Soluble IL-6R alpha upregulated IL-6, MMP-1 and MMP-2 secretion in bone marrow stromal cells

IL-6 mediates its activity through a cell surface receptor composed of a signal transducing protein, CD130, and a ligand-binding protein which exists in membrane-bound form (CD126) or in soluble form (sIL-6R alpha). Interestingly, sIL-6R alpha combined with IL-6 is able to interact with CD130 leading to the intracellular cascade of activation. In the present study, using flow cytometry, we show that stromal cells from human bone marrow (BMSC) express CD130 but not CD126. We demonstrate that BMSC are responsive to IL-6 only in the presence of exogenous sIL-6R alpha. Indeed, exogenous sIL-6R alpha induces in BMSC the production of its own ligand, IL-6, and of both MMP-1 and MMP-2, two matrix metalloproteinases involved in bone resorption and in tumour spreading, respectively. Since myeloma cells release sIL-6R alpha in the close vicinity of BMSC, these data suggest a role for this factor in the pathophysiology of multiple myeloma, a B-cell malignancy dependent on IL-6 for its growth and characterized by bone destruction.     

The role of interleukin-6 and interleukin-6/interleukin-6 receptor-alpha complex in the pathogenesis of multiple myeloma

Multiple myeloma (MM) is a plasma-cell disorder in which malignant plasma cells accumulate in the bone marrow and usually produce a monoclonal immunoglobulin. Usual presenting features of overt MM include recurrent osteolytic lesions, bacterial infections, anemia and renal insufficiency. MM is responsible for about 1 percent of all cancer-related deaths in Western countries. Its epidemiologic pattern remains obscure, and its cause unknown [1]. The presence of somatic mutations within the immunoglobulin genes of myeloma cells indicate that the putative myeloma-cell precursors have been stimulated by antigens within germinal centers and are either memory B cells or migrating plasmablasts. Myeloma cells proliferate slowly in the bone marrow and display a weak apoptotic index in vivo [2]. This suggest that some defects in the apoptotic process could be involved in this neoplasia. Interleukin-6 (IL-6) is known to be an essential survival factor of myeloma cells and to protect them from apoptosis induced by different stimuli (e.g. dexamethasone, CD95, serum starvation, gamma-irradiation). More recently, important works have been devoted to the biology of the soluble form of the IL-6R alpha i.e., sIL-6R alpha. These works give IL-6/sIL-6R alpha complex an important role in the biology of IL-6. The purpose of the current review is to emphasize the role of this complex in the pathogenesis of MM.     

The role of transsignalling via the agonistic soluble IL-6 receptor in human diseases

The activation of cells that do not express the membrane bound interleukin-6 6 receptor (IL-6R) by IL-6 and the soluble IL-6 receptor (sIL-6R) is termed transsignalling. Transsignalling may be an pathogenetic factor in human diseases as diverse as multiple myeloma (MM), Castleman's disease, prostate carcinoma, Crohn's disease, systemic sclerosis, Still's disease, osteoporosis and cardiovascular diseases. IL-6 and sIL-6R may directly or indirectly enhance their own production on endothelial or bone marrow stromal cells. Positive feedback autocrine loops thus created in affected organs may either cause or maintain disease progression. In autoimmune or vasculitic disease, the ability of the IL-6/sIL-6R complex to inhibit apoptosis of autoreactive T-cells may be central to the development of tissue specific autoimmunity. The anti-apoptotic effect of the IL-6/sIL-6R complex may be involved in tumour genesis and resistance to chemotherapy.Only in rare cases, where counterregulation has failed, there is a notable systemic effect of IL-6/sIL-6R. Appropriate animal models are necessary to establish the pathogenetic role of the IL-6/sIL-6R complex. A specific treatment option for diseases influenced by the sIL-6R could be based on gp130-Fc, a soluble gp130 (sgp130) linked to the Fc-fragment of IgG1. gp130-Fc has shown efficacy in vivo in animal models of Crohn's disease.     

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.     

Hyaluronic acid induces survival and proliferation of human myeloma cells through an interleukin-6-mediated pathway involving the phosphorylation of retinoblastoma protein

Originating from a post-switch memory B cell or plasma cell compartment in peripheral lymphoid tissues, malignant myeloma cells accumulate in the bone marrow of patients with multiple myeloma. In this favorable microenvironment their growth and survival are dependent upon both soluble factors and physical cell-to-cell and cell-to-extracellular matrix contacts. In this report we show that hyaluronan (HA), a major nonprotein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, is a survival and proliferation factor for human myeloma cells. The effect of HA is mainly mediated through a gp 80-interleukin 6 (IL-6) receptor pathway by a CD44-independent mechanism, suggesting that HA retains and concentrates IL-6 close to its site of secretion, thus favoring its autocrine activity. In addition, we show that HA-mediated survival and proliferation of myeloma cells is associated with a down-regulation in the expression of p27(kip1) cyclin-dependent kinase inhibitor and a hyperphosphorylation of the retinoblastoma protein (pRb). These data suggest that HA could be an important component in the myeloma cell physiopathology in vivo by potentiating autocrine and/or paracrine IL-6 activities.     

Serum proinflammatory mediators at different periods of therapy in patients with multiple myeloma

Multiple myeloma (MM) is a malignant disease characterized by the clonal proliferation of plasma cells within the bone marrow. Several cytokines have been demonstrated to be involved in the control of growth, progression, and dissemination of MM. We determined serum levels of interleukin-1beta (IL-1beta), soluble interleukin-2 receptor (sIL-2R), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), and C-reactive protein (CRP) in 14 newly diagnosed MM patients. The median age of the patients was 63.4 +/- 10.8 years and all of the patients were stage III (classified according to the Durie-Salmon classification). The same parameters were measured in 15 healthy controls. In addition, we also examined the effects of vincristine-adriamycin-dexamethasone (VAD) therapy on the same parameters and mediators as well as the relationship among the parameters in the same patient groups. The serum concentrations of TNF-alpha, IL-1beta, sIL-2R, IL-6, IL-8, and CRP (18.6 +/- 3.7 pg/mL, 10.1 +/- 2.8 pg/mL, 730 +/- 220 U/mL, 11.4 +/- 3.3 pg/mL, 23.9 +/- 8.3 pg/mL, and 49.9 +/- 19.5 mg/dL, resp) were significantly higher in newly diagnosed MM patients than in healthy controls (P < .0001). All of the parameters were found to be significantly reduced after chemotherapy. In conclusion, we found that after the VAD therapy, the level of these cytokines which are thought to play an important role in the pathogenesis of MM was significantly suppressed. This is the first study demonstrating strong impact of VAD treatment on circulating mediators of sIL-2R and IL-8 levels parameters.     

Cytokines in Gaucher's disease.

Gaucher's disease (GD) is characterized by hepatosplenomegaly, bone marrow infiltration, osteonecrosis, which may all be associated with the presence of pathological macrophages that contain undegraded glycosphingolipids. Levels of serum cytokines, which are soluble products of mononuclear phagocytes (MNP), were evaluated in 24 GD patients. Levels of interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and soluble interleukin-2 receptor (sIL-2R) in GD patients were significantly higher than in normal controls. We attempted to correlate cytokine levels with disease severity. Type I GD patients with more severe clinical manifestations had significantly higher levels of IL-1beta, IL-1Ra and IL-6, relative to type I patients with milder disease. Three patients homozygous for the 1448C mutation with neuropathic type III disease, had significantly higher levels of sIL-2R than type I patients or controls. We speculate that cytokine over-expression may relate to the pathophysiology of some of the clinical manifestations of GD. Thus, the elevated IL-1beta, TNF-alpha and IL-6 levels may induce the bone manifestations, the neutrophil chemotaxis and the increased incidence of hyper-gammaglobulinemia present in GD patients.     

Gp130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL-6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.     

CD28 expressed on malignant plasma cells induces a prosurvival and immunosuppressive microenvironment

Interactions between the malignant plasma cells of multiple myeloma and stromal cells within the bone marrow microenvironment are essential for myeloma cell survival, mirroring the same dependence of normal bone marrow-resident long-lived plasma cells on specific marrow niches. These interactions directly transduce prosurvival signals to the myeloma cells and also induce niche production of supportive soluble factors. However, despite their central importance, the specific molecular and cellular components involved remain poorly characterized. We now report that the prototypic T cell costimulatory receptor CD28 is overexpressed on myeloma cells during disease progression and in the poor-prognosis subgroups and plays a previously unrecognized role as a two-way molecular bridge to support myeloid stromal cells in the microenvironment. Engagement by CD28 to its ligand CD80/CD86 on stromal dendritic cell directly transduces a prosurvival signal to myeloma cell, protecting it against chemotherapy and growth factor withdrawal-induced death. Simultaneously, CD28-mediated ligation of CD80/CD86 induces the stromal dendritic cell to produce the prosurvival cytokine IL-6 (involving novel cross-talk with the Notch pathway) and the immunosuppressive enzyme IