Cell-specific paracrine actions of IL-6 family cytokines from bone,marrow and muscle that control bone formation and resorption

Bone renews itself and changes shape throughout life to account for the changing needs of the body; this requires co-ordinated activities of bone resorbing cells (osteoclasts), bone forming cells (osteoblasts) and bone’s internal cellular network (osteocytes). This review focuses on paracrine signaling by the IL-6 family of cytokines between bone cells, bone marrow, and skeletal muscle in normal physiology and in pathological states where their levels may be locally or systemically elevated. These functions include the support of osteoclast formation by osteoblast lineage cells in response to interleukin 6 (IL-6), interleukin 11 (IL-11), oncostatin M (OSM) and cardiotrophin 1 (CT-1). In addition it will discuss how bone-resorbing osteoclasts promote osteoblast activity by secreting CT-1, which acts as a “coupling factor” on osteocytes, osteoblasts, and their precursors to promote bone formation. OSM, produced by osteoblast lineage cells and macrophages, stimulates bone formation via osteocytes. IL-6 family cytokines also mediate actions of other bone formation stimuli like parathyroid hormone (PTH) and mechanical loading. CT-1, OSM and LIF suppress marrow adipogenesis by shifting commitment of pluripotent precursors towards osteoblast differentiation. Ciliary neurotrophic factor (CNTF) is released as a myokine from skeletal muscle and suppresses osteoblast differentiation and bone formation on the periosteum (outer bone surface in apposition to muscle). Finally, IL-6 acts directly on marrow-derived osteoclasts to stimulate release of “osteotransmitters” that act through the cortical osteocyte network to stimulate bone formation on the periosteum. Each will be discussed as illustrations of how the extended family of IL-6 cytokines acts within the skeleton in physiology and may be altered in pathological conditions or by targeted therapies.     

The βc receptor family – Structural insights and their functional implications

Granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5 are members of a small family of cytokines that share a beta receptor subunit (βc). These cytokines regulate the growth, differentiation, migration and effector function activities of many hematopoietic cells in bone marrow, blood and sites of inflammation. Excessive or aberrant signaling can result in chronic inflammatory conditions and myeloid leukemias. The crystal structures of the GM-CSF ternary complex, the IL-5 binary complex and the very recent IL-3 receptor alpha subunit build upon decades of structure–function studies, giving new insights into cytokine–receptor specificity and signal transduction. Selective modulation of receptor function is now a real possibility and the structures of the βc receptor family are being used to discover novel and disease-specific therapeutics.     

A panoramic review and in silico analysis of IL-11 structure and function

Human Interleukin (IL)-11 is a multifunctional cytokine, recognized for its thrombopoietic effects for more than two decades; clinically, IL-11 is used in the treatment of thrombocytopenia. IL-11 shares structural and functional similarities with IL-6, a related family member. In recent years, there has been a renewed interest in IL-11, because its distinct biological activities associated with cancers of epithelial origin and inflammatory disorders have been revealed. Although the crystal structure of IL-11 was resolved more than two years, a better understanding of the mechanisms of IL-11 action is required to further extend the clinical use of IL-11. This review will discuss the available structural, functional, and bioinformatics knowledge concerning IL-11 and will summarize its relationship with several diseases.     

Interleukin-15 increases glucose uptake in skeletal muscle An antidiabetogenic effect of the cytokine

Previous studies have demonstrated that interleukin-15 (IL-15) has important anabolic effects on muscle protein metabolism. In the present investigation we have analysed the effects of IL-15 on glucose metabolism in skeletal muscle. Administration of a single dose of the cytokine (100 μg/kg body weight) resulted in a 32% increase on glucose uptake (as measured by the uptake of 2-deoxyglucose) in skeletal muscle. The effects observed on glucose uptake were direct since in vitro incubations of rat EDL muscles in the presence of the cytokine resulted in a 30% increase in glucose uptake. Similarly, IL-15 increased glucose uptake in C2C12 cell cultures, this being related with an increase in both glucose oxidation to CO₂ and the incorporation into muscle lipid. The effects of the cytokine were associated with an increase in GLUT-4 mRNA, suggesting a higher effect in insulin sensitivity. In conclusion, the data presented here indicate that IL-15 facilitates glucose metabolism in skeletal muscle and, therefore, a possible role of the cytokine as an antidiabetogenic drug merits future investigations.     

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.     

Satellite cells derived from obese humans with type 2 diabetes and differentiated into myocytes in vitro exhibit abnormal response to IL-6

Obesity and type 2 diabetes are associated with chronically elevated systemic levels of IL-6, a pro-inflammatory cytokine with a role in skeletal muscle metabolism that signals through the IL-6 receptor (IL-6Rα). We hypothesized that skeletal muscle in obesity-associated type 2 diabetes develops a resistance to IL-6. By utilizing western blot analysis, we demonstrate that IL-6Rα protein was down regulated in skeletal muscle biopsies from obese persons with and without type 2 diabetes. To further investigate the status of IL-6 signaling in skeletal muscle in obesity-associated type 2 diabetes, we isolated satellite cells from skeletal muscle of people that were healthy (He), obese (Ob) or were obese and had type 2 diabetes (DM), and differentiated them in vitro into myocytes. Down-regulation of IL-6Rα was conserved in Ob myocytes. In addition, acute IL-6 administration for 30, 60 and 120 minutes, resulted in a down-regulation of IL-6Rα protein in Ob myocytes compared to both He myocytes (P<0.05) and DM myocytes (P<0.05). Interestingly, there was a strong time-dependent regulation of IL-6Rα protein in response to IL-6 (P<0.001) in He myocytes, not present in the other groups. Assessing downstream signaling, DM, but not Ob myocytes demonstrated a trend towards an increased protein phosphorylation of STAT3 in DM myocytes (P = 0.067) accompanied by a reduced SOCS3 protein induction (P<0.05), in response to IL-6 administration. Despite this loss of negative control, IL-6 failed to increase AMPKα2 activity and IL-6 mRNA expression in DM myocytes. There was no difference in fusion capacity of myocytes between cell groups. Our data suggest that negative control of IL-6 signaling is increased in myocytes in obesity, whereas a dysfunctional IL-6 signaling is established further downstream of IL-6Rα in DM myocytes, possibly representing a novel mechanism by which skeletal muscle function is compromised in type 2 diabetes.     

SOCS3 deletion in B cells alters cytokine responses and germinal center output

B cell behavior is fine-tuned by internal regulatory mechanisms and external cues such as cytokines and chemokines. Suppressor of cytokine signaling 3 (SOCS3) is a key regulator of STAT3-dependent cytokine responses in many cell types and has been reported to inhibit CXCL12-induced retention of immature B cells in the bone marrow. Using mice with SOCS3 exclusively deleted in the B cell lineage (Socs3(Δ/Δ)mb1cre(+)), we analyzed the role of SOCS3 in the response of these cells to CXCL12 and the STAT3-inducing cytokines IL-6 and IL-21. Our findings refute a B cell-intrinsic role for SOCS3 in B cell development, because SOCS3 deletion in the B lineage did not affect B cell populations in naive mice. SOCS3 was strongly induced in B cells stimulated with IL-21 and in plasma cells exposed to IL-6. Its deletion permitted excessive and prolonged STAT3 signaling following IL-6 stimulation of plasma cells and, in a T cell-dependent immunization model, reduced the number of germinal center B cells formed and altered the production of Ag-specific IgM and IgE. These data demonstrate a novel regulatory signal transduction circuit in plasma cells, providing, to our knowledge, the first evidence of how these long-lived, sessile cells respond to the external signals that mediate their longevity.     

Stem cell based therapy for skeletal muscle diseases

The use of stem cells to repair and replace damaged skeletal muscle cells in chronic, debilitating muscle diseases such as the muscular dystrophies holds great promise. Different stem cell populations, both of embryonic and adult origin display the potential to generate skeletal muscle cells and have been studied in animal models of muscular dystrophy. These include muscle derived satellite cells; bone marrow derived mesenchymal stem cells, muscle or bone marrow side population cells, circulating CD133+ cells and cells derived from blood vessel walls such as mesoangioblasts or pericytes. The design of effective stem cell based therapies requires a detailed understanding of the molecules and signaling pathways which determine myogenic lineage commitment and differentiation. We discuss the great strides that have been made in delineating these pathways and how a better understanding of muscle stem cell biology has the potential to lead to more effective stem cell based therapies for skeletal muscle regeneration for devastating muscle diseases.     

IL-6-induced skeletal muscle atrophy.

Chronic, low-level elevation of circulating interleukin (IL)-6 is observed in disease states as well as in many outwardly healthy elderly individuals. Increased plasma IL-6 is also observed after intense, prolonged exercise. In the context of skeletal muscle, IL-6 has variously been reported to regulate carbohydrate and lipid metabolism, increase satellite cell proliferation, or cause muscle wasting. In the present study, we used a rodent local infusion model to deliver modest levels of IL-6, comparable to that present after exercise or with chronic low-level inflammation in the elderly, directly into a single target muscle in vivo. The aim of this study was to examine the direct effects of IL-6 on skeletal muscle in the absence of systemic changes in this cytokine. Data included cellular and molecular markers of cytokine and growth factor signaling (phosphorylation and mRNA content) as well as measurements to detect muscle atrophy. IL-6 infusion resulted in muscle atrophy characterized by a preferential loss of myofibrillar protein (-17%). IL-6 induced a decrease in the phosphorylation of ribosomal S6 kinase (-60%) and STAT5 (-33%), whereas that of STAT3 was increased approximately twofold. The changes seen in the IL-6-infused muscles suggest alterations in the balance of growth factor-related signaling in favor of a more catabolic profile. This suggests that downregulation of growth factor-mediated intracellular signaling may be a mechanism contributing to the development of muscle atrophy induced by elevated IL-6.     

TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling

Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.     

Effect of bone morphogenetic protein-6 on haemopoietic stem cells and cytokine production in normal human bone marrow stroma

Normal human bone marrow stroma cells include stem cells for both haemopoietic and osteochondrogenic lineages and express both bone morphogenetic protein (BMP) type I and type II receptors. As a member of the TGF-beta super-family, BMP-6 binds to both BMP type I and type II receptors and is involved in the developmental processes of renal and hepatic systems as well as of human foetal intestine. Also, BMP-6 induces osteoblastic differentiation of pluripotent mesenchymal cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP-6 on human cobblestone-area-forming cells (CAFC), that represent the functional primitive repopulating haemopoietic stem cell in long-term bone marrow culture. Also, the effect of BMP-6 on marrow stroma production of interleukin-6, -11 and their common receptor gp130 that is expressed in haemopoietic stem cells and is indispensable for their proliferation and tri-lineage differentiation was examined. Moreover, the effect of BMP-6 on marrow stroma release of soluble adhesion molecule VCAM-1 mediating the primitive haemopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP-6 treatment from 88+/-10 per 10(5)cells in control cultures in a dose dependent manner to only 48+/-3 per 10(5)cells in 50 ng/ml BMP-6-treated cultures, P< 0.01. Quantitative ELISA measurement revealed 50 ng/ml BMP-6 was able to significantly reduce IL-6 and IL-11 production from marrow stroma, P< 0.01. Also, BMP-6 significantly increased soluble gp130 release by 7.4-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The profound rapid increase in this natural antagonist of human IL-6 cytokine family may reduce the gp130 signaling. Also, the soluble VCAM-1 released increased by two-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The marked increase in the soluble form may exert an antagonist effect on the function of VCAM-1 (ligand for VLA4). Recently, blocking the VLA4/VCAM-1 adhesion pathway was shown to mobilise haemopoietic CD34 positive cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G-CSF-mobilised blood CD34 positive cells than on bone marrow CD34 positive cells before mobilisation in the same normal donors. Since BMP are currently being used in clinical trials for bone repair and fracture healing, the present results suggest a possible role for BMP-6 in mobilising CD34 positive cells for transplantation. Further in vitro tests are required to evaluate this potential mobilising role of BMP-6 in human long-term bone marrow culture.     

