Neutrophil dynamics in surgical wounds – A novel role of interleukin-7☆

Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (T(reg)) and effector (T(eff)) T cells, whereby access to IL-2 can either increase the survival of T(eff) cells or the suppressive capacity of T(reg) cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of T(reg) cells, namely the specific suppression of survival signals for weakly activated T(eff) cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of T(reg) suppression.

Interleukin 6 and Haemostasis

IL-1 receptor-associated kinase (IRAK) is phosphorylated, ubiquitinated, and degraded upon interleukin-1 (IL-1) stimulation. In this study, we showed that IRAK can be ubiquitinated through both Lys-48- and Lys-63-linked polyubiquitin chains upon IL-1 induction. Pellino 3b is the RING-like motif ubiquitin protein ligase that promotes the Lys-63-linked polyubiquitination on IRAK. Pellino 3b-mediated Lys-63-linked IRAK polyubiquitination competed with Lys-48-linked IRAK polyubiquitination for the same ubiquitination site, Lys-134 of IRAK, thereby blocking IL-1-induced IRAK degradation. Importantly, the negative impact of Pellino 3b on IL-1-induced IRAK degradation correlated with the inhibitory effect of Pellino 3b on the IL-1-induced TAK1-dependent pathway, suggesting that a positive role of IRAK degradation in IL-1 induced TAK1 activation. Taken together, our results suggest that Pellino 3b acts as a negative regulator for IL-1 signaling by regulating IRAK degradation through its ubiquitin protein ligase activity.

Obligatory Role for Interleukin-13 in Obstructive Lesion Development in Airway Allografts

Inflammatory cytokines such as interleukin-1 and tumor necrosis factor-alpha modulate a transcription factor cascade in the liver to induce and sustain an acute and systemic defense against foreign entities. The transcription factors involved include NF-kappaB, STAT, and CCAAT/enhancer-binding protein (C/EBP). Whether the NFAT group of transcription factors (which was first characterized as playing an important role in cytokine gene expression in the adaptive response in immune cells) participates in the acute-phase response in hepatocytes is not known. Here, we have investigated whether NFAT is part of the transcription factor cascade in hepatocytes during inflammatory stress. We report that interleukin-1 or tumor necrosis factor-alpha increases expression of and activates NFATc2. C/EBP-mediated NFATc2 induction is temporally required for expression of type IIA secretory phospholipase A2. NFATc2 is also required for expression of phospholipase D1 and the calcium-binding protein S100A3. Thus, a C/EBP-NFATc2 transcription factor cascade provides an additional means to modulate the acute-phase response upon stimulation with inflammatory cytokines.

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.

Binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) stimulates Src family kinases, tyrosine phosphorylation of several proteins, conversion of Ras to its active GTP-bound form, and eventually c-fos, c-jun, and c-myc induction. The IL-2R beta chain plays a crucial role in IL-2R signaling. Within the cytoplasmic domain of the beta chain, a region essential for mitogenesis and another involved in binding the Src family kinase Lck have been defined. The beta chain itself is tyrosine-phosphorylated upon IL-2 stimulation. Since the adapter protein Shc acts upstream of Ras and is involved in T cell receptor-mediated Ras activation, we examined the role of Shc in IL-2 signaling. Shc was found to be tyrosine-phosphorylated upon IL-2 stimulation in CTLL-20 cells. After its phosphorylation, Shc interacted with another adapter protein, Grb2, and, via Grb2, with the Ras GTP/GDP exchange factor mSOS. After IL-2 stimulation, Shc also associated with the IL-2R beta chain. Thus, during IL-2 signaling, the interaction of Shc with the IL-2R beta chain and its simultaneous association with Grb2 and mSOS may couple IL-2R stimulation to Ras signaling.

