin-1beta-converting enzyme inhibitor on ischemic acute renal failure in murine models1

To study the effect of selective interleukin-1beta-converting enzyme (ICE, caspase-1) inhibitor on ischemic acute renal failure (ARF). Mouse models of ischemic ARF were treated with the specific ICE inhibitor AC-YVAD-CMK. A renal function assay and renal morphological studies were employed to estimate the renal protective effect of AC-YVAD-CMK. The survival rate of mouse models was also analyzed by a time series test. Furthermore, renal ICE activity, mature interleukin-18 (IL-18) protein expression and interferon-gamma (IFN-gamma) mRNA expression were also detected by fluorescent enzyme-linked immunosorbent assay (ELISA), ELISA, and semi-quantitative reverse transcription-polymerase chain reaction, respectively. The levels of blood urea nitrogen (BUN) and serum creatinine (Scr) increased remarkably in the model controls compared with the sham-operated groups (P<0.01). Typical renal tubular necrosis was found in the model controls. Renal ICE activity, mature IL-18 protein expression, and IFN-gamma mRNA expression were also increased significantly in the model controls compared with the sham-operated groups. The levels of BUN and Scr in the AC-YVAD-CMK therapy group were decreased significantly compared with the untreated model controls (P<0.01). Renal tubulointerstitial lesion was also attenuated significantly (P<0.05). AC-YVAD-CMK therapy alleviated the clinical features of ARF, and increased the survival rate (P<0.01). Furthermore, AC-YVAD-CMK therapy also decreased ICE activity, mature IL-18 protein expression, and IFN-gamma mRNA expression in renal tissue (P<0.05). The selective ICE inhibitor AC-YVAD-CMK can effectively protect the kidney from acute ischemic lesions. This protective effect is associated with decreased renal ICE activity and suppressed IL-18 maturation and IFN-gamma mRNA transcription.

Interleukin 18 (IL-18) upregulation in acute rejection of kidney allograft

Interleukin 18 (IL-18) is a potent proinflammatory cytokine involved in the host defence by upregulating both innate and acquired immune responses and may be of particular importance also in mechanisms of kidney allograft rejection. Immunohistochemical staining of protocol biopsies showed constitutive IL-18 expression in the epithelium of distal tubules with the induction of immunoreactivity in acute rejection patients where also proximal tubules, infiltrating leukocytes, and endothelium were strongly positive. Furthermore, serum levels of IL-18 were significantly elevated in patients with acute rejection of kidney allograft (1247 ± 389 pg/l) as compared to patients with uncomplicated outcome of kidney transplantation (444 ± 164 pg/l) and subjects with acute tubulointerstitial nephropathy (385 ± 155 pg/l, p < 0.0001 for both comparisons). Tissue culture model of renal epithelial cells expressed IL-18 mRNA constitutively and released mature IL-18 in response to TNF-α and IFN-γ. We assume that upregulation of epithelial IL-18 plays an important role in immune and immunopathological reactions in renal parenchyma and contributes to rejection mechanisms of kidney allograft.