Areum Kwak

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.

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.

Cytokine-like Activity of Liver Type Fatty Acid Binding Protein (L-FABP) Inducing Inflammatory Cytokine Interleukin-6

It has been reported that fatty acid binding proteins (FABPs) do not act only as intracellular mediators of lipid responses but also have extracellular functions. This study aimed to investigate whether extracellular liver type (L)-FABP has a biological activity and to determined serum L-FABP levels in patients with end-stage renal disease (ESRD). We isolated L-FABP complementary deoxyribonucleic acid (cDNA) from the Huh7 human hepatocarcinoma cell line and expressed the recombinant L-FABP protein in Escherichia coli. A549 lung carcinoma and THP-1 monocytic cells were stimulated with the human recombinant L-FABP. Human whole blood cells were also treated with the human recombinant L-FABP or interleukin (IL)-1α. IL-6 levels were measured in cell culture supernatants using IL-6 enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in A549, THP-1 cells, and whole blood cells. The blood samples of healthy volunteers and patients with ESRD were taken after an overnight fast. The serum levels of L-FABP in healthy volunteers and ESRD patients were quantified with L-FABP ELISA. The values of L-FABP in patients with ESRD were significantly lower than those in the control group. Our results demonstrated the biological activity of L-FABP in human cells suggesting L-FABP can be a mediator of inflammation.

Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts

Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12-EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells, a cervical cancer cell line. The entry of LDP12-EGFP was partially blocked by amiloride, while cytochalasin D or methyl-β-cyclodextrin slightly increased the uptake. LDP12-EGFP shows efficient transduction in mouse blastocysts, but not in oocytes, two-cell-stage, or morula-stage-preimplantation embryos. LDP12-mediated delivery of EGFP-LC3, a widely used marker of autophagic activation, is successful in HeLa cells and mouse blastocysts, as it enters cells and exhibits a signature punctate pattern. The lipidation of EGFP-LC3 also normally occurs after transduction, suggesting that the transduced protein retains the functional characteristics. Collectively, we show that LDP12-driven protein delivery is a fast and convenient method applicable to mouse blastocysts and reproductive cancer cells.

IL-32-induced Inflammatory Cytokines Are Selectively Suppressed by α1-antitrypsin in Mouse Bone Marrow Cells

The induction of interleukin (IL)-32 in bone marrow (BM) inflammation is crucial in graft versus host disease (GvHD) that is a common side effect of allogeneic BM transplantation. Clinical trials on α-1 antitrypsin (AAT) in patients with GvHD are based on the preliminary human and mouse studies on AAT reducing the severity of GvHD. Proteinase 3 (PR3) is an IL-32-binding protein that was isolated from human urine. IL-32 primarily induces inflammatory cytokines in myeloid cells, probably due to PR3 expression on the membrane of the myeloid lineage cells. The inhibitory activity of AAT on serine proteinases may explain the anti-inflammatory effect of AAT on GvHD. However, the anti-inflammatory activity of AAT on BM cells remains unclear. Mouse BM cells were treated with IL-32γ and different inflammatory stimuli to investigate the anti-inflammatory activity of AAT. Recombinant AAT-Fc fusion protein inhibited IL-32γ-induced IL-6 expression in BM cells, but failed to suppress that induced by other stimuli. In addition, the binding of IL-32γ to PR3 was abrogated by AAT-Fc. The data suggest that the specific anti-inflammatory effect of AAT in mouse BM cells is due to the blocking of IL-32 binding to membrane PR3.

Species Specific Antiviral Activity of Porcine Interferon-α8 (IFNα8)

Interferons (IFNs) have been known as antiviral genes and they are classified by type 1, type 2, and type 3 IFN. The type 1 IFN consists of IFNα, IFNβ, IFNτ, and IFNω whereas the type 2 IFN consists of only IFNγ, which is a key cytokine driving T helper cell type 1 immunity. IFNλ belongs to the type 3 IFN, which is also known as IL-28 and IL-29 possessing antiviral activities. Type 1 IFN is produced by viral infection whereas type 2 IFN is induced by mitogenic or antigenic T-cell stimuli. The IFNτ of bovine was first discovered in an ungulate ruminant recognition hormone. IFNτ belongs to the type 1 IFN with the common feature of type 1 IFN such as antiviral activity. IFNs have been mostly studied for basic research and clinical usages therefore there was no effort to investigate IFNs in industrial animals. Here we cloned porcine IFNα8 from peripheral blood mononuclear cells of Korean domestic pig (Sus scrofa domestica). The newly cloned IFNα8 amino acid sequence from Korean domestic pig shares 98.4% identity with the known porcine IFNα8 in databank. The recombinant porcine IFNα8 showed potent antiviral activity and protected bovine Madin-Darby bovine kidney epithelial (MDBK) cells from the cytopathic effect of vesicular stomatitis virus, but it failed to protect human Wistar Institute Susan Hayflick (WISH) cells and canine Madin-Darby canine kidney epithelial-like (MDCK) cells. The present study demonstrates species specific antiviral activity of porcine IFNα8.

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1β, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1β but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1β activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/β. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.

Development of an interleukin (IL)-33 sandwich ELISA kit specific for mature IL-33

ABSTRACT Interleukin (IL)-33 is an inflammatory cytokine and belongs to the IL-1 family of cytokines. There are eleven members of the IL-1 family of cytokines and all have important roles in host defense against infections. Their levels are increased during infection and in various auto-inflammatory diseases. IL-33 is also associated with autoimmune diseases such as asthma, atopic dermatitis, rheumatoid arthritis, and atherosclerosis. IL-33 receptors consist of IL-1R4 and IL-1R3 to induce both Th1 and Th2 type immune response. Here we present the development of monoclonal antibodies (mAbs) against human mature IL-33. Recombinant human mature IL-33 protein was expressed in E. coli and purified by multi-step affinity chromatography. The human IL-33 activity was examined in HMC-1 and Raw 264.7 cells. Mice were immunized with the biologically active mature IL-33 to generate mAb against IL-33. The anti-IL-33 mAb (clone/4) was used as a capture antibody for a sandwich enzyme-linked immunosorbent assay (ELISA). This assay detects mature IL-33 with a high sensitivity (80 pg/mL) but does not recognize the biologically inactive precursor IL-33. This article describes the methods for a newly developed IL-33 ELISA kit that is specific for mature IL-33 and may be used to analyze bioactive mature IL-33 in various immunological diseases.