A Robust Quadruple Protein-Based Indirect ELISA for Detection of Antibodies to African Swine Fever Virus in Pigs.

African swine fever (ASF) emerged in domestic pigs and wild boars in China in 2018 and rapidly spread to neighboring Asian countries. Currently, no effective vaccine or diagnostic tests are available to prevent its spread. We developed a robust quadruple recombinant-protein-based indirect enzyme-linked immunosorbent assay (QrP-iELISA) using four antigenic proteins (CD2v, CAP80, p54, and p22) to detect ASF virus (ASFV) antibodies and compared it with a commercial kit (IDvet) using ASFV-positive and -negative serum samples. The maximum positive/negative value was 24.033 at a single antigen concentration of 0.25 µg/mL and quadruple ASFV antigen combination of 1 µg/mL at a 1:100 serum dilution. Among 70 ASFV-positive samples, 65, 67, 65, 70, 70, and 14 were positive above the cut-offs of 0.121, 0.121, 0.183, 0.065, 0.201, and 0.122, for CD2v, CAP80, p54, p22-iELISA, QrP-iELISA, and IDvet, respectively, with sensitivities of 92.9%, 95.7%, 92.9%, 100%, 100%, and 20%, respectively, all with 100% specificity. The antibody responses in QrP-iELISA and IDvet were similar in pigs infected with ASFV I. QrP-iELISA was more sensitive than IDvet for early antibody detection in pigs infected with ASFV II. These data provide a foundation for developing advanced ASF antibody detection kits critical for ASF surveillance and control.

Development of an indirect ELISA against African swine fever virus using two recombinant antigens, partial p22 and p30.

African swine fever (ASF) is a highly fatal viral disease affecting pigs. It is caused by the ASF virus (ASFV), and causes serious economic losses to the swine industry worldwide, including in Korea. Commercially available enzyme-linked immunosorbent assay (ELISA) kits for detecting anti-ASFV antibodies are used for the diagnosis and surveillance of ASF. In this study, an ELISA was developed to detect anti-ASFV antibodies using two recombinant proteins, p22 and p30, from genotype II ASFV. Recombinant transmembrane domain-deleted p22 (p22∆TM) and p30 were expressed in E.coli vector system pET32a and mixed for use as antigens in indirect ELISA. The p22∆TM/p30-based indirect ELISA was validated using 31 sera from genotype I ASFV-infected pigs and 1133 sera from uninfected pigs. Area under the curve of this test was 0.999 [95 % concentration interval 0.992-1.000], and sensitivity and specificity were 93.5 % and 99.8 %, respectively. The between run coefficient of variation for internal quality control serum was 6.61 %. In the seroconversion analysis, the p22∆TM/p30-based indirect ELISA showed equal or better ability to detect antibodies in pigs experimentally challenged with ASFV p72 genotypes I and II (p < 0.05). In conclusion, the p22∆TM/p30-based indirect ELISA is a reliable diagnostic method for detecting anti-ASFV antibodies.

Evaluation of swine protection with foot-and-mouth disease O1/Campos and O/Primorsky/2014 vaccines against the O Mya-98 lineage virus from East Asia.

Two type O commercial vaccines, the O1/Campos and O/Primorsky/2014 vaccines, were studied to evaluate the in vivo efficacy in pigs against heterologous virus challenge with the O/SKR/Jincheon/2014 virus (O/SEA/Mya-98 lineage) isolated in Korea in 2014. The in vivo challenge results indicated that both vaccines induced a high heterologous virus neutralization test (VNT) titer by a single injection and successfully protected specific pathogen-free (SPF) pigs from challenge infection. To determine the optimal vaccination age, a field trial with each vaccine was conducted with three one-shot-vaccinated groups that were injected at 8, 12, or 14 weeks of age and one two-shot-vaccinated group that was injected at 8 and 12 weeks of age in the pig farms. In these field trials, the improved serological performance at 20 and 24 weeks of age expected with vaccination at 12 or 14 weeks of age was not observed, although improved serological results were expected as the result of decreasing interference of maternally derived antibodies (MDAs), as MDAs waned with age. In addition, delayed vaccination resulted in MDA depletion at 14 weeks of age. Therefore, the optimal age for primary vaccination with two different formulated vaccines was 8 weeks old in pigs, considering that MDAs could provide a protective immunity against foot-and-mouth disease (FMD) infection. Prolonged significantly higher VNT titers of immunized pigs were demonstrated in the two-shot-vaccinated groups. In total, the effectiveness of the two vaccines was demonstrated through efficacy tests and field trials in pigs.

Isolation and Genetic Characterization of African Swine Fever Virus from Domestic Pig Farms in South Korea, 2019.

On 17 September 2019, the first outbreak of African swine fever in a pig farm was confirmed in South Korea. By 9 October, 14 outbreaks of ASF in domestic pigs had been diagnosed in 4 cities/counties. We isolated viruses from all infected farms and performed genetic characterization. The phylogenetic analysis showed that all of fourteen ASFV isolates in South Korea belong to genotype II and serogroup 8. Additionally, all isolates had an intergenic region (IGR) II variant with additional tandem repeat sequences (TRSs) between the I73R and I329L genes and showed characteristics of central variable region (CVR) 1 of the B602L gene and IGR 1 of MGF 505 9R/10R genes. These are identical to the genetic characteristics of some European isolates and Chinese isolates.

The interference effect of maternally-derived antibodies on the serological performance of pigs immunized with a foot-and-mouth disease oil emulsion vaccine.

To control foot-and-mouth disease (FMD) outbreaks that originated in Jincheon County in South Korea between 2014 and 2015, several commercial vaccines were studied for their efficacy and serological performance in the field. In this study, the efficacy of the O SKR 7/10 vaccine was evaluated by challenge with the FMD virus (FMDV) O/Jincheon/SKR/2014 (O Jincheon), which has the same O/SEA/Mya-98 lineage as the O/SKR/7/10 strain that was isolated in 2010 in South Korea, in FMD-seronegative pigs. Full protection against the O Jincheon virus was demonstrated as early as 14 days postvaccination, which was explained by the strong serological relationship (r1 value: ≥ 0.92) between the O Jincheon and O SKR 2010 viruses. However, in the field trial, no satisfactory serological elevations against FMDV were observed, even in the double-vaccinated groups. Therefore, it can be concluded that the O SKR 7/10 vaccine may need to be improved to overcome the interference effects from the high levels of maternally-derived antibodies generated due to the mandatory nationwide vaccination of sows in South Korea.

Cross-protective efficacy of the O1 Manisa + O 3039 bivalent vaccine and the O 3039 monovalent vaccine against heterologous challenge with FMDV O/Jincheon/SKR/2014 in pig.

After massive foot-and-mouth disease (FMD) outbreaks originated from Jincheon County from Dec. 2014 to Apr. 2015, the effectiveness of the previous FMD vaccine containing only the O1 Manisa as the O antigen, O1 Manisa + A Malaysia 97 + Asia 1 Sharmir trivalent vaccine, was questioned in South Korea, and a change in the O antigen in FMD vaccines was demanded to control the FMD caused by FMDV O/Jincheon/SKR/2014, the O Jincheon strain. Therefore, the efficacies of O1 Manisa + O 3039 bivalent vaccine and O 3039 monovalent vaccine were studied for cross-protection against heterologous challenge with the O Jincheon strain. In this study, the efficacy of the O1 Manisa + O 3039 bivalent vaccine was better than that of the O 3039 monovalent vaccine, even though the serological relationship (r1 value) between O Jincheon and O 3039 was matched according to the OIE Terrestrial Manual. According to serological test results from vaccinated specific pathogen free pigs, virus neutralization test titers against Jincheon were good estimates for predicting protection against challenge. A field trial of the O1 Manisa + O 3039 bivalent vaccine was performed to estimate the possibility of field application in conventional pig farms, especially due to concerns about the effect of maternally derived antibodies (MDA) in field application of the FMD vaccine. According to the result of the field trial, the O1 Manisa + O 3039 bivalent vaccine was considered to overcome MDA. The results of the efficacy and field trials indicated that the O1 Manisa + O3039 vaccine could be suitable to replace previous FMD vaccines to control the FMD field situation caused by O Jincheon FMDV.

Financial Impact of Foot-and-mouth disease outbreaks on pig farms in the Republic of Korea, 2014/2015.

The financial impact of foot-and-mouth disease (FMD) that occurred in 180 piggeries (100 farrow-to-finish and 80 fattening farms) confirmed infected during the 2014/2015 epidemic in the Republic of Korea was estimated at the farm level. The median loss due to slaughtering of pigs prior to their expected market weights was US$ 71.8 (uncovered compensation-compensation loss) plus US$ 57.3 (foregone net gain) per pig. Median loss per farm was US$ 27,487 (55.6% of total loss) for compensation and US$ 15,925 (44.4%) for foregone net gain. The total loss per farm (median, 25th-75th percentile) was US$ 43,822 (9,767-115,893), which represented 49.4% (11.5-112.8) of the annual net gain of pig farms. The total financial loss in 180 FMD outbreak pig farms was US$ 25.2 million, which was nearly one-half of the control cost (US$ 58.3 million) spent by the Korean government on this epidemic. The findings in this study should help planning to help reduce the impact at the farm level in the Republic of Korea in the future.

