Eunsom Kim

The Interleukin-1α Precursor is Biologically Active and is Likely a Key Alarmin in the IL-1 Family of Cytokines

Among the 11 members of the IL-1 family cytokines, the precursors of IL-1α, IL-1β, and IL-33 have relatively long N-terminal pro-sequences of approximately 100 amino acid residues prior to the N-terminus of the mature forms. Compared to the mature forms secreted from the cell, 80–90% of the primary translation product is in the intracellular compartment in the precursor form. However, the precursors are readily released from cells during infections but also with non-infectious conditions such a hypoxia and trauma. In this setting, the precursors act rapidly as “alarmins” in the absence of a processing mechanism to remove the pro-sequence and generate a mature form. In the case of IL-1α, the release of the precursor activates adjacent cells via receptor-mediated signaling. However, there are no data comparing the specific activity of the IL-1α precursor to the mature form. In the present study, we compared the precursor and mature forms of recombinant human IL-1α, IL-1β, and IL-33 proteins on the induction of cytokines from A549 cells as well as from human peripheral blood mononuclear cells (PBMC). Similar to the mature form, the IL-1α precursor was active in inducing IL-6 and TNFα, whereas the precursor forms of IL-1β and IL-33 were not active. On PBMC, precursor and mature IL-1α at 0.04 and 0.2 nM were equally active in inducing IL-6. Given the fact that during necrotic cell death, the IL-1α precursor is released intact and triggers IL-1 receptors on tissue macrophages, these data identify the precursor form of IL-1α as a key player in sterile inflammation.

Identification of Constitutively Active Interleukin 33 (IL-33) Splice Variant

IL-33/IL-1F11 is a new member of the IL-1 family ligand and provokes T helper-type immune responses. IL-33 is the ligand of ST2 and IL-1 receptor accessory protein (IL-1RAcP) that triggers nuclear factor-κ light chain enhancer of activated B cells (NF-κB) and MAPK signaling. We discovered a novel short splice variant of IL-33 that was termed spIL-33. The new spIL-33 lacks exon 3 containing a proposed caspase-1 cleavage site. We isolated spIL-33 cDNA from the Huh7 human hepatocarcinoma cell line and expressed the recombinant spIL-33 protein in Escherichia coli. The recombinant spIL-33 and pro-IL-33 were not cleaved by caspase-1, unlike IL-18 (IL-1F4). The recombinant spIL-33 was constitutively active, and spIL-33-induced inflammatory cytokine production was caspase-1-independent in HMC-1 and Raw 264.7 cells. The recombinant spIL-33 induced the phosphorylation of IL-1 receptor-associated kinase (IRAK1), NF-κB, p38 MAPK, p44/42 MAPK, and JNK in a time- and dose-dependent manner. Anti-ST2 monoclonal antibody specifically blocked the spIL-33-induced cytokine production. In this study, we identified and characterized a new IL-33 splice variant, which was a constitutively active IL-33 isoform. The existence of constitutively active spIL-33 suggests that the biological activity of IL-33 could be triggered by diverse stimulations during immune responses. Further investigation of the spIL-33 expression pattern may contribute to understanding the involvement of IL-33 in inflammatory disorders.

Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity

Background: The maturation process of IL-33 (IL-1F11) remains unclear. Results: IL-33 ligand affinity column isolates neutrophil proteinase 3. Conclusion: PR3 is an IL-33-processing enzyme. Significance: PR3 has a dual function in IL-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-1β (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.

Characterizing antiviral mechanism of interleukin-32 and a circulating soluble isoform in viral infection

Interleukin-32 (IL-32) is an inflammatory cytokine, and its activity is associated with various auto-inflammatory disorders as well as infectious pathogens such as Mycobacterium tuberculosis, and viral infections. However, the precise antiviral mechanism of IL-32 remains unclear. We assessed the IL-32 level in the sera of H1N1 influenza A patients and IL-32 level was significantly elevated. Next we examined the antiviral activity of recombinant IL-32γ (rIL-32γ) with WISH cells infected by vesicular stomatitis virus (VSV) but no antiviral activity was observed. Therefore we investigated the supernatant of rIL-32-treated THP-1 cells since this cell line effectively responded to rIL-32γ. The supernatant of rIL-32-treated THP-1 cell possessed an antiviral effect and in addition, an agonistic monoclonal antibody further enhanced a specific antiviral activity of rIL-32γ. The fractionation and mass spectrometer analysis of the THP-1 cell supernatant revealed that the antiviral activity of rIL-32γ is via a THP-1 cell-produced factor, transferrin, rather than the direct effects of rIL-32γ on epithelial cells. We also characterized a secreted soluble IL-32γ protein in serum of IL-32γ transgenic mouse (TG), but not in that of IL-32α TG. The present results suggest that IL-32γ expression and its genetic variation in individual could be an important aspect of viral infections.

Effect of Recombinant α1-Antitrypsin Fc-Fused (AAT-Fc)Protein on the Inhibition of Inflammatory Cytokine Production and Streptozotocin-Induced Diabetes

Abstractα1-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.