Introduction Although there are different causes of low back pain, intervertebral disc (IVD) degeneration is one of them. In healthy discs, nociceptive nerve fibers and mechanoreceptors penetrate up to the outer annulus fibrosus (AF). In contrast, the endplates are heavily innervated. However, neither the inner AF nor the nucleus pulposus (NP) are innervated. Discogenic pain can occur due to degenerated discs acquiring cracks and fissures (annulogenic pain) or endplate damage (vertebrogenic pain) resulting in increased nerve fibers that penetrate the inner AF and NP. The increased expression of neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) has been identified in human and animal models of degenerating IVDs. 1 Proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) have been shown to trigger the expression of NGF, BDNF, and substance P in human NP and AF cells. In related studies, bone morphogenetic protein (BMP) was shown to suppress innervation while overexpression of the BMP inhibitor noggin resulted in a significant increase in nerve fibers. 2 However, the use of growth factors in clinical practice is limited by their high cost. Using synthetic peptides, such as link N, which stimulate BMP signaling, can circumvent this cost. The purpose of the present study was to evaluate the effect of link N on neurotrophins and substance P by human IVD cells stimulated with proinflammatory cytokines as well as in injured bovine IVDs. Materials and Methods Lumbar IVDs were obtained through organ donations within 6 hours after death. The procedure was approved by the local Research Ethics Board. Cells were isolated from NP and AF regions of the discs by sequential digestion with pronase followed by collagenase IA digestion for NP and collagenase II digestion for AF, respectively. They were cultured in monolayers and stimulated with TNF-α (100 ng/mL) and IL-1β (10 ng/mL) in the presence or absence of link N (1 μg/mL) for 48 hours. Total RNA was isolated and gene expression was measured using reverse-transcriptase polymerase chain reaction. The release of substance P into the culture media was measured after cells were stimulated by TNF-α (100-ng/mL) and IL-1β (10 ng/mL) in the presence or absence of link N (1 μg/mL) at different time points (1, 2, 4,and 24 hours). Coccygeal IVDs from the tails of adult bovine steers (20-25 months) were used for disc isolation. Four discs with cartilage end plates were isolated and were treated (control, capsaicin [1.5 μg/mL], punctured with a 16G needle, link N [10 μg/mL] treated) after preconditioning for 24 hours in complete DMEM. Disc culture media was collected at different time points for analysis. Substance P in the media was concentrated by solid phase extraction and was assayed by ELISA. Results Link N is known to induce proteoglycan synthesis by isolated disc cells and in degenerate rabbit and human discs, and to enhance chondrogenesis of MSCs in vitro. It is, however, not known if link N can suppress inflammatory mediators and neurotransmitters in disc degeneration. Without intervention or with link N supplementation alone only trace amounts of NGF gene expression in human lumbar disc cells from AF regions was observed. When human lumbar disc cells from AF regions were exposed to TNF-α for 48 hours, NGF gene expression was observed to increase significantly ( p < 0.05). However, supplementing 1.0 µg/mL link N to this media led to a significant downregulation of NGF gene expression. Further, link N significantly suppressed substance P release from punctured bovine discs after 24 hours of treatment as compared with the untreated punctured disc ( p < 0.05) ( Fig. 1 ). We also showed that link N can suppress TNF-α and IL-1β-induced messenger RNA levels of brain-derived neurotrophic factor (BDNF) and TAC 1 in AF cells. Thus, link N appears to reduce inflammatory mediators and neurotransmitters in human AF cells from normal and degenerated discs, as well as in injured bovine IVDs. [Figure: see text] Conclusion Link N is a promising agent for biological repair of degenerated human discs but an affective treatment for discogenic pain will also depend on stopping inflammation. Previous studies have shown that BMPs can suppress peripheral innervation in the skin. 2 Because BMPs and link N both share the Smad 1/5 signaling pathway, it was not unreasonable to assume that link N could also suppress inflammatory mediators associated with discogenic pain. The present study indicates that link N can also suppress NGF, BDNF, and TAC 1 in human disc cells as well as substance P release in injured bovine discs. This suggests that link N has the potential to inhibit pain induced by neuronal innervation caused by disc degeneration. Disc degeneration is often associated with low back pain; link N represents a potential economical growth factor with beneficial effects on disc repair. It would be of clinical significance to see if link N has any potential in reducing the pain caused by neuronal invasion during disc degeneration. Disclosure of Interest None declared References García-Cosamalón J, del Valle ME, Calavia MG, et al. Intervertebral disc, sensory nerves and neurotrophins: who is who in discogenic pain? J Anat 2010;217(1):1–15 Guha U, Gomes WA, Samanta J, Gupta M, Rice FL, Kessler JA. Target-derived BMP signaling limits sensory neuron number and the extent of peripheral innervation in vivo. Development 2004;131(5):1175–1186

IL-13 in asthma and allergic disease: Asthma phenotypes and targeted therapies

TAK1 (transforming growth factor beta-activated kinase 1) is a serine/threonine kinase that is a mitogen-activated protein kinase kinase kinase and an essential intracellular signaling component in inflammatory signaling pathways. Upon stimulation of cells with inflammatory cytokines, TAK1 binds proteins that stimulate autophosphorylation within its activation loop and is thereby catalytically activated. This activation is transient; it peaks within a couple of minutes and is subsequently down-regulated rapidly to basal levels. The mechanism of down-regulation of TAK1 has not yet been elucidated. In this study, we found that toxin inhibition of type 2A protein phosphatases greatly enhances interleukin 1 (IL-1)-dependent phosphorylation of Thr-187 in the TAK1 activation loop as well as the catalytic activity of TAK1. From proteomic analysis of TAK1-binding proteins, we identified protein phosphatase 6 (PP6), a type-2A phosphatase, and demonstrated that PP6 associated with and inactivated TAK1 by dephosphorylation of Thr-187. Ectopic and endogenous PP6 co-precipitated with TAK1, and expression of PP6 reduced IL-1 activation of TAK1 but did not affect osmotic activation of MLK3, another MAPKKK. Reduction of PP6 expression by small interfering RNA enhances IL-1-induced phosphorylation of Thr-187 in TAK1. Enhancement occurred without change in levels of PP2A showing specificity for PP6. Our results demonstrate that PP6 specifically down-regulates TAK1 through dephosphorylation of Thr-187 in the activation loop, which is likely important for suppressing inflammatory responses via TAK1 signaling pathways.