H5N8 Highly Pathogenic Avian Influenza in the Republic of Korea: Epidemiology During the First Wave, from January Through July 2014.

OBJECTIVES: This study describes the outbreaks of H5N8 highly pathogenic avian influenza (HPAI) in Korea during the first wave, from January 16, 2014 through July 25, 2014. Its purpose is to provide a better understanding of the epidemiology of H5N8 HPAI. METHODS: Information on the outbreak farms and HPAI positive wild birds was provided by the Animal and Plant Quarantine Agency. The epidemiological investigation sheets for the outbreak farms were examined. RESULTS: During the 7-month outbreak period (January-July 2014), H5N8 HPAI was confirmed in 212 poultry farms, 38 specimens from wild birds (stools, birds found dead or captured). Ducks were the most frequently infected poultry species (159 outbreak farms, 75.0%), and poultry in 67 (31.6%) outbreak farms was asymptomatic. CONCLUSION: As in the previous four H5N1 epidemics of HPAI that occurred in Korea, this epidemic of H5N8 proved to be associated with migratory birds. Poultry farms in Korea can hardly be free from the risk of HPAI introduced via migratory birds. The best way to overcome this geographical factor is to reinforce biosecurity to prevent exposure of farms, related people, and poultry to the pathogen.

Highly pathogenic avian influenza virus (H5N8) in domestic poultry and its relationship with migratory birds in South Korea during 2014.

Highly pathogenic H5N8 avian influenza viruses (HPAIVs) were introduced into South Korea during 2014, thereby caused outbreaks in wild birds and poultry farms. During the 2014 outbreak, H5N8 HPAIVs were isolated from 38 wild birds and 200 poultry farms (up to May 8, 2014). To better understand the introduction of these viruses and their relationships with wild birds and poultry farm, we analyzed the genetic sequences and available epidemiological data related to the viruses. Genetic analysis of 37 viruses isolated from wild birds and poultry farms showed that all of the isolates belonged to clade 2.3.4.6 of the hemagglutinin (HA) gene, but comprised two distinct groups. During the initial stage of the outbreak, identical isolates from each group were found in wild birds and poultry farms near Donglim Reservoir, which is a resting site for migratory birds, thereby indicating that two types of H5N8 HPAIVs were introduced into the lake at the same time. Interestingly, the one group of H5N8 HPAIV predominated around Donglim Reservoir, and the predominant virus was dispersed by wild birds among the migratory bird habitats in the western region of South Korea as time passed, and it was also detected in nearby poultry farms. Furthermore, compared with the results of the annual AIV surveillance of captured wild birds, which has been performed since 2008, more HPAIVs were isolated and H5 sero-prevalence was also detected during the 2014 outbreak. Overall, our results strongly suggest that migratory birds played a key role in the introduction and spread of viruses during the initial stage of the 2014 outbreak.

IL-32γ overexpression accelerates streptozotocin (STZ)-induced type 1 diabetes.

Interleukin-32 (IL-32) is a cytokine produced by T lymphocytes, natural killer (NK) cells, monocytes and epithelial cells. There are five splicing variants (α, ß, γ, δ, and ε) and IL-32γ is the most active isoform. We generated human IL-32γ transgenic (IL-32γ TG) mice, displaying a high level of IL-32γ expression in the pancreas. We investigated the effect of IL-32γ on streptozotocin (STZ)-induced type 1 diabetes model using IL-32γ TG mice. After a suboptimal diabetogenic dose of STZ administration, IL-32γ TG mice showed significantly increased blood glucose level comparing with that of wild type (WT) mice at day 5. Inflammatory cytokines levels such as, IL-6, TNFα, IFNγ and IL-1ß, in pancreas and liver lysates were accessed by a specific cytokine ELISA. The proinflammatory cytokines were significantly enhanced in the pancreas of IL-32γ TG mice comparing to that of WT mice whereas those cytokines levels in liver of IL-32γ TG and WT mice were not changed by STZ. These data indicate that the overexpression of IL-32γ contributes to initial islet ß-cells injury and inflammation in pancreas and aggravates STZ-induced type 1 diabetes.