Circulating IL-33 level is associated with the progression of lung cancer

OBJECTIVES Interleukin (IL)-33 protects against infection and inflammation; however, few studies have explored the relevance of IL-33 in lung cancer patients. We evaluated relation of plasma IL-33 levels with development and progression of lung cancer. MATERIALS AND METHODS A total of 160 patients with lung cancer and 160 controls with normal lungs were enrolled. Plasma IL-33 levels were measured using a specific sandwich ELISA; these levels were followed-up in 18 patients who underwent surgery and in 14 patients treated with chemotherapy. Malignant lesions and normal lung tissues from 10 cancer patients were subjected to immunohistochemical staining for IL-33. RESULTS IL-33 levels were significantly lower in cancer patients than normal controls (0.08 vs. 0.38 ng/mL, p=0.005). Among cancer patients, IL-33 decreased in a stage-dependent manner from 0.76 ng/mL in stage I patients to 0.25 ng/mL in those with stage II, 0.08 ng/mL in those with stage III, and 0.08 ng/mL in those with stage IV (p=0.002). The levels were higher at stage I (p=0.041) and markedly lower at stages III and IV than those of controls (p=0.005 and p=0.001, respectively). A similar pattern was observed when IL-33 levels were analyzed by T stage; the levels were 0.39 ng/mL at T1/T2 vs. 0.08 ng/mL at T3/T4 (p=0.001). However, no difference was noted when stage N1 levels were compared with N2 and N3 levels (p=0.058), or between stage M0 and M1 levels (p=0.147). IL-33 levels gradually decreased after surgical resection of malignant lesions (from 1.075 to 0.756 ng/mL, p=0.006), but were unchanged after chemotherapy (0.705 vs. 0.829 ng/mL, p=0.875). On immunohistochemical staining, bronchial epithelial and vascular endothelial cells of normal lung tissues mainly expressed IL-33. CONCLUSIONS Plasma IL-33 levels are associated inversely with progression of lung cancer. The observed decreases may be attributed to lung volume reduction containing bronchial epithelium and vascular endothelium as the sources of IL-33.

Interleukin-32 gamma specific monoclonal antibody and developing IL-32 specific ELISA.

Cytokines are essential coordinators of defensive immune responses for resolving the invasion of pathogens such as bacteria, virus, and fungi. However, dysregulated cytokines are the main cause of various autoinflammatory immune disorders such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis. Interleukin-32 (IL-32) is a recently described cytokine and characterized as a proinflammatory cytokine. IL-32 stimulates monocytes and macrophages to induce important proinflammatory cytokines (IL-1β, IL-6, and TNFα) and chemokines (IL-8 and MIP-2) by activating the NF-κB and p38 mitogen-activated protein (MAP) kinase pathways. The biological activities of IL-32 are associated with epidemic pathogens, Mycobacterium tuberculosis, influenza A virus, and human immunodeficiency virus (HIV). IL-32 is transcribed as six alternative splice variants (α, β, γ, δ, ɛ, and ζ), with IL-32γ being the most active isoform. However, it is unclear which isoform is related to specific disease activities since there are no high quality antibodies available to measure circulating IL-32 in biological samples of patients. Therefore, we developed specific anti-human IL-32γ monoclonal antibodies from recombinant human IL-32γ, which was expressed in Escherichia coli. The IL-32γ specific monoclonal antibodies recognized IL-32 in cell culture supernatants and serum of IL-32γ transgenic mice. The newly developed IL-32γ monoclonal antibodies will be a useful tool to measure IL-32 level in serum samples of various inflammatory diseases. These monoclonal antibodies will be helpful in investigating the precise function of IL-32 in immune responses and in autoinflammatory diseases.

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, β, 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-1β, 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 β-cells injury and inflammation in pancreas and aggravates STZ-induced type 1 diabetes.

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.

Proinsulin Shares a Motif with Interleukin-1α (IL-1α) and Induces Inflammatory Cytokine via Interleukin-1 Receptor 1

Although it has been established that diabetes increases susceptibility to infections, the role of insulin (INS) in the immune response is unknown. Here, we investigated the immunological function of INS. Proinsulin dimer (pINSd) was a potent immune stimulus that induced inflammatory cytokines, but mature INS was unable to induce an immune response. An affinity-purified rabbit polyclonal antibody raised against mature IL-1α recognized IL-1α and pINS but failed to detect mature INS and IL-1β. Analysis of the pINS sequence revealed the existence of an INS/IL-1α motif in the C-peptide of pINS. Surprisingly, the INS/IL-1α motif was recognized by monoclonal antibody raised against IL-1α. Deleting the INS/IL-1α motif in pINSd and IL-1α changed their activities. To investigate the pINSd receptor, the reconstitution of IL-1 receptor 1 (IL-1R1) in Wish cells restored pINSd activity that was reversed by an IL-1R antagonist. These data suggested that pINSd needs IL-1R1 for inflammatory cytokine induction. Mouse embryo fibroblast cells of IL-1R1-deficient mice further confirmed that pINSd promotes immune responses through IL-1R1.