Cutting edge: The ST2 ligand IL-33 potently activates and drives maturation of human mast cells

IL-33, the natural ligand of the IL-1 receptor family member ST2L, is known to enhance experimental allergic-type inflammatory responses by costimulating the production of cytokines from activated Th2 lymphocytes. Although ST2L has long been known to be expressed by mast cells, its role in their biology has not been explored. In this study we report that IL-33 directly stimulates primary human mast cells (MCs) to produce several proinflammatory cytokines and chemokines and also exerts a permissive effect on the MCs response to thymic stromal lymphopoietin, a recently described potent MCs activator. IL-33 also acts both alone and in concert with thymic stromal lymphopoietin to accelerate the in vitro maturation of CD34(+) MC precursors and induce the secretion of Th2 cytokines and Th2-attracting chemokines. Taken together, these results suggest that IL-33 may play an important role in mast cell-mediated inflammation and further emphasize the role of innate immunity in allergic diseases.     

Ets-1 activates the DRA promoter in B cells.

The X box in promoters of class II major histocompatibility complex genes plays a crucial role in the B-cell-specific and gamma interferon-inducible expression of these genes. The sequence TTCC is located in the pyrimidine tract which extends 5' to and partially overlaps the X box of the DRA promoter. This sequence resembles the core binding site for the Ets family of DNA-binding proteins. In this study, we demonstrate that mutations within the pyrimidine tract which change the TTCC motif, but do not affect the binding of regulatory factor X to the X box, decrease the activity of the DRA promoter in B cells. Furthermore, using electrophoretic mobility shift assays and cotransfection experiments, we demonstrate that Ets-1, but not Ets-2 or PU.1, functionally interacts with the pyrimidine tract and activates the DRA promoter.     

Alloantigen presentation by B cells. Requirement for IL-1 and IL-6

We have evaluated the requirements for non-activated allogeneic B cells to present alloantigen in primary MLC. We found that the generation of an MLR was dependent on the addition of exogenous IL-1 and IL-6 to the cultures; neither lymphokine by itself was capable of significantly stimulating the response. Our results suggest that the concentration of these lymphokines present in a standard MLR culture may serve to regulate the magnitude of the proliferative response. The B cells that co-stimulated with IL-1 and IL-6 were found to be contained almost exclusively within the large, low density population. Presumably, large low density B cells have attained a state in which they are particularly effective at presentation of alloantigen. Together our findings indicate that three components are required for Ag presentation in primary MLC: class II MHC-positive stimulator cells that have attained the appropriate state of differentiation and the presence of both IL-1 and IL-6.     

Loureirin B activates GLP-1R and promotes insulin secretion in Ins-1 cells

Loureirin B (LB) is a natural product derived from Sanguis draconis, which has hypoglycaemic effects. In order to research the possible target of LB in the treatment of diabetes, molecular docking was used to simulate the interaction between LB and potential targets, and among them, glucagon-like peptide-1 receptor (GLP-1R) had the optimal results. Further, spectroscopy and surface plasmon resonance (SPR) experiments were applied to detect the interaction between LB and GLP-1R. Ultimately, after GLP-1R siRNA interfering the expression of GLP-1R in Ins-1 cell, the promoting insulin secretion of LB was weaken, which directly proved that GLP-1R plays an important role. These results show that LB promotes insulin secretion of Ins-1 cells through GLP-1R. Hence, the strategy of LB as a prodrug will provide a potential approach for non-peptide GLP-1R agonist.     

Type I IL-1 receptor blockade exacerbates murine listeriosis.

It was found that IL-1 is produced in livers and spleens of mice shortly after the i.v. injection of a sublethal or lethal Listeria monocytogenes inoculum. In sublethally infected mice, IL-1 was present in infected livers and spleens by the end of the first day of infection. Thereafter, the amounts of IL-1 in these organs increased and decreased in concordance with bacterial numbers. IL-1 was not present in the peripheral circulation of mice during sublethal listeriosis, but was present in the blood late in lethal infection. Evidence showing that IL-1 plays a role in antibacterial resistance early in listeriosis was obtained through the use of 35F5 mAb that binds to the murine type I IL-1R and functions to block IL-1 alpha and IL-1 beta actions. Blockade of the type I IL-1R by the 35F5 mAb results in greatly enhanced bacterial growth in the livers and spleens of mice that had received a sublethal Listeria inoculum. Consistent with the exacerbation of listeriosis caused by 35F5 mAb, but in contrast to the effect of 35F5 mAb in other murine models, 35F5 mAb-treated mice exhibit markedly elevated levels of IL-6 in their circulation and infected organs.     

Differential binding of IL-1 alpha and IL-1 beta to receptors on B and T cells

The interleukin 1 receptors (IL-1R) on the human B lymphoma RAJI and on the murine thymoma EL4-6.1 have been characterized. Equilibrium binding analysis using both 125I-labeled IL-1 alpha and IL-1 beta showed that RAJI cells have a higher number of binding sites/cell for IL-1 beta (2400, Kd 2.2 nM) than for IL-1 alpha (316, Kd 0.13 nM). On the other hand, EL4-6.1 cells have more receptors/cell for IL-1 alpha (22 656, Kd 1 nM) than for IL-1 beta (2988, Kd 0.36 nM). Dexamethasone (DXM) induced on RAJI cells a time-dependent increase in binding sites for both IL-1 beta and IL-1 alpha without affecting their binding affinities. However, while receptor-bound 125I-IL-1 alpha was displaced with equal efficiency by both IL-1 forms, only unlabeled IL-1 beta could effectively displace 125I-IL-1 beta. Cross-linking experiments indicated that RAJI cells have a predominant IL-1R of about 68 kDa, while EL4-6.1 cells have an IL-1-binding polypeptide of 80 kDa. These results suggest that B and T cells possess structurally different IL-1R with distinct binding properties for IL-1 alpha and IL-1 beta.     

IL-1 activates two separate signal transduction pathways in T helper type II cells

We have investigated the signal transduction pathways mediated by IL-1 in the Th 2 cell line D10.A, and we have made the following findings. Interaction of IL-1 with its receptor leads to the translocation of protein kinase C (PKC) from the cytosol to the membrane, phosphorylation of the 80-kDa protein that is substrate for PKC, as well as an increase in the level of cAMP. In addition, IL-1 induced IL-5 mRNA expression in these cells. We have established that the IL-5 gene is activated in D10.A cells in response to either phorbol esters or 8-Br cAMP, and that the two agents act as cofactors. IL-1 is able to synergize with phorbol esters and is additive with 8-Br cAMP for IL-5 mRNA expression. There are two possibilities to explain these results: 1) D10.A cells express two types of functional IL-1R, each linked to an independent signal transduction pathway; or 2) these cells have only one kind of IL-1R which, upon ligand interaction, mediates the activation of both the PKC and the adenylate cyclase pathway.     

Identification of initiator B cells, a novel subset of activation-induced deaminase-dependent B-1-like cells that mediate initiation of contact sensitivity

Contact sensitivity (CS) is related to delayed-type hypersensitivity and is a well-characterized prototype of T cell-mediated inflammation. However, the inflammatory response associated with CS is additionally dependent on Ag-specific IgM produced by a subpopulation of B cells in response to sensitization. Upon re-exposure to hapten, this IgM mediates rapid vascular activation and subsequent recruitment of proinflammatory T cells to the local site. Interference with this pathway prevents the full development of the classic delayed inflammatory response and is therefore termed the "CS initiation" pathway. In this study, we show that CS initiation is defective in mice deficient in activation-induced deaminase, an enzyme central to the process of somatic hypermutation. Using adoptive transfer experiments, we demonstrate that the defect is specific to a B-1-like population of B cells and that transfer of WT cells reconstitutes CS initiation mechanisms in deficient recipients. We went on to identify a novel subpopulation of Ag-binding B cells in the spleens of sensitized mice that possess initiation activity (CD19(+)CD5(+)Thy-1(int)IgM(high)IgD(high)) that we name "initiator B cells." Analysis of BCR H chain genes isolated from these cells revealed evidence of activation-induced deaminase-mediated somatic hypermutation. The sensitivity of CS initiation to very low amounts of sensitizing hapten suggests that the responsible B cells have increased IgM receptor gene mutations enabling selection to generate Abs with sufficient affinity to mediate the response.     

Up-regulation of IL-33 expression in various types of murine cells by IL-3 and IL-4

The crucial roles of the novel cytokine IL-33 in allergic, inflammatory, infectious and autoimmune diseases are becoming characterized. However, the cytokines which regulate IL-33 expression and secretion are still largely unknown. In this study, IL-3 and IL-4 were found to up-regulate IL-33 mRNA expression in mouse peritoneal exudate cells by a two-color DNA microarray and further confirmed by real time PCR and ELISA. IL-3 and IL-4 synergistically promote IL-33 mRNA expression and IL-33 intracrine in the heterogeneous cell populations as peritoneal exudates cells, bone marrow cells and splenic cells. IL-3 and IL-4 also induced IL-33 introcrine in the peritoneal exudate cells from the macrophage-deficient op/op mice, suggesting that macrophage is not the only target of IL-3 and IL-4 in the heterogeneous peritoneal exudate cells. Furthermore, IL-3 and IL-4 were verified to promote the IL-33 intracrine in the homogeneous cell population as fibroblasts and mast cells. These results indicate that up-regulation of IL-33 expression by IL-3 and IL-4 is not a feature particular to a specific type of cells. Up to 100 cytokines were screened, but none of them stimulated the secretion or release of IL-33 in the culture system. In summary, we confirm for the first time that IL-3 and IL-4 are critical for IL-33 intracrine in murine cells of various types, indicating that IL-3 and IL-4 may play an important role in the constitutive expression of IL-33 in vivo.     

Expression of IL-1 receptors on human peripheral B cells

The expression of IL-1R on human peripheral B cells was analyzed by the binding assay with 125I-labeled human rIL-1 alpha and by the flow cytofluorometry by the use of FITC-conjugated IL-1 alpha. The proliferation and the differentiation of B cells stimulated with Staphylococcus aureus Cowan I (SAC) in the presence of T cell-derived factors were dependent on IL-1. By the binding experiment with 125I-labeled IL-1 alpha, B cells expressed only few IL-1R without any stimulations. When they were stimulated with SAC, IL-1R on B cells began to increase by only 1 h, reached the maximum level at 6 h. The binding of 125I-labeled IL-1 alpha to B cells was inhibited by the addition of either cold IL-1 alpha or IL-1 beta suggesting that IL-1R on B cells reactive for IL-1 alpha and IL-1 beta were identical. By Scatchard plot analysis, the existence of two classes of IL-1R on B cells was found. A major class of IL-1R (320 molecules/cell) has a lower affinity (Kd = 3.8 x 10(-10) M) and a minor class of IL-1R (70 molecules/cell) has a higher affinity (Kd = 4.4 x 10(-12) M). When B cells were stimulated with SAC, both lower and higher affinity IL-1R were increased to 1960 molecules/cell and 300 molecules/cell, respectively. Furthermore, IL-1R on B cells were also detected with FITC-conjugated IL-1 alpha by a flow cytofluorometer. Only 3 to 5% of B cells expressed IL-1R without any stimulations. When B cells were stimulated with SAC, IL-1R-positive B cells were increased to 20%. The addition of anti-class II antibodies inhibited B cell proliferation and differentiation induced with SAC, IL-1, and T cell-derived factors. Anti-class II antibodies also inhibited the number of IL-1R on B cells. These results suggest that the expression of IL-1R was induced as the initial stage of B cell activation and that class II Ag play an important role for the expression of IL-1R on B cells.     

A complicated liaison: IL-33 and IL-33R in arthritis pathogenesis

Interruption of cytokine signaling, by targeting either the cytokine itself or its cellular receptor, is a mainstay in the therapy for patients with rheumatic diseases. Interleukin (IL)-33, a member of the IL-1 cytokine family, has emerged as an important mediator of inflammatory responses. In a side-by-side examination of IL-33-deficient and IL-33 receptor (IL-33R)-deficient mice in the K/BxN serum transfer model, arthritis was ameliorated in the IL-33R knockout (KO) mice but not in the IL-33 KO mice. These findings complement previous knowledge on IL-33R signaling, demonstrating that the IL-33R cross-activates other signaling pathways in addition to IL-33-mediated signals. The results reported by Martin and colleagues in a previous issue of Arthritis Research & Therapy underline the clinical relevance of IL-33R cross-signaling and further illustrate that targeting a cytokine receptor (IL-33R) can have completely different clinical outcomes than targeting the respective cytokine.     

Murine B1 B cells require IL-5 for optimal T cell-dependent activation

T helper cell-driven activation of murine B cells has been shown to depend upon CD40-CD40 ligand (CD40L) interactions and a defined set of cytokines. These observations are primarily based on the use of conventional B cells obtained from the spleen. Therefore, it is presently unclear whether all mature B cell subsets found in the mouse have an equal dependence upon CD40-CD40L interactions and use the same T cell-derived cytokines. The present study tested the response of splenic follicular and marginal zone as well as peritoneal B2 and B1 B cells to Th cell stimulation. Splenic and peritoneal B cell subsets were sort purified based on CD23 expression, and cultured with rCD40L and cytokines or Th2 cells. The results demonstrate that follicular, marginal zone, and peritoneal B2 B cells require CD40-CD40L interactions and preferentially use IL-4 for optimal proliferation, differentiation, and isotype switching. In contrast, peritoneal B1 B cells use IL-5 in conjunction with CD40-CD40L interactions for maximal Th cell-dependent responses. Furthermore, B1 B cells are capable of proliferating, differentiating, and isotype switching in the absence of CD40-CD40L interactions. B1 B cells are able to respond to Th2 clones in the presence of anti-CD40L mAb as well as to Th2 clones derived from CD40L(-/-) mice. The CD40-CD40L-independent response of B1 B cells is attributable to the presence of both IL-4 and IL-5, and may explain the residual Ab response to T cell-dependent Ags in CD40L- or CD40-deficient mice, and in X-linked hyper-IgM (X-HIM) patients.     

IL-11 activates human endothelial cells to resist immune-mediated injury

IL-11, a gp130-signaling cytokine, is protective in several in vivo models of immune-mediated and inflammatory injury. HUVECs express IL-11 receptor alpha-chain and gp130. Human IL-11 causes rapid (2-10 min) tyrosine phosphorylation of gp130. IL-11 at 0.1 and 10 ng/ml induces tyrosine phosphorylation of STAT3 and STAT1, respectively, although maximal responses require 50 ng/ml. Phospho-STAT3 and phospho-STAT1 levels peak rapidly (2.5 min) and disappear by 60 min. The p42 and p44 mitogen-activated protein kinases (MAPKs) are phosphorylated in response to 0.3 ng/ml IL-11 with maximal activation at 30 ng/ml IL-11. Phosphorylation of p42 and p44 MAPKs, which can be prevented by a mitogen-activated protein/extracellular signal-related kinase kinase-1 inhibitor, peaks by 15-20 min and largely disappears by 40 min. IL-11 does not activate NF-kappaB nor does it inhibit NF-kappaB activation by TNF. Similarly, IL-11 neither induces E-selectin or ICAM-1 nor blocks induction by TNF. Although IL-11 does not alter class I MHC complex molecule expression, pretreatment with 0.5 ng/ml IL-11 partially protects HUVECs against lysis by allospecific class I MHC-restricted cytolytic T lymphocytes or by anti-class I MHC Ab plus heterologous C. IL-11-induced cytoprotection is protein synthesis dependent and may depend on mitogen-activated protein/extracellular signal-related kinase kinase-1. Our results indicate that low (i.e., STAT3- and MAPK-activating) concentrations of IL-11 confer resistance to immune-mediated injury in cultured HUVECs without inhibiting proinflammatory responses.     

Cell-mediated immunity: role of IL-3 and IL-6 in the regulation of contact sensitivity reaction

Delayed type hypersensitivity reaction (DTH) consists of a sequential cascade of steps depending on different types of T cells, as well as mast cells, endothelial cells and macrophages. Recently it has been shown that CD4+ TH1 lymphocytes ("inflammatory type") play a central role in DTH reaction. Activated TH1 cells produce a characteristic pattern of cytokines: IL-2, IL-3, TNF-beta, IFN-gamma. Using the contact sensitivity (CS) reaction on mice as a model system, the role of cytokines in the regulation of DTH is presented, particularly the significance of IL-3 and IL-6. The recent data can be interpreted to show that IL-6 released by activated macrophages (APC cells) in the induction phase of the CS reaction probably stimulate CD8+ T suppressor cells. These in turn inhibit the production of IL-2 and IL-3 by CD4+ TH1 cells followed by a state of unresponsiveness.     

IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex

IL-33 (IL-1F11) is a recently described member of the IL-1 family of cytokines that stimulates the generation of cells, cytokines, and Igs characteristic of a type 2 immune response. IL-33 mediates signal transduction through ST2, a receptor expressed on Th2 and mast cells. In this study, we demonstrate that IL-33 and ST2 form a complex with IL-1R accessory protein (IL-1RAcP), a signaling receptor subunit that is also a member of the IL-1R complex. Additionally, IL-1RAcP is required for IL-33-induced in vivo effects, and IL-33-mediated signal transduction can be inhibited by dominant-negative IL-1RAcP. The implications of this shared usage of IL-1RAcP by IL-1(alpha and beta) and IL-33 are discussed.     

Activation of vascular endothelial cells by synovial fibrosis promotes Netrin-1-induced sensory nerve sprouting and exacerbates pain sensitivity

Synovial fibrosis is one of the most dominant histopathological changes in osteoarthritis of the knee (KOA), and activation of vascular endothelial cells in synovial fibrosis is both an important factor in mediating pain in KOA and a major contributor to the generation of pain signals. At the same time, angiogenesis and nerve fibres are more likely to underlie the pathology of pain induced by synovial fibrosis. In the present study, we established a co-culture model of human umbilical vein endothelial cells (HUVECs) with dorsal root ganglion (DRG) and detected tissue and cellular Netrin-1, vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), growth-associated protein-43 (GAP43), colorectal cancer deleted (DCC), uncoordinated 5 (UNC5), and the related expression of calcitonin gene-related peptide (CGRP), substance P (SP) and nerve growth factor (NGF) in supernatant by ELISA to investigate the intervention of vascular endothelial cell activation on sensory nerve sprouting exacerbating peripheral pain sensitivity and to investigate the effect of Netrin-1 from the perspective of Netrin-1 secretion to illustrate its effector mechanism.     

IL-33 induces antigen-specific IL-5+ T cells and promotes allergic-induced airway inflammation independent of IL-4

Type 2 cytokines (IL-4, IL-5, and IL-13) play a pivotal role in helminthic infection and allergic disorders. CD4(+) T cells which produce type 2 cytokines can be generated via IL-4-dependent and -independent pathways. Although the IL-4-dependent pathway is well documented, factors that drive IL-4-independent Th2 cell differentiation remain obscure. We report here that the new cytokine IL-33, in the presence of Ag, polarizes murine and human naive CD4(+) T cells into a population of T cells which produce mainly IL-5 but not IL-4. This polarization requires IL-1R-related molecule and MyD88 but not IL-4 or STAT6. The IL-33-induced T cell differentiation is also dependent on the phosphorylation of MAPKs and NF-kappaB but not the induction of GATA3 or T-bet. In vivo, ST2(-/-) mice developed attenuated airway inflammation and IL-5 production in a murine model of asthma. Conversely, IL-33 administration induced the IL-5-producing T cells and exacerbated allergen-induced airway inflammation in wild-type as well as IL-4(-/-) mice. Finally, adoptive transfer of IL-33-polarized IL-5(+)IL-4(-)T cells triggered airway inflammation in naive IL-4(-/-) mice. Thus, we demonstrate here that, in the presence of Ag, IL-33 induces IL-5-producing T cells and promotes airway inflammation independent of IL-4.     

Overexpression of Bcl(XL) in B cells promotes Th1 response and exacerbates collagen-induced arthritis

B cells play a pathogenic or regulatory role in many autoimmune diseases through production of autoantibodies, cytokine production, and Ag presentation. However, the mechanisms that regulate these B cell functions under different autoimmune settings remain unclear. In the current study, we found that when B cells overexpress an antiapoptotic gene, Bcl(XL), they significantly increased production of IFN-gamma and enhanced Th1 response. Consistently, Bcl-x(L) transgenic mice developed more severe and sustained collagen-induced arthritis due to the enhanced Th1 response. The production of autoantibodies in Bcl(XL) transgenic mice was comparable to that in wild-type mice. Thus, our results indicate a novel role of Bcl(XL) in regulating B cell functions and immune responses. In patients with rheumatoid arthritis, arthritogenic B cells often up-regulate Bcl(XL) expression, which may not only render B cells resistant to apoptosis but also alter the ability of the autoreactive B cells to produce cytokines and modulate the inflammatory response. This may have therapeutic implications if Bcl(XL) expression can be down-regulated in autoreactive B cells.     

IL-21 isoform is a membrane-bound ligand and activates directly interacted cells

IL-21 is a pleiotropic cytokine that regulates the function of T cells, B cells, natural killer cells, and myeloid cells. We previously identified an IL-21 isoform, IL-21iso, in humans and mice, and found that IL-21iso was secreted in much smaller amounts than conventional IL-21. In this study, we determined that secreted IL-21iso also has less signaling activity than IL-21. However, the amounts of intracellular IL-21 or IL-21iso, and the level of STAT3 phosphorylation induced by the two IL-21 forms, were similar. IL-21-sensitive reporter cells co-cultured with cells producing IL-21iso showed STAT3 activation, apoptosis, and proliferation. However, when IL-21iso-producing cells were cultured in a transwell chamber, which prevented direct contact with the IL-21-sensitive cells, no IL-21iso-induced signaling was observed. Though IL-21iso is secreted in smaller amounts and has less potent signaling activity than IL-21, IL-21iso acts both on IL-21iso-bearing cells and other IL-21-sensitive cells through direct interactions probably without being secreted. Thus, IL-21iso’s regulation of immune cells may be limited to the immediate proximity around the IL-21iso-producing cells, in regions such as immune organs or inflammation sites.