Interleukin-32γ transgenic mice resist LPS-mediated septic shock.

Interleukin-32 (IL-32) is a cytokine and inducer of various proinflammatory cytokines such as TNFα, IL-1ß, and IL-6 as well as chemokines. There are five splicing variants (α, ß, γ, delta, and epsilon) and IL-32γ is the most active isoform. We generated human IL-32γ transgenic (IL-32γ TG) mice to express high level of IL-32γ in various tissues, including immune cells. The pathology of sepsis is based on the systemic inflammatory response that is characterized by upregulating inflammatory cytokines in whole body, particularly in response to gram-negative bacteria. We investigated the role of IL-32γ in a mouse model of experimental sepsis by using lipopolysaccharides (LPS). We found that IL-32γTG mice resisted LPS-induced lethal endotoxemia. IL-32γ reduced systemic cytokines release after LPS administration but not the local immune response. IL-32γTG increased neutrophil influx into the initial foci of the primary injected site, and prolonged local cytokines and chemokines production. These results suggest that neutrophil recruitment in IL-32γTG occurred as a result of the local induction of chemokines but not the systemic inflammatory cytokine circulation. Together, our results suggest that IL-32γ enhances an innate immune response against local infection but inhibits the spread of immune responses, leading to systemic immune disorder.

Interleukin-32γ suppresses allergic airway inflammation in mouse models of asthma.

Asthma is a chronic airway inflammatory disease typically associated with T helper cell type 2 (Th2) cytokines. IL-32, first reported as an inducer of tumor necrosis factor (TNF)-α, is an inflammatory cytokine involved in various autoinflammatory diseases, viral infection, and cancer-related inflammation. However, the role of IL-32γ in asthma has not been clearly elucidated. In this study, the levels of IL-32γ in sputum from patients with asthma were measured by ELISA, and IL-32γ function was investigated in murine models of asthma with human IL-32γ-overexpressed transgenic (IL-32γ TG) mice. The therapeutic effect of recombinant IL-32γ (rIL-32γ) on allergic inflammation was also evaluated through bronchoalveolar lavage fluid analysis and histopathologic examinations. Sputum IL-32γ levels from patients with asthma were lower than those from healthy control subjects. In an acute mouse model of asthma, IL-32γ TG mice exhibited significantly reduced airway inflammation compared with that in wild-type mice. The production of Th1 cytokines, such as TNF-α and IFN-γ, and Th2 cytokines, such as IL-4, IL-5, and IL-13, was decreased in the lungs of IL-32γTG mice. On the contrary, the expression of IL-10 and IL-10-producing CD11b(+) monocytic cells was significantly increased in the lungs of ovalbumin-sensitized IL-32γ TG mice. In addition, rIL-32γ treatment revealed a suppressive effect on the airway inflammation in a chronic mouse model of asthma. The results of this study suggest that IL-32γ may have a preventive role in the development of allergic airway inflammation and could be a potential novel therapeutic target for bronchial asthma.

Effect of recombinant α1-antitrypsin Fc-fused (AAT-Fc)protein on the inhibition of inflammatory cytokine production and streptozotocin-induced diabetes.

α1-Antitrypsin (AAT) is a member of the serine proteinase inhibitor family that impedes the enzymatic activity of serine proteinases, including human neutrophil elastase, cathepsin G and neutrophil proteinase 3. Here, we expressed recombinant AAT by fusing the intact AAT gene to the constant region of IgG1 to generate soluble recombinant AAT-Fc protein. The recombinant AAT-Fc protein was produced in Chinese hamster ovary (CHO) cells and purified using mini-protein A affinity chromatography. Recombinant AAT-Fc protein was tested for antiinflammatory function and AAT-Fc sufficiently suppressed tumor necrosis factor (TNF)-α-induced interleukin (IL)-6 in human peripheral blood mononuclear cells (PBMCs) and inhibited cytokine-induced TNFα by different cytokines in mouse macrophage Raw 264.7 cells. However, AAT-Fc failed to suppress lipopolysaccharide-induced cytokine production in both PBMCs and macrophages. In addition, our data showed that AAT-Fc blocks the development of hyperglycemia in a streptozotocin-induced mouse model of diabetes. Interestingly, we also found that plasma-derived AAT specifically inhibited the enzymatic activity of elastase but that AAT-Fc had no inhibitory effect on elastase activity.

The inhibitory function of Fc-ST2 depends on cell type; IL-1RAcP and ST2 are necessary but insufficient for IL-33 activity.

IL-33 (IL-1F11) is a member of IL-1 family ligand, which stimulates the production of inflammatory cytokines. IL-33 receptor complex is comprised of IL-1 receptor accessory protein (IL-1RAcP) and ST2 that are activated by IL-33 ligand binding. ST2 is a ligand-binding chain of the IL-33 receptor component, and the soluble ST2 form possesses antagonistic activity. Here, we expressed the extracellular domain of ST2-fused to the immunoglobulin of IgG1 constant region in order to generate a soluble recombinant Fc-ST2. Human and mouse recombinant Fc-ST2 protein were expressed in Chinese hamster ovary cells and purified using a mini-protein A affinity chromatography. The recombinant Fc-ST2 protein was used to examine inhibitory function in IL-33-induced cytokine production in different cell types. The human Fc-ST2 abolished IL-33-induced IL-8 production in human mast cells, but mouse Fc-ST2 failed to inhibit IL-33-induced TNFα production in mouse Raw 264.7 macrophage cells. We further investigated the expression of IL-33 receptor component with various cell lines. IL-33 receptors expression pattern and Fc-ST2 inhibitory activity in different cell types suggest that IL-1RAcP and ST2 are necessary but insufficient for IL-33 activity. Our results suggest that an additional receptor component may participate in the biological activity of IL-33.

BIRB 796 has Distinctive Anti-inflammatory Effects on Different Cell Types.

The pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin (IL)-1ß are crucial mediators involved in chronic inflammatory diseases. Inflammatory signal pathways regulate inflammatory cytokine expression-mediated by p38 mitogen activated protein kinase (p38MAPK). Therefore, considerable attention has been given to p38MAPK as a target molecule for the development of a novel anti-inflammatory therapeutics. BIRB 796, one of p38MAPK inhibitor, is a candidate of therapeutic drug for chronic inflammatory diseases. In this study, we investigated the effect of BIRB 796 on inflammatory cytokine productions by lipopolysaccharide (LPS) in different immune cell types. BIRB 796 reduced LPS-mediated IL-8 production in THP-1 cells but not in Raw 264.7 cells. Further analysis of signal molecules by western blot revealed that BIRB 796 sufficiently suppressed LPS-mediated phosphorylation of p38MAPK in both cell types whereas it failed to block inhibitor of kappa B (I-κB) degradation in Raw 264.7 cells. Taken together, these results suggest that the anti-inflammatory function of BIRB 796 depends on cell types.

Elevated interleukin-32 expression in granulomatosis with polyangiitis.

OBJECTIVE: To evaluate the role of IL-32 in granulomatosis with polyangiitis (GPA) patients and the relationship between IL-32 and disease activity, as PR3 has the ability to bind and activate IL-32, which has been described as a novel cytokine that induces inflammatory cytokines. METHODS: We investigated the level of IL-32, PR3, TNF-α and IL-6 in GPA patients by using ELISA. Northern blot was used to analyse the level of IL-32 mRNA in leucocytes of GPA patients. The intracellular colocalization of IL-32 and PR3 in leucocytes was examined by IF staining. RESULTS: We observed that IL-32 and PR3 levels in GPA patients were increased significantly when compared with normal individuals and each was tightly associated (P < 0.001). Northern blot analysis revealed that the mRNA level of IL-32 was prominently elevated in leucocytes of GPA patients. The intracellular colocalization of IL-32 and PR3 in leucocytes from GPA patients vs normal individuals was verified by IF staining. CONCLUSION: IL-32 level was elevated in GPA patients but its level was changed by treatment response. IL-32 could be an index in GPA and play a role in the aetiology of GPA.

Recombinant Fc-IL-18BPc isoform inhibits IL-18-induced cytokine production.

IL-18 is a pro-inflammatory cytokine that is produced from T cells and NK cells. IL-18 has been implicated in the pathogenesis of various inflammatory and cardiovascular diseases. IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18 that possesses higher affinity to IL-18 than that of the IL-18 receptor alpha chain on the cell surface. Human isoform a and c among four isoforms of IL-18BPs have an inhibitory effect on IL-18-induced cytokines whereas mouse IL-18BP isoforms exist only in two isoforms: c and d. Fc-fusion protein is a molecule in which the immunoglobulin Fc is fused genetically to a protein of interest, such as an extracellular domain of a receptor, ligand, or enzyme. In this study, we expressed and purified human Fc-IL-18BPa and c isoforms from CHO-DG44 cells and their biological activities were compared to each other. This is the first time that expressed recombinant human Fc-IL-18BPc has been examined for its biological activity on IL-18-induced IFNγ in human PBMC and IL-6 in A549/IL-18Rß.

Monoclonal antibodies against recombinant AtHOS15.

Histone modifications are important components of transcriptional regulation and chromatin-based regulatory processes. In addition, WD40-repeat protein and several other components are involved in these functions. Here we present the development of monoclonal antibodies (MAbs) against Arabidopsis HOS15, a WD40-repeat protein. Mice were immunized with recombinant HOS15 (rHOS15) protein for generating MAbs via the classic hybridoma production technique. We confirmed the specific activity of anti-HOS15 MAbs by tobacco transient expression assays. Based on immunoprecipitation assays, the anti-HOS15 MAb was able to detect endogenous HOS15 in Arabidopsis wild-type plants, but not in hos15 mutant plants. Finally, the anti-HOS15 MAbs are highly sensitive for detecting endogenous HOS15 protein. They can be used for immunological and immunoprecipitation assays to support other experimental strategies. In particular, the anti-HOS15 MAbs will be essential tools to investigate the role of HOS15 in the regulation of tolerance to environmental stresses in plants.

Clinical characteristics and the expression profiles of inflammatory cytokines/cytokine regulatory factors in asymptomatic patients with nodular gastritis.

BACKGROUND: Nodular gastritis (NG) has been reported in adult dyspeptic patients, whereas information on NG in asymptomatic patients is limited. AIMS: To evaluate the prevalence, clinico-epidemiological characteristics, and expression profiles of inflammatory cytokines or cytokine regulatory factors of NG in asymptomatic adults. METHODS: A cross-sectional study was conducted prospectively using 2,579 consecutive asymptomatic subjects who underwent screening esophagogastroduodenoscopy. The expression of inflammatory cytokines or cytokine regulatory factors in the gastric mucosa of NG patients was evaluated using immunofluorescence staining. RESULTS: NG was diagnosed in 52 patients (2.0%) and showed a predilection for females (M:F = 1:1.89) and young adults (median age: 34 years; range: 25-51 years). All NG patients were positive for Helicobacter pylori infection. Based on multivariate analysis, the risk of NG was increased in patients younger than 40 years (OR, 7.57; 95% CI, 3.76-15.24) and of the female gender (OR, 2.12; 95% CI; 1.05-4.28). Immunofluorescent staining for interleukin (IL)-1ß, IL-10, IL-18, IL-18 binding protein, IL-32, IL-33, and neutrophil proteinase 3 (PR3) was performed on cryosections of gastric mucosa. Interestingly, the expression of PR3 was highly increased in the gastric biopsies from asymptomatic NG patients but was expressed infrequently in the controls. CONCLUSIONS: Asymptomatic NG is associated with H. pylori infection, and a predilection for this condition exists in young females. The PR3 expression of gastric mucosa might play an important role in the pathogenesis of NG.

Contradictory functions (activation/termination) of neutrophil proteinase 3 enzyme (PR3) in interleukin-33 biological activity.

IL-1 family ligand does not possess a typical hydrophobic signal peptide and needs a processing enzyme for maturation. The maturation process of IL-33 (IL-1F11), a new member of the IL-1 family ligand, remains unclear. Precursor IL-33 ligand affinity column isolates neutrophil proteinase 3 (PR3) from human urinary proteins. PR3 is a known IL-1 family ligand-processing enzyme for IL-1ß (IL-1F2) and IL-18 (IL-1F4), including other inflammatory cytokines. We investigated PR3 in the maturation process of precursor IL-33 because we isolated urinary PR3 by using the precursor IL-33 ligand affinity column. PR3 converted inactive human and mouse precursor IL-33 proteins to biological active forms; however, the increase of PR3 incubation time abrogated IL-33 activities. Unlike caspase-1-cleaved precursor IL-18, PR3 cut precursor IL-33 and IL-18 at various sites and yielded multibands. The increased incubation period of PR3 abated mature IL-33 in a time-dependent manner. The result is consistent with the decreased bioactivity of IL-33 along with the increased PR3 incubation time. Six different human and mouse recombinant IL-33 proteins were expressed by the predicted consensus amino acid sequence of PR3 cleavage sites and tested for bioactivities. The human IL-33/p1 was highly active, but human IL-33/p2 and p3 proteins were inactive. Our results suggest the dual functions (activation/termination) of PR3 in IL-33 biological activity.