Kate L E Phillips

Tumor necrosis factor α- and interleukin-1β-dependent induction of CCL3 expression by nucleus pulposus cells promotes macrophage migration through CCR1.

OBJECTIVE To investigate tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. METHODS Quantitative reverse transcription-polymerase chain reaction and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, CCAAT/enhancer binding protein (C/EBPβ), and MAPK on cytokine-mediated CCL3 promoter activity. The effect of NP-conditioned medium on macrophage migration was measured using a Transwell system. RESULTS An increase in CCL3 expression and promoter activity was observed in NP cells after TNFα or IL-1β treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBPβ on the CCL3 promoter was confirmed through gain-of-function and loss-of-function studies. Notably, cotransfection with p50 completely blocked cytokine- and p65-dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with short hairpin RNA for p65 (shp65) and shIKKβ significantly decreased the TNFα-dependent increase in CCL3 expression. Analysis of degenerated human NP tissue samples showed that CCL3, but not CCL4, expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNFα or IL-1β promoted their migration. Pretreatment of macrophages with an antagonist of CCR1, the primary receptor for CCL3 and CCL4, blocked cytokine-mediated migration. CONCLUSION Our findings indicate that TNFα and IL-1β modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPβ signaling. The CCL3-CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs.

Interleukin-1 receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration

Objectives Interleukin 1 (IL-1) is potentially important in the pathogenesis of intervertebral disc (IVD) degeneration; increasing production of matrix degradation enzymes and inhibiting matrix synthesis. Although IL-1 polymorphisms have been linked to increased risk of IVD degeneration, it is still unclear whether IL-1 drives IVD degeneration in vivo or is a secondary feature of degeneration. Here, we investigated whether IVD degeneration could be induced spontaneously by the removal of the natural inhibitor of IL-1 (IL-1 receptor antagonist) in mice that lack a functional IL-1rn gene. Methods Histological staining and immunohistochemistry was performed on BALB/c IL-1rn+/+ and IL-1rn−/− mice to examine degeneration and to localise and detect IL-1, matrix metalloproteinases (MMP)3, MMP7, a disintigrin and MMP with thrombospondin motifs (ADAMTS)4 protein production. In addition, IVD cells were isolated using collagenase and proliferation potential determined. Results IL-1rn−/− knockout mice displayed typical features of human disc degeneration: loss of proteoglycan and normal collagen structure and increased expression of matrix degrading enzymes: MMP3; MMP7 and ADAMTS4. Histological grade of degeneration increased in IL-1rn−/− mice which was more evident within older mice. In addition IVD cells isolated from IL-1rn−/− mice displayed reduced proliferation potential. Conclusions Here, we show that IL-1rn−/− mice develop spinal abnormalities that resemble characteristic features associated with human disc degeneration. The current evidence is consistent with a role for IL-1 in the pathogenesis of IVD degeneration. The imbalance between IL-1 and IL-1Ra which is observed during human IVD degeneration could therefore be a causative factor in the degeneration of the IVD, and as such, is an appropriate pharmaceutical target for inhibiting degeneration.

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc.

IntroductionThe aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.MethodsReal-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.ResultsLDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.ConclusionsOur data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.

Inflammatory Cytokines Induce NOTCH Signaling in Nucleus Pulposus Cells IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION

Background: The regulation of NOTCH signaling under inflammatory conditions in the nucleus pulposus is unknown. Results: Expression of select NOTCH pathway genes, including NOTCH2 and NOTCH signaling, is regulated by IL-1β and TNF-α. Conclusion: Inflammatory cytokines promote NOTCH signaling in disc. Significance: NOTCH signaling may play a role in pathogenesis of disc disease. The objective of the study was to investigate how inflammatory cytokines, IL-1β, and TNF-α control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/β2/δ, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38γ played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1β as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1β and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.

Hydroxyurea therapy in UK children with sickle cell anaemia: A single-centre experience

Despite the demonstrated efficacy of hydroxyurea therapy, children with sickle cell anaemia in the UK are preferentially managed with supportive care or transfusion. Hydroxyurea is reserved for children with severe disease phenotype. This is in contrast to North America and other countries where hydroxyurea is widely used for children of all clinical phenotypes. The conservative UK practice may in part be due to concerns about toxicity, in particular marrow suppression with high doses, and growth in children.

Non‐tumour bone marrow lymphocytes correlate with improved overall survival in childhood acute lymphoblastic leukaemia

Composition of tumour immune cell infiltrates correlates with response to treatment and overall survival (OS) in several cancer settings. We retrospectively examined immune cells present in diagnostic bone marrow aspirates from paediatric patients with B‐cell acute lymphoblastic leukaemia. Our analysis identified a sub‐group (∼30% of patients) with >2.37% CD20 and >6.05% CD7 expression, which had 100% OS, and a sub‐group (∼30% of patients) with ≤2.37% CD20 and ≤6.05% CD7 expression at increased risk of treatment failure (66.7% OS, P < 0.05). Immune cell infiltrate at diagnosis may predict treatment response and could provide a means to enhance immediate treatment risk stratification.

Human Degenerate Discs Show Increased Activation of Intracellular Signalling Pathways of IL-1

Introduction Intervertebral disc degeneration is implicated in 40% of low back pain cases. Interleukin 1 (IL-1) is known to be important in the pathogenesis of intervertebral disc (IVD) degeneration, here we investigated the intracellular signaling pathways activated by IL-1 and determined the activation status of these pathways in native IVD tissues highlighting potential pathways for new therapies. Materials and Methods Human nucleus pulposus cells (NP) removed during discetomy for nerve root pain were stimulated with IL-1 for 30 minutes. The activation of ERK1/2, p38, c-jun, and IκB were determined using cell-based enzyme-linked immunosorbent assays, in addition pNFκB localisation in stimulated cells was determined using immunohistochemistry. Localisation of phosphorylated c-jun, p38, ERK1/2, and NFκB together with IL-1 was investigated within paraffin embedded sections of human IVD to investigate the presence of active pathways in vivo. Pretreatment with inhibitors of p38, c-jun, and NFκB for 30 minutes before stimulation with IL-1 (10 ng/mL) for 48 hours was investigated to determine the ability of individual signaling inhibitors to block the catabolic responses induced by IL-1. Results IL-1-induced activation of p38, MAPK, ERK ½, JNK/c-jun, and NFκB signaling pathways in human NP cells. IVD tissue samples displayed immunopositive staining for phosphorylated c-jun, ERK1/2, NFκB, and p38, immunopositivity was significantly increased within degenerate discs, particularly in those discs which also expressed high levels of IL-1. Inhibition of individual signaling pathways demonstrated differential regulation of the pleathora of IL-1-induced catabolic events, with different signaling molecules shown to regulate matrix metalloproteases, cytokines, and factors involved in innervation and angiogenesis of the disc. Conclusion Here, we have shown that the signaling pathways activated by IL-1 in vitro display increased activation in vivo during disc degeneration. Inhibitors of these pathways demonstrated multiple signaling pathways were involved in the pleathora of IL-1 actions. Thus, inhibition of signaling pathways could be a novel mechanism to inhibit catabolic processes which could hold promise to inhibit degeneration at early stages of disease but also create the correct tissue niche to promote regeneration of the disc. Disclosure of Interest None declared

Role of Semaphorins in Angiogenesis and Innervation in Human Intervertebral Disc Degeneration

Introduction Although the intervertebral disc (IVD) is considered the largest aneural structure within the human body, during degeneration nociceptive nerves are seen to accompany blood vessels into the IVD yet it remains unclear how this process occurs. Semaphorins are axonal guidance molecules known to repulse nerves by altering the actin cytoskeleton of growth cones. Semaphorin 3A (sema3A) has recently been localized in healthy IVDs, suggesting an inhibitory role toward neural and vascular ingrowth. This study aimed to first identify the presence of the class 3 semaphorins and sema4D shown to be involved in promoting angiogenesis and their receptors within nucleus pulposus(NP) cells and an endothelial cell line and determine whether their expression could be altered by cytokine treatment. Materials and Methods Real-time PCR (RT-PCR) was performed to investigate gene expression of class 3 semaphorins; Sema3A-3F, sema4D, their receptors plexin A1-A4 and plexin D1 and neuropilin-1 (NRP-1) and NRP-2 in directly extracted RNA from human NP cells and NP cells cultured in alginate for 2weeks before treatment for 48hours with interleukin (IL)-1, IL-6, or tumor necrosis factor α(TNFα) at 0 to 100ng/mL. Similarly, HDMEC-1 endothelial cells were treated with IL-1, IL-6, or TNFα at 0 and 10ng/mL for 48hours, after which mRNA expression was investigated by RT-PCR. Results Expression of semaphorins and their receptors were present in normal and degenerate disc samples. In human NP cells, IL-6 inhibited semaphorin expression, whereas IL-1 and TNFα significantly increased sema3D over 10-fold (p≤0.05). IL-1 upregulated all plexins, conversely TNFα significantly decreased plexin A2. NRP-2 showed a significant increase in response to IL-1. While NRP-1 was significantly decreased in response to IL-1. In HDMEC-1 cells IL-1 significantly increased sema3C and sema3D while TNF significantly increased sema3C. TNF significantly increased NRP-2 expression, however, NRP-1 was decreased by all cytokines. Plexin receptor expression was increased upon IL-6 and TNF treatment whereas IL-1 caused significant decrease in plexin A3 expression. Conclusion Here, we have demonstrated that several cytokines known to be upregulated during disc degeneration and disc prolapse, regulate expression of certain members of class 3 semaphorins, as well as their receptors in human NP cells and HDMEC-1 cells. This data suggests that the presence of these factors within the degenerate disc may be responsible for modulating the ingrowth of blood vessels and promote nerve ingrowth leading to discogenic pain. Disclosure of Interest None declared

Differential Intracellular Signaling Pathways Are Activated by Catabolic and Anabolic Factors in the Ivd, and these Could be Targeted in Selected Therapeutic Approaches

Introduction Intervertebral disk (IVD) degeneration is a major cause of lower back pain. Evidence suggests that inflammatory cytokines produced by chondrocyte-like cells within the IVD during degeneration are integral in the pathogenesis of this disease, with a particular role for IL-1 hypothesized. Targeting intracellular signaling mechanisms of IL-1 could provide a mechanism to inhibit multiple catabolic cytokines simultaneously. Many cytokines share intracellular signaling pathways and thus inhibiting signaling rather than direct receptors could prevent compensatory actions occurring. Here we investigated signaling pathways activated by IL-1 together with an anabolic factor to identify differential pathways. Materials and Methods Human nucleus pulposus (NP) cells were extracted via collagenase digestion and expanded in monolayer culture, prior to transfer to alginate bead culture for 2 weeks to enable redifferentiation. Following redifferentiation cells were treated with either IL-1 (10 ng/mL) or CDMP-1 (10 ng/ml) for 30 minutes, protein extracted and R&D proteome array deployed to investigate potential differential signaling molecules. In addition, NFkB activation was investigated using immuno-fluorescence and activation of pERK, pP38 MAPK, and AKT were investigated using BD phosflow techniques. In addition, cells were pretreated with inhibitors of signaling pathways: SB203580 (p38 MAPK) and Helenalin (NFkB) to investigate effects on matrix and matrix degrading enzyme gene expression following stimulation with CDMP and IL-1. Results R&D proteome array enabled the investigation of 46 signaling molecules simultaneously following and identified a number of differentially activated pathways, including p38MAPK (Fig. 1). IL-1 induced p38MAPK, NFkB, AKT, and ERK pathways were confirmed using BD phosflow and immunofluorescence techniques. Inhibition of NFkB pathway using helenine reduced the inhibitory effect on aggrecan induced by IL-1, but helenine alone also induced an inhibition of aggrecan induction by CDMP. Conclusion The catabolic cytokine IL-1 has been implicated in the pathogenesis of IVD degeneration which leads to low back pain. The identification of differential intracellular signaling pathways between anabolic and catabolic factors could provide new methods for modulating the catabolic response, and preventing further IVD degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared