Tear levels of IL-7, IL-1α, and IL-1ß may differentiate between IgG4-related disease and Sjögren's syndrome.

We aim to assess and compare a cytokine and chemokine profile in tears from patients with IgG4-related disease (IgG4-RD) and Sjögren's syndrome (SS), and to see if this profile could aid in differentiating these two diseases. We included 10 patients with IgG4-RD who met the Comprehensive Diagnostic Criteria for IgG4-RD and 17 patients who met the AECG criteria for primary SS. The Schirmer-I test was carried out using two standardized sterile tear strips, which were then immediately frozen at - 86 °C until assayed. The tears were extracted from the strips after they had been defrosted using a buffer containing 0.5 M NaCl and 0.5% Tween-20. The amounts (pg/ml) of the following cytokines and chemokines were then measured using luminometry: IFN-γ, TNF-α, G-CSF, IL-1-α, IL-1ß, IL-4, IL-7, IL-12p40, IL-12p70, IL-13, IL-17A, CCL2, CCL3, CCL4, CCL11, and CXCL10. In the IgG4-RD group, seven patients had lacrimal gland involvement, five had dry eye symptoms, and six had a positive Schirmer-I test. In the SS group, 16 (94.1%) had dry eyes and all had a positive Schirmer-I test. We were able to differentiate between both diseases using levels of IL-7, IL-1α, and IL-1ß; in particular, the IL-7/IL-1α and IL-7/IL-1ß ratios had the best discriminatory potential, with cut-off values of 0.32 (AUC: 0.93, sensitivity: 94%, specificity: 80%, p = 0.0003) and 12.55 (AUC: 0.96, sensitivity: 94%, specificity: 90%, p = 0.0001), respectively. Our results suggest that IL-7, IL-1α, and IL-1ß tear levels could help differentiate IgG4-RD from SS. Key Points • The lacrimal gland is frequently involved in IgG4-RD and SS. This characteristic makes both diseases mimics of one another. • Patients with IgG4-RD and SS have different profiles of tear cytokines and chemokines. • Tear IL-7, IL-1α, and IL-1ß levels may serve as helpful biomarkers in separating IgG4-RD from SS.

IL-1α Processing, Signaling and Its Role in Cancer Progression.

Interleukin-1α (IL-1α) is a major alarmin cytokine which triggers and boosts the inflammatory responses. Since its discovery in the 1940s, the structure and bioactivity of IL-1α has been extensively studied and emerged as a vital regulator in inflammation and hematopoiesis. IL-1α is translated as a pro-form with minor bioactivity. The pro-IL-1α can be cleaved by several proteases to generate the N terminal and C terminal form of IL-1α. The C terminal form of IL-1α (mature form) has several folds higher bioactivity compared with its pro-form. IL-1α is a unique cytokine which could localize in the cytosol, membrane, nucleus, as well as being secreted out of the cell. However, the processing mechanism and physiological significance of these differentially localized IL-1α are still largely unknown. Accumulating evidence suggests IL-1α is involved in cancer pathogenesis. The role of IL-1α in cancer development is controversial as it exerts both pro- and anti-tumor roles in different cancer types. Here, we review the recent development in the processing and signaling of IL-1α and summarize the functions of IL-1α in cancer development.

A sensitive and selective approach for detection of IL 1α cancer biomarker using disposable ITO electrode modified with epoxy-substituted polythiophene polymer.

A highly sensitive and label-free electrochemical immunosensor for sensitive detection of interleukin 1α cancer biomarker was described by using epoxy-substituted polythiophene polymer modified disposable indium tin oxide electrode. This conjugated polymer was synthesized to fabricate this immunosensor for the first time and it offered a good biosensing platform for anti-IL 1α antibody immobilization due to epoxy groups present on the side groups of the polymer. Furthermore, the epoxy-substituted polythiophene polymer coated indium tin oxide electrode was used for the determination of IL 1α antigen for the first time. Electrode fabrication stages were characterized by using electrochemical (electrochemical impedance spectroscopy and cyclic voltammetry) and morphological (scanning electron microscopy and atomic force microscopy) techniques. Under optimum experimental conditions, impedimetric responses of the immunosensor increased with the increasing of IL 1α antigen concentration, and the proposed immunosensor displayed a wide linear detection range from 0.01 pg/mL to 5.5 pg/mL with a detection limit of 3.4 fg/mL. The proposed immunosensor exhibited outstanding performance including excellent reproducibility, good repeatability, high selectivity and long storage stability. With the advantages of simple operation, low-cost fabrication and label-free format, the suggested immunosensor was expected to have potential applications for IL 1α cancer biomarker detection.

Interleukin-1 alpha increases anti-tumor efficacy of cetuximab in head and neck squamous cell carcinoma.

BACKGROUND: Despite the high prevalence of epidermal growth factor receptor (EGFR) overexpression in head and neck squamous cell carcinomas (HNSCCs), incorporation of the EGFR inhibitor cetuximab into the clinical management of HNSCC has not led to significant changes in long-term survival outcomes. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that EGFR inhibition activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the clinical implications of activating this pathway are unclear in the context of cetuximab therapy. Given the role of IL-1 signaling in anti-tumor immune response, we hypothesized that increases in IL-1α levels would enhance tumor response to cetuximab. METHODS: Parental and stable myeloid differentiation primary response gene 88 (MyD88) and IL-1 receptor 1 (IL-1R1) knockdown HNSCC cell lines, an IL-1R antagonist (IL-1RA), neutralizing antibodies to IL-1α and IL-1ß, and recombinant IL-1α and IL-1ß were used to determine cytokine production (using ELISA) in response to cetuximab in vitro. IL-1 pathway modulation in mouse models was accomplished by administration of IL-1RA, stable overexpression of IL-1α in SQ20B cells, administration of rIL-1α, and administration of a polyanhydride nanoparticle formulation of IL-1α. CD4+ and CD8+ T cell-depleting antibodies were used to understand the contribution of T cell-dependent anti-tumor immune responses. Baseline serum levels of IL-1α were measured using ELISA from HNSCC patients treated with cetuximab-based therapy and analyzed for association with progression free survival (PFS). RESULTS: Cetuximab induced pro-inflammatory cytokine secretion from HNSCC cells in vitro which was mediated by an IL-1α/IL-1R1/MyD88-dependent signaling pathway. IL-1 signaling blockade did not affect the anti-tumor efficacy of cetuximab, while increased IL-1α expression using polyanhydride nanoparticles in combination with cetuximab safely and effectively induced a T cell-dependent anti-tumor immune response. Detectable baseline serum levels of IL-1α were associated with a favorable PFS in cetuximab-based therapy-treated HNSCC patients compared to HNSCC patients with undetectable levels. CONCLUSIONS: Altogether, these results suggest that IL-1α in combination with cetuximab can induce a T cell-dependent anti-tumor immune response and may represent a novel immunotherapeutic strategy for EGFR-positive HNSCCs.

IL-1RA VNTR and IL-1α 4845G>T polymorphisms and risk of idiopathic male infertility in Iranian men: A case-control study and an in silico analysis.

This study aimed to investigate the association of IL-1RA VNTR and IL-1α 4845G>T polymorphisms with idiopathic male infertility followed by an in silico analysis. In a case-control study, we collected blood samples from 230 infertile and 230 healthy men. Genotyping of IL-1RA VNTR was performed by PCR whereas IL-1α 4845G>T was genotyped by polymerase chain reaction-restriction fragment length polymorphism. An in silico approach was employed for the detection of IL-1RA VNTR and IL-1α 4845G>T effects on some molecular aspects of IL-1RA and IL-1α respectively. The result of our genetic association study for IL-1α 4845G>T revealed that there was a significant association between GT genotype, TT genotype, T allele and idiopathic male infertility. Although there was no significant association between IL-1RA VNTR and male infertility in the overall analysis. However, subgroup analysis revealed that the subjects with VNTR 4R/5R genotype were at a higher risk of oligozoospermia. Furthermore, 4845TT genotype, and 4845T allele were associated with oligozoospermia, asthenozoospermia and nonobstructive azoospermia. Bioinformatics analysis showed that IL-1RA VNTR may affect the splicing pattern of IL-1RA. Moreover, IL-1α 4845G>T has a significant effect on RNA structure and protein function. Based on our findings, both IL-1RA VNTR and IL-1α 4845G>T polymorphisms could be considered as potential biomarkers for screening of susceptible individuals.

Structural basis for IL-1α recognition by a modified DNA aptamer that specifically inhibits IL-1α signaling.

IL-1α is an essential cytokine that contributes to inflammatory responses and is implicated in various forms of pathogenesis and cancer. Here we report a naphthyl modified DNA aptamer that specifically binds IL-1α and inhibits its signaling pathway. By solving the crystal structure of the IL-1α/aptamer, we provide a high-resolution structure of this critical cytokine and we reveal its functional interaction interface with high-affinity ligands. The non-helical aptamer, which represents a highly compact nucleic acid structure, contains a wealth of new conformational features, including an unknown form of G-quadruplex. The IL-1α/aptamer interface is composed of unusual polar and hydrophobic elements, along with an elaborate hydrogen bonding network that is mediated by sodium ion. IL-1α uses the same interface to interact with both the aptamer and its cognate receptor IL-1RI, thereby suggesting a novel route to immunomodulatory therapeutics.The cytokine interleukin 1α (IL-1α) plays an important role in inflammatory processes. Here the authors use SELEX to generate a modified DNA aptamer which specifically binds IL-1α, present the structure of the IL-1α/aptamer complex and show that this aptamer inhibits the IL-1α signaling pathway.

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-1ß. 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.

Generation and characterization of ABT-981, a dual variable domain immunoglobulin (DVD-Ig(TM)) molecule that specifically and potently neutralizes both IL-1α and IL-1ß.

Interleukin-1 (IL-1) cytokines such as IL-1α, IL-1ß, and IL-1Ra contribute to immune regulation and inflammatory processes by exerting a wide range of cellular responses, including expression of cytokines and chemokines, matrix metalloproteinases, and nitric oxide synthetase. IL-1α and IL-1ß bind to IL-1R1 complexed to the IL-1 receptor accessory protein and induce similar physiological effects. Preclinical and clinical studies provide significant evidence for the role of IL-1 in the pathogenesis of osteoarthritis (OA), including cartilage degradation, bone sclerosis, and synovial proliferation. Here, we describe the generation and characterization of ABT-981, a dual variable domain immunoglobulin (DVD-Ig) of the IgG1/k subtype that specifically and potently neutralizes IL-1α and IL-1ß. In ABT-981, the IL-1ß variable domain resides in the outer domain of the DVD-Ig, whereas the IL-1α variable domain is located in the inner position. ABT-981 specifically binds to IL-1α and IL-1ß, and is physically capable of binding 2 human IL-1α and 2 human IL-1ß molecules simultaneously. Single-dose intravenous and subcutaneous pharmacokinetics studies indicate that ABT-981 has a half-life of 8.0 to 10.4 d in cynomolgus monkey and 10.0 to 20.3 d in rodents. ABT-981 exhibits suitable drug-like-properties including affinity, potency, specificity, half-life, and stability for evaluation in human clinical trials. ABT-981 offers an exciting new approach for the treatment of OA, potentially addressing both disease modification and symptom relief as a disease-modifying OA drug.

Cell membrane penetrating function of the nuclear localization sequence in human cytokine IL-1α.

Cytokines are released from the cell, bind to their receptors, and affect cellular responses. The precursor form of interleukin 1 alpha (pIL-1α) has a nuclear localization sequence (NLS) that causes it to be localized to the nucleus and regulate specific gene expression. The amino acids of the NLS are basic amino acid-rich sequences, as is the cell penetrating peptide (CPP), which has been widely studied as a way to deliver macromolecules into cells. Here, we hypothesized that the NLS in pIL-1α (pIL-1αNLS) can penetrate the cell membrane and it could deliver macromolecules such as protein in vivo. We characterized cell membrane penetration ability of pIL-1αNLS or its tandem repeated form (2pIL-1αNLS) to enhance its intracellular delivery efficiency. 2pIL-1αNLS showed comparable protein delivery efficiency to TAT-CPP and it mediates endocytosis following heparan sulfate interaction. 2pIL-1αNLS conjugated enhanced green fluorescence protein was localized to the nucleus and the cytoplasm. Intra-peritoneal administration of 2pIL-1αNLS conjugated dTomato protein showed remarkable in vivo intracellular delivery efficiency in various tissues including spleen, liver, and intestine in mice. Moreover, cytotoxicity of 2pIL-1αNLS was not observed even at 100 µM. Our results demonstrate cell membrane-penetrating function of NLS in pIL-1α, which can be used as a safe therapeutic macromolecular delivery peptide.

N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

Interleukin-1 gene cluster variants and recurrent pregnancy loss among North Indian women: retrospective study and meta-analysis.

The aim of this study was to determine whether or not interleukin-1 alpha (IL-1a), interleukin-1 beta (IL-1b) and IL-1 receptor antagonist (IL-1RA) polymorphisms are associated with risk of unexplained recurrent pregnancy loss (RPL) among North Indian women. This retrospective case­control study examine 200 well-characterized RPL cases for IL-1 gene cluster variants, determined by restriction fragment length polymorphism-PCR. The observed allele, genotype and haplotype distributions were compared with those obtained from 300 ethnically matched negative controls. Invariant distribution of IL-1 gene cluster single-nucleotide polymorphisms was observed among RPL cases and controls. Meta-analysis of IL-1b _511, +3953 and IL-1RN 86-bp variable number tandem repeat from the reported literature and this study did not reveal any significant association with the risk of RPL. In conclusion, no significant difference between RPL and control groups was observed at the allele, genotype or haplotype levels when tested for association using the dominant, recessive and additive models of inheritance for IL-1 gene cluster variants. As far as is known, this is the first report from India pertaining to IL-1 gene cluster variants' association with the risk of RPL from North India.

Comparative analysis of receptor binding by chicken and human interleukin-1ß.

Interleukin-1ß (IL-1ß) is an important cytokine in the immune system. Mammalian and avian IL-1ßs share only 31-35% sequence identity, and the function of avian IL-1ßs is less well understood by comparison. Although chicken and mammalian IL-1ßs have similar tertiary structures, these ILs differ significantly with respect to receptor activation. Analysis of the structures and sequences of IL-1ßs reveals that the major differences lie in loops. Modeling docking of chicken IL-1ß to its receptor reveals that these variable loops are critical for receptor binding. Molecular dynamics simulations of the IL-1ßs reveal significant changes in the dynamic range of motion upon receptor binding. Loops 3 and 9 of the unbound chicken IL-1ß had greater fluctuations compared with the other loops. Upon binding, the flexibility of these loops, which directly contact the receptor, markedly decreases. Taken together, these results suggest that receptor binding leads to not only favorable enthalpy but also lower conformational entropy.

1H, 13C and 15N backbone and side chain resonance assignments of human interleukin 1alpha.

As part of our NMR structure determination of the human Interleukin-1alpha, we report nearly complete NMR chemical shift assignments for the (1)H, (13)C and (15)N nuclei.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare.

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare.

Fragmentation of proteins in cartilage treated with interleukin-1: specific cleavage of type IX collagen by matrix metalloproteinase 13 releases the NC4 domain.

Degradation of bovine nasal cartilage induced by interleukin-1 (IL-1) was used to study catabolic events in the tissue over 16 days. Culture medium was fractionated by two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE). Identification of components by peptide mass fingerprinting revealed released fragments representing the NC4 domain of the type IX collagen alpha1 chain at days 12 and 16. A novel peptide antibody against a near N-terminal epitope of the NC4 domain confirmed the finding and indicated the presence of one of the fragments already at day 9. Mass spectrometric analysis of the two most abundant fragments revealed that the smallest one contained almost the entire NC4 domain cleaved between arginine 258 and isoleucine 259 in the sequence -ETCNELPAR258-COOH NH2-ITP-. A larger fragment contained the NC4 domain and the major part of the COL3 domain with a cleavage site between glycine 400 and threonine 401 in COL3 (-RGPPGPPGPPGPSG400-COOH NH2-TIG-). The presence of multiple collagen alpha1 (IX) N-terminal sequences demonstrates that the released molecules were cleaved at sites very close to the original N terminus either prior to or due to IL-1 treatment. Matrix metalloproteinase 13 (MMP-13) is active and cleaves fibromodulin in the time interval studied. Cartilage explants treated with MMP-13 were shown to release collagen alpha1 (IX) fragments with the same sizes and with the same cleavage sites as those obtained upon IL-1 treatment. These data describe cleavage by an MMP-13 activity toward non-collagenous and triple helix domains. These potentially important degradation events precede the major loss of type II collagen.

Molecular cloning, overexpression and characterization of human interleukin 1alpha.

Interleukin-1 alpha (IL-1alpha) regulates a wide range of important cellular processes. In this study for the first time, we report the cloning, expression, biophysical, and biological characterization of the human interleukin-1alpha. Human IL-1alpha has been expressed in Escherichia coli in high yields ( approximately 4mg per liter of the bacterial culture). The protein was purified to homogeneity ( approximately 98% purity) using affinity chromatography and size exclusion chromatography. Results of the steady-state fluorescence and 2D NMR experiments show that the recombinant IL-1alpha is in a folded conformation. Far-UV circular dichroism (CD) data suggest that IL-1alpha is an all beta-sheet protein with a beta-barrel architecture. Isothermal titration calorimetry (ITC) experiments show that the recombinant IL-1alpha binds strongly (K(d) approximately 5.6 x 10(-7) M) to S100A13, a calcium binding protein that chaperones the in vivo release of IL-1alpha into the extracellular compartment. Recombinant IL-1alpha was observed to exhibit strong cytostatic effect on human umbilical vascular endothelial cells. The findings of the present study not only pave way for an in-depth structural investigation of the molecular mechanism(s) underlying the non-classical release of IL-1alpha but also provide avenues for the rational design of potent inhibitors against IL-1alpha mediated pathogenesis.

Structural differences between the putative carbohydrate-recognition domains of human IL-1 alpha, IL-1 beta and IL-1 receptor antagonist obtained by in silico modeling.

In a previous report (Cebo et al. J Biol Chem 276 (2001) 5685-5691), it was established that biologically active recombinant human IL-1alpha and IL-1beta had different carbohydrate-binding properties. IL-1alpha recognized a di-antennary N-glycan with two alpha2-3-linked sialic acid residues, whereas IL-1beta recognized the GM(4), a alpha2-3-linked sialylated glycosphingolipid. These different carbohydrate-binding properties of two interleukins binding to the same receptor (IL-1R) could explain why these molecules had different biological effects and cell specificities. Molecular modeling of the ligands and in silico docking experiments defined putative carbohydrate-recognition domains localized in the same area of the two molecules, a domain different from that defined as the type I IL-1R binding domain. The calculated pattern of hydrogen bonding and of van der Waals interactions fulfilled the essential features observed for calcium-independent lectins (mammalian, viral or bacterial). The analysis of the same domain of the third members of this family of molecules, the IL-1R-antagonist, indicated it did not fulfill the criteria for carbohydrate-recognition domains. It is proposed that its role as a pure antagonist is due to the absence of lectin activity and consequently explained its inability to associate IL-1R with other surface molecular complexes necessary for signaling.

[Stress-protective effect of the synthetic ACTH-like peptide leucocorticotropin].

We found that the tritium-labeled synthetic ACTH-like octapeptide leucocorticotropin corresponding to the 81-88 sequence of the precursor of human interleukin-1alpha ([3H]GKVLKKRR) is bound by the ACTH receptor of rat adrenal cortex with a high affinity and specificity (Kd 2.2 +/- 0.1 nM). This peptide was shown to exert no effect on the adenylate cyclase activity of the membranes of rat adrenal cortex in the concentration range from 1 to 1000 nM. Leucocorticotropin administration three times at doses of 10-20 microg/animal did not change the level of hydroxycorticosteroids (11-HOCS) in the rat adrenal glands in the absence of temperature action. At the same time, the peptide abolishes (at a dose of 20 microg/animal, three times) or significantly decreases (at a dose of 10 microg/animal, three times) the dramatic increase in the 11-HOCS content in the adrenal glands occurring in the case of cold or heat shock. Thus, leucocorticotropin normalizes the 11-HOCS level in the rat adrenal cortex during stress. The stress-protective effect of the peptide is mediated through the ACTH receptor.