To characterize the expression of interleukin-7 (IL-7) and IL-7 receptor (IL-7R) in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages, endothelial cells, and synovial tissue fibroblasts in RA. Expression of IL-7 and IL-7R in RA and normal synovial tissue was demonstrated by immunohistochemistry. Expression and regulation of IL-7 and IL-7R in RA peripheral blood in vitro-differentiated macrophages, RA synovial tissue fibroblasts, and human microvascular endothelial cells (HMVECs) were determined by real-time reverse transcription-polymerase chain reaction and/or flow cytometry. Enzyme-linked immunosorbent assay was used to examine production of proangiogenic factors by IL-7-activated macrophages, RA fibroblasts, and endothelial cells. IL-7 and IL-7R were coexpressed on RA synovial tissue lining and sublining macrophages and endothelial cells. Expression of IL-7 and its receptor was significantly elevated in RA synovial fluid and peripheral blood macrophages as well as RA fibroblasts, compared to normal cells. Toll-like receptor 4 ligation (with lipopolysaccharide) and tumor necrosis factor α (TNFα) stimulation modulated expression of IL-7 and IL-7R on RA macrophages and HMVECs. However, in RA fibroblasts, lipopolysaccharide and TNFα activation increased expression of IL-7R only. IL-7 also mediated RA pathogenesis by inducing production of potent proangiogenic factors from macrophages and endothelial cells. We have identified, for the first time, regulators of IL-7 and IL-7R expression in RA fibroblasts, RA peripheral blood in vitro-differentiated macrophages, and endothelial cells. Our results document a novel role of IL-7 in RA angiogenesis.

Background: Interleukin-2 (IL-2) stimulation had been reported as having a beneficial impact to the expression of CD56bright, CD56dim, and interferon-γ (IFN-γ) in the case of immunological dysfunction diseases. However, in the case of systemic lupus erythematosus (SLE), the role of IL-2 had never been investigated. The objective of this study was to assess the impact of IL-2 on the expression of CD56bright, CD56dim, and IFN-γ in SLE patients. Methods: An experimental study was conducted by involving peripheral blood mononuclear cells isolated from six SLE patients. The study consisted of four groups based on IL-2 stimulation: D0 (0U/ml), D1 (50U/ml), D2 (150U/ml), and D3 (250U/ml); and they were then cultured for 72 hours. The levels of CD56bright and CD56dim were measured by FACSMelodyTM, while the levels of IFN-γ were measured using ELISA. Results: In group D0, D1, D2, and D3; the levels of CD56bright were 57.27±37.27, 241.16±64.41, 256.94±50.95, and 259.37±36.44 x1000 cells/mm3 respectively. Moreover, the levels of CD56dim were 812.85±167.37, 631.98±129.90, 616.42±157.97, and 615.90±155.57 x1000 cells/mm3 respectively. On the other hand, the levels of IFN-γ were 24.01±2.56, 26.09±4.79, 30.11±5.34, and 32.43±7.14 pg/ml respectively. Our analysis elucidated that the administration of IL-2 provided potential impact to the levels of CD56bright, but not to the levels of CD56dim and IFN-γ. Our findings indicated that the increased dosage of IL-2 resulted in a more significant impact on CD56bright. Conclusions: Our study clarifies that IL-2 provides a beneficial impact on CD56bright expression in SLE patients.

Shc associates with the IL-3 receptor β subunit, SHIP and Gab2 following IL-3 stimulation: Contribution of Shc PTB and SH2 domains

We recently reported that interleukin-3 (IL-3) stimulation of the murine IL-3-responsive cell line, B6SUtA1, results in the rapid phosphorylation of the beta subunit of the IL-3 receptor (IL-3R), not only on tyrosine residues but on serine/threonine (Ser/Thr) residues as well. Since this occurred even at 4 degrees C, it suggested that a Ser/Thr-specific kinase might be closely associated with the IL-3R. To test this possibility, IL-3R complexes were isolated with anti-IL-3R (alpha IL-3R) antibodies, and in vitro phosphorylation studies were undertaken. These revealed the presence of a 110-kDa protein that was heavily phosphorylated in vitro on serine and threonine residues and that bound selectively to gamma-ATP-Sepharose beads. Moreover, this protein, which was not the 110-kDa subunit of phosphatidylinositol 3-kinase, was tyrosine phosphorylated in response to IL-3 and was specifically labeled in vitro with azido-[32P]ATP. These data, together with in vitro kinase inhibitor studies, suggest that an as yet uncharacterized H7- and staurosporine-sensitive 110-kDa Ser/Thr kinase may be constitutively associated with the IL-3R and activated following IL-3 stimulation. A comparison of IL-3R and erythropoietin receptor complexes suggests that this 110-kDa protein may be preferentially associated with the IL-3R.

Interleukin-3 regulates the activity of the LYN protein-tyrosine kinase in myeloid-committed leukemic cell lines