Receptor expression is essential for proliferation induced by dimerized Jak kinases

Two members of Jak kinases, Jak1 and Jak3, are associated with the cytoplasmic domains of the interleukin-2 (IL-2) receptor (IL-2R) β chain (IL-2Rβ) and the common cytokine receptor γ chain (γc), respectively, and accumulating evidence indicates their functional importance in IL-2 signaling. Here, I showed that coumermycin-induced chemical heterodimerization of Jak1 and Jak3 but not homodimerization of Jak1 or Jak3 induces cell proliferation of an IL-2R-reconstituted cell line. In this regard, expression of IL-2Rβ was essential for cell proliferation by chemical heterodimerization of Jak1 and Jak3, indicating that dimerized Jak1 and Jak3 induce heterodimerization of IL-2Rβ and γc, which may activate receptor-bound signaling molecules. Previous reports using chemical dimerization suggest that dimerization of Jak kinases is sufficient to induce cell proliferation. The present study indicates that re-evaluation of this conclusion is necessary and that interpretation of functional analysis of signaling molecules using chemical dimerizers needs more careful assessment.     

The Soluble Interleukin-6 Receptor Is a Mediator of Hematopoietic and Skeletal Actions of Parathyroid Hormone

Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6^+/+ and IL-6^−/− mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6^−/− compared with IL-6^+/+ bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6^−/− earlier than IL-6^+/+ marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6^−/− marrow. In the skeletal system, although intermittent PTH administration to IL-6^+/+ and IL-6^−/− mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-β1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.     

Soluble interleukin-18 receptor complex is a novel biomarker in rheumatoid arthritis

Introduction

There has been no report in the literature of a soluble form of interleukin (IL)-18 receptor α (IL-18Rα). In this study, we evaluated the levels and characteristics of soluble IL-18Rα (sIL-18Rα) in the sera of patients with rheumatoid arthritis (RA) and compared these results to control populations.

Methods

The sIL-18Rα complex was isolated from pooled human blood serum using an anti-IL-18Rα monoclonal antibody affinity column. The purified sIL-18Rα was then examined using Western blot analysis and used in experiments to evaluate the effects on an IL-18-responsive natural killer (NK) human cell line, NK0. An enzyme-linked immunosorbent assay was developed, and sera from 145 patients with RA, 6 patients with adult-onset Still's disease, 31 patients with osteoarthritis (OA), 39 patients with systemic lupus erythematosus (SLE) and 67 controls were tested, along with levels of immunoglobulin M, rheumatoid factor, anticyclic citrullinated peptide antibody, IL-18, IL-13 and interferon (IFN)-γ. Area under the receiver operating characteristic curve (ROC-AUC) analysis was used to evaluate the diagnostic utility of the sIL-18Rα complex.

Results

The isolated sIL-18Rα complex can be associated with IL-18 and the soluble form of the IL-18Rβ chain. The sIL-18Rα complex bound to the surface to the NK0 cell line, antagonized the stimulatory effects of IL-18 and IL-2 on the NK0 cell line and inhibited IFN-γ production by the cells. The serum levels of sIL-18Rα complex in RA (186.0 ± 33.5 ng/mL, n = 145) and adult-onset Still's disease (98.2 ± 8.9 ng/mL, n = 6) were significantly (P < 0.001) higher than those in the healthy controls (52.3 ± 8.5 ng/mL, n = 67), OA (38.6 ± 5.4 ng/mL, n = 31), SLE (44.6 ± 3.2 ng/mL, n = 39). The serum level of sIL-18Rα complex was not significantly different between RA and adult-onset Still's disease patients. The serum levels of IL-18, IL-13 and IFN-γ in the RA patients were significantly (P < 0.01) higher than in OA and SLE patients as well as healthy controls. ROC-AUC analysis of the serum concentration of sIL-18Rα indicated that it was significantly diagnostic of RA. Moreover, a tumor necrosis factor inhibitor, etanercept, significantly (P < 0.0001) decreased levels of sIL-18Rα in the sera of 29 RA patients 6 months after treatment.

Conclusions

The sIL-18Rα complex could be a potentially useful biomarker for the diagnosis of RA.     

Crystal structure of a soluble decoy receptor IL-22BP bound to interleukin-22

Interleukin-22 (IL-22) plays an important role in the regulation of immune and inflammatory responses in mammals. The IL-22 binding protein (IL-22BP), a soluble receptor that specifically binds IL-22, prevents the IL-22/interleukin-22 receptor 1 (IL-22R1)/interleukin-10 receptor 2 (IL-10R2) complex assembly and blocks IL-22 biological activity. Here we present the crystal structure of the IL-22/IL-22BP complex at 2.75 Å resolution. The structure reveals IL-22BP residues critical for IL-22 binding, which were confirmed by site-directed mutagenesis and functional studies. Comparison of IL-22/IL-22BP and IL-22/IL-22R1 crystal structures shows that both receptors display an overlapping IL-22 binding surface, which is consistent with the inhibitory role played by IL-22 binding protein.

Structured summary

MINT-7010533: IL-22 BP (uniprotkb:Q969J5) and IL-22 (uniprotkb:Q9GZX6) bind (MI:0407) by X-ray crystallography (MI:0114)     

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.     

人IL-6 /sIL-6R 结合分子模型的建立及在药物筛选中的应用

目的：建立人IL-6 /sIL-6R 结合的分子模型,用于筛选IL-6 /sIL-6R的抑制剂。方法：将人IL-6基因克隆至原核表达载体pET28a（+）中表达IL-6蛋白,western blot及人IL-6检测试剂盒分析鉴定表达蛋白。同法将人sIL-6R在pET15b载体中表达,纯化并用western blot检测目的蛋白。依据ELISA原理建立IL-6 /sIL-6R 结合的分子模型,并通过改变IL-6、sIL-6R及IL-6 antibody的浓度来优化该模型,用于IL-6 /sIL-6R拮抗药物的筛选。结果：人IL-6可在载体PET28a（+）中高效表达,且经western blot鉴定正确,人IL-6检测试剂盒检测显示具有较高的免疫活性。sIL-6R在PET15b中表达,western blot鉴定正确。通过对IL-6 /sIL-6R结合的分子模型的优化,得到其最佳条件为：IL-6R 1?g/well, IL-6 500ng/well, IL-6 antibody 1?g/well。应用该模型筛选发现有些化合物可显著抑制IL-6与其受体的结合。结论：成功构建IL-6 /sIL-6R结合的分子模型,为高通量筛选IL-6拮抗剂提供平台。     

Different dynamics of IL-15R activation following IL-15 cis- or trans-presentation

Interleukin (IL)-15 is a cytokine critical for the homeostasis and the function of NK cells, NK-T cells, and memory CD8+ T cells. IL-15 signals are delivered through the IL-15Rβ and the common γ (γ(c)) receptor chains. The third receptor chain, IL-15Rα, confers specificity and high affinity for the cytokine. While IL-15 can activate with high affinity the trimeric receptor expressed by a target cell (cis-presentation), IL-15Rα is also known to trans-present IL-15 with high affinity to target cells expressing the IL-15Rβ/γ(c) complex. In order to compare the IL-15 cis- and trans-presentation processes, and using a T cell line expressing both IL-15Rα/β/γ(c) and IL-15Rβ/γ(c), we analyzed cell surface receptor chain down-modulation, cytokine internalization and signaling responses induced either with IL-15 (cis-presentation) or with RLI, a protein resulting from fusion between IL-15 and an extended IL-15Rα sushi domain, that mimics trans-presentation. Whereas IL-15 bound with high affinity to IL-15Rα/β/γ(c), RLI bound with a similar high affinity to IL-15Rβ/γ(c). The kinetics of cell surface IL-15R down-modulation were slower following RLI treatment than after IL-15 treatment, as were the kinetics of RLI internalization, which was slower than that of IL-15. IL-15 and RLI dose-dependently induced the activation of similar signaling pathways. However, the kinetics and duration of these activations were markedly different, RLI-induced signaling, being slower, but more prolonged than that induced by IL-15, although the final proliferative responses at 48?h were similar. These findings collectively indicate that IL-15 cis- and trans-presentation mechanisms lead to different dynamics of receptor activation and signal transduction, with cis-presentation inducing fast and transient responses, and trans-presentation inducing slower, more persistent ones. They provide clues for a better understanding of how IL-15 action is controlled, and how it plays a key role in the coordination between innate and adaptative immunity.     

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.     

Intracellular free zinc up-regulates IFN-γ and T-bet essential for Th1 differentiation in Con-A stimulated HUT-78 cells

Zinc deficiency impairs cellular immunity. Up-regulation of mRNA levels of IFN-γ, IL-12Rβ2, and T-bet are essential for Th₁ differentiation. We hypothesized that zinc increases Th₁ differentiation via up-regulation of IFN-γ and T-bet expression. To test this hypothesis, we used zinc-deficient and zinc-sufficient HUT-78 cells (a Th₀ cell line) under different condition of stimulation in this study. We also used TPEN, a zinc-specific chelator, to decrease the bioavailability of zinc in the cells. We measured intracellular free zinc, cytokines, and the mRNAs of T-bet, IFN-γ, and IL-12Rβ2. In this study, we show that in zinc-sufficient HUT-78 cells, mRNA levels of IFN-γ, IL-12Rβ2, and T-bet in PMA/PHA-stimulated cells were increased in comparison to zinc-deficient cells. Although intracellular free zinc was increased slightly in PMA/PHA-stimulated cells, Con-A-stimulated cells in 5 μM zinc medium showed a greater sustained increase in intracellular free zinc in comparison to cells incubated in 1 μM zinc. The cells pre-incubated with TPEN showed decreased mRNA levels of IFN-γ and T-bet mRNAs in comparison to cells without TPEN incubation. We conclude that stimulation of cells by Con-A via TCR, release intracellular free zinc which functions as a signal molecule for generation of IFN-γ and T-bet, and IL-12Rβ2 mRNAs required for Th₁ cell differentiation. These results suggest that zinc increase Th₁ cell differentiation by up-regulation of IFN-γ and T-bet, and IL-12Rbβ2 mRNAs.     

The response of interleukin-6 and soluble interleukin-6 receptor isoforms following intermittent high intensity and continuous moderate intensity cycling

As interleukin-6 (IL-6), its soluble receptor (sIL-6R), and the IL-6/sIL-6R complex is transiently elevated in response to prolonged moderate-intensity exercise, this study investigated how these levels would be modulated by an acute bout of high-intensity intermittent (HIIT) exercise in comparison to continuous moderate-intensity exercise (MOD). This study also investigated the expression of the differentially spliced sIL-6R (DS-sIL-6R) in response to exercise. Eleven healthy males completed two exercise trials matched for external work done (582 ± 82 kJ). During MOD, participants cycled at 61.8 (2.6)% VO_2peak for 58.7 (1.9) min, while HIIT consisted of ten 4-min intervals cycling at 87.5 (3.4)% [(V)\dot]O_2peak \dot{V}{{\hbox{O}}_{2{\rm{peak}}}} separated by 2-min rest. Blood samples were collected pre-exercise, post-exercise, and 1.5, 6, and 23 h post-exercise. Plasma IL-6, sIL-6R, IL-6/sIL-6R complex, and DS-sIL-6R levels were measured by enzyme-linked immunosorbent assay. HIIT caused a significantly greater increase in IL-6 than MOD (P = 0.018). Both MOD and HIIT resulted in an increase in sIL-6R and IL-6/sIL-6R complex (P < 0.001), however, this was not significantly different between trials. Soluble IL-6R peaked at 6 h post-exercise in both trials. DS-sIL-6R increased significantly with exercise (P = 0.02), representing 0.49% of the total sIL-6R increase. This investigation has demonstrated that the IL-6 response is greater after intermittent high-intensity exercise than comparable moderate-intensity exercise; however, increased IL-6/sIL-6R complex nor sIL-6R was different between HIIT and MOD. The current study has shown for the first time that elevated sIL-6R after HIIT exercise is derived from both proteolytic cleavage and differential splicing.     

IL-15 can signal via IL-15Rα, JNK, and NF-κB to drive RANTES production by myeloid cells

IL-15 plays many important roles within the immune system. IL-15 signals in lymphocytes via trans presentation, where accessory cells such as macrophages and dendritic cells present IL-15 bound to IL-15Rα in trans to NK cells and CD8(+) memory T cells expressing IL-15/IL-2Rβ and common γ chain (γ(c)). Previously, we showed that the prophylactic delivery of IL-15 to Rag2(-/-)γ(c)(-/-) mice (mature T, B, and NK cell negative) afforded protection against a lethal HSV-2 challenge and metastasis of B16/F10 melanoma cells. In this study, we demonstrated that in vivo delivery of an adenoviral construct optimized for the secretion of human IL-15 to Rag2(-/-)γ(c)(-/-) mice resulted in significant increases in spleen size and cell number, leading us to hypothesize that IL-15 signals differently in myeloid immune cells compared with lymphocytes, for which IL-15/IL-2Rβ and γ(c) expression are essential. Furthermore, treatment with IL-15 induced RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells, but the presence of γ(c) did not increase bone marrow cell sensitivity to IL-15. This IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells occurred independently of the IL-15/IL-2Rβ and Jak/STAT pathways and instead required IL-15Rα signaling as well as activation of JNK and NF-κB. Importantly, we also showed that the trans presentation of IL-15 by IL-15Rα boosts IL-15-mediated IFN-γ production by NK cells but reduces IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) myeloid bone marrow cells. Our data clearly show that IL-15 signaling in NK cells is different from that of myeloid immune cells. Additional insights into IL-15 biology may lead to novel therapies aimed at bolstering targeted immune responses against cancer and infectious disease.     

Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor β1 (IL-12Rβ1) and the cytokine-specific receptors IL-12Rβ2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rβ1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rβ2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rβ1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rβ2 are known, and two regions of p40 critical for binding to IL-12Rβ1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rβ1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23-dependent signaling and did not bind to IL-12Rβ1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rβ1, but not binding to IL-23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12Rβ1, indicating binding of homodimeric p40 to IL-12Rβ1 is comparable to the interaction of IL-23/IL-12 and IL-12Rβ1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rβ1. Structural insights into cytokine–cytokine receptor binding are important to develop novel therapeutic strategies.     

Soluble cytokine receptors (sIL-2Rα, sIL-2Rβ) induce subunit-specific behavioral responses and accumulate in the cerebral cortex and basal forebrain

Soluble cytokine receptors are normal constituents of body fluids that regulate peripheral cytokine and lymphoid activity. Levels of soluble IL-2 receptors (sIL-2R) are elevated in psychiatric disorders linked with autoimmune processes, including ones in which repetitive stereotypic behaviors and motor disturbances are present. However, there is no evidence that sIL-2Rs (or any peripheral soluble receptor) induce such behavioral changes, or that they localize in relevant brain regions. Here, we determined in male Balb/c mice the effects of single peripheral injections of sIL-2Rα or sIL-2Rβ (0-2 μg/male Balb/c mouse; s.c.) on novelty-induced ambulatory activity and stereotypic motor behaviors. We discovered that sIL-2Rα increased the incidence of in-place stereotypic motor behaviors, including head up head bobbing, rearing/sniffing, turning, and grooming behavior. A wider spectrum of behavioral changes was evident in sIL-2Rβ-treated mice, including increases in vertical and horizontal ambulatory activity and stereotypic motor movements. To our knowledge, this is the first demonstration that soluble receptors induce such behavioral disturbances. In contrast, soluble IL-1 Type-1 receptors (0-4 μg, s.c.) didn't appreciably affect these behaviors. We further demonstrated that sIL-2Rα and sIL-2Rβ induced marked increases in c-Fos in caudate-putamen, nucleus accumbens and prefrontal cortex. Anatomical specificity was supported by the presence of increased activity in lateral caudate in sIL-2Rα treated mice, while sIL-2Rβ treated mice induced greater c-Fos activity in prepyriform cortex. Moreover, injected sIL-2Rs were widely distributed in regions that showed increased c-Fos expression. Thus, sIL-2Rα and sIL-2Rβ induce marked subunit- and soluble cytokine receptor-specific behavioral disturbances, which included increases in the expression of ambulatory activity and stereotypic motor behaviors, while inducing increased neuronal activity localized to cortex and striatum. These findings suggest that sIL-2Rs act as novel immune-to- brain messengers and raise the possibility that they contribute to the disease process in psychiatric disorders in which marked increases in these receptors have been reported.     

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling.     

Pharmacologic IL-6Rα inhibition in cholangiocarcinoma promotes cancer cell growth and survival

Biliary tract cancer (BTC) represents a malignant tumor of the biliary tract including cholangiocarcinoma (CCA) and the carcinoma of the gallbladder (GBC) with a 5-year survival rate between 5 and 18% due to late diagnosis and rapid disease progression. Chronic inflammation is one of the main risk factors for CCA and GBC in particular. IL-6, as a mediator of inflammation, can act through a membrane-bound receptor alpha-chain (mIL-6R, “IL-6 classic signaling”) or via soluble forms (sIL-6R, “IL-6 trans-signaling”). However, little is known about the impact on cellular responses of IL-6 trans-signaling on BTC. We analyzed primary tumors as whole sections and as tissue microarrays, and also searched The Cancer Genome Atlas database. Compared to non-neoplastic, non-inflamed gallbladder tissue, IL-6Rα was downregulated in GBC, and this correlated with the patients' overall survival. Furthermore, different CCA cell lines and compounds for activation (IL-6 and Hyper-IL-6) or inhibition (Tocilizumab and sgp130Fc) of IL-6 classic signaling and trans-signaling were used to determine their effects on cellular processes between the two modes of IL-6 signaling. Inhibition of IL-6 trans-signaling by sgp130Fc reduced CCA cell line viability and apoptosis, whereas migration and proliferation were increased. We conclude that IL-6Rα expression is a good prognostic marker for GBC, and that the blocking of IL-6 trans-signaling and activation of IL-6 classic signaling have tumor promoting activity. These findings warrant the exclusion of patients with GBC or other malignancies associated with bile metabolism from IL-6R inhibitor therapy.     

A purified protein from Salmonella typhimurium inhibits high-affinity interleukin-2 receptor expression on CTLL-2 cells

Previously, we have demonstrated that the immunosuppression induced by the purified substance Salmonella typhimurium-derived inhibitor of T-cell proliferation (STI) involves T-cell non-responsiveness to interleukin-2 (IL-2). In the present study, it was found that STI inhibited the growth of CTLL-2 cells, which are an IL-2-dependent cytotoxic T-cell line. Analysis of IL-2 receptor (IL-2R) function showed that STI inhibited high-affinity receptor expression and internalization by CTLL-2 cells. Furthermore, FACS analysis demonstrated that STI inhibited both β chain and γ chain expression of IL-2R on the cells. These results suggest that the suppression of T-cell proliferation induced by STI results from a defect in IL-2R function.     

Recombinant human heterodimeric IL-15 complex displays extensive and reproducible <Emphasis Type="Italic">N</Emphasis>- and <Emphasis Type="Italic">O</Emphasis>-linked glycosylation

Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and β-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.     

Increased levels of soluble interleukin-6 receptor and CCL3 in COPD sputum

Background

COPD patients have increased numbers of macrophages and neutrophils in the lungs. Interleukin-6 (IL-6) trans-signaling via its soluble receptor sIL-6R, governs the influx of innate immune cells to inflammatory foci through regulation of the chemokine CCL3. We hypothesized that there would be enhanced levels of IL-6, sIL-6R and CCL3 in COPD sputum.

Methods

59 COPD patients, 15 HNS and 15 S underwent sputum induction and processing with phosphate buffered saline to obtain supernatants for IL-6, sIL-6R and CCL3 analysis. Cytoslides were produced for differential cell counting and immunocytochemistry (COPD; n = 3) to determine cell type surface expression of the CCL3 receptors CCR5 and CCR1.

Results

COPD patients expressed higher levels (p < 0.05) of sIL-6R and CCL3 compared to controls (sIL-6R medians pg/ml: COPD 166.4 vs S 101.1 vs HNS 96.4; CCL3 medians pg/ml: COPD 117.9 vs S 0 vs HNS 2.7). COPD sIL-6R levels were significantly correlated with sputum neutrophil (r = 0.5, p < 0.0001) and macrophage (r = 0.3, p = 0.01) counts. Immunocytochemical analysis revealed that CCR5 and CCR1 were exclusively expressed on airway macrophages.

Conclusion

Enhanced airway generation of sIL-6R may promote IL-6 trans-signaling in COPD. Associated upregulation of CCL3 may facilitate the recruitment of macrophages into the airways by ligation of CCR1 and CCR5.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0103-4) contains supplementary material, which is available to authorized users.