Cross-reactivity of antibody against SARS-coronavirus nucleocapsid protein with IL-11

Infection of SARS-associated coronavirus (SARS-CoV) induced a strong anti-nucleocapsid (anti-N) antibody response. However, the pathophysiological significance of the anti-N antibodies in SARS pathogenesis is largely unknown. To profile the anti-N antibodies, a phage-displayed scFv library was prepared from mice immunized with heat-inactivated SARS-CoV-infected Vero E6 cell lysate. Specific anti-N scFvs were isolated by panning against a recombinant nucleocapsid protein and reactivity was confirmed with phage-ELISA. Sequence analysis indicated that two of the isolated anti-N scFv clones were identical and displayed a high homology with an scFv specific for interleukin 11 (IL-11), an anti-inflammatory cytokine derived from bone marrow stroma cells. In a neutralization assay, IL-11-induced STAT 3 phosphorylation in rat intestinal epithelial IEC-18 cells was completely suppressed by the anti-N scFv clone L9N01.     

IL-6 receptor independent stimulation of human gp130 by viral IL-6

The genome of human herpes virus 8, which is associated with Kaposi's sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.     

The IL-1 receptor accessory protein (AcP) is required for IL-33 signaling and soluble AcP enhances the ability of soluble ST2 to inhibit IL-33

Interleukin (IL)-33 (or IL-1F11) was recently identified as a ligand for the orphan IL-1 receptor family member T1/ST2 (ST2). IL-33 belongs to the IL-1 cytokine family and, upon binding to ST2, induces intracellular signals similar to those utilized by IL-1. The effects of other IL-1 family cytokines are mediated by their binding to a specific receptor and the recruitment of a co-receptor required for elicitation of signaling. The aim of this study was to characterize the co-receptor involved in IL-33 signaling. Immunoprecipitation confirmed that IL-33 specifically binds ST2 and revealed that cellular IL-1 receptor accessory protein (AcP) associates with ST2 in a ligand-dependent manner. Receptor binding measurements demonstrated that the affinity of mouse (m)IL-33 for ST2 is increased by 4-fold in presence of AcP. IL-33 dose-dependently stimulated IL-6 secretion from wild-type (WT) mast cells, while no effect of IL-33 was observed with mast cells derived from AcP-deficient mice. Finally, soluble (s)ST2-Fc and sAcP-Fc acted synergistically to inhibit IL-33 activity. These observations identify AcP as a shared co-receptor within the IL-1 family that is essential for IL-33 signaling and suggest a novel role for sAcP in modulating the activity of IL-33.     

多发性骨髓瘤患者骨髓单个核细胞Treg、Th17和血清IL-6、IL-10与临床分期以及治疗效果的关系分析

摘要 目的：探讨多发性骨髓瘤（MM）患者骨髓单个核细胞调节性T细胞（Treg）、辅助性T细胞（Th17）和血清白细胞介素-6（IL-6）、白细胞介素-10（IL-10）与临床分期以及治疗效果的关系。方法：选择2016年3月至2020年12月河北医科大学第一医院收治的MM患者60例为研究对象,检测并对比不同Durie-Salmon分期患者的骨髓单个核细胞Treg、Th17、Treg/Th17及血清IL-6、IL-10水平;患者入院后均给予常规治疗,根据疗效分为有效组和无效组,比较两组治疗前后骨髓单个核细胞Treg、Th17、Treg/Th17及血清IL-6、IL-10水平;分析Treg、Th17、Treg/Th17及血清IL-6、IL-10与MM患者Durie-Salmon分期、治疗效果的相关性。结果：MM患者骨髓单个核细胞Treg、Treg/Th17及血清IL-6、IL-10水平III期组高于II期组,II期组高于I期组（P＜0.05）。有效组治疗后骨髓单个核细胞Treg、Treg/Th17水平及血清IL-6、IL-10水平较治疗前明显降低（P＜0.05）;治疗后,骨髓单个核细胞Treg、Treg/Th17及血清IL-6、IL-10水平无效组高于有效组（P＜0.05）。骨髓单个核细胞Treg、Treg/Th17及血清IL-6、IL-10水平与MM患者Durie-Salmon分期呈正相关,与治疗效果呈负相关（P＜0.05）;骨髓单个核细胞Th17水平与MM患者的Durie-Salmon分期、治疗效果无明显的相关性（P＞0.05）。结论：骨髓单个核细胞Treg、Treg/Th17水平及血清IL-6、IL-10水平与MM患者肿瘤临床分期、治疗效果密切相关,检测其水平可对MM的临床治疗及预后起到一定评估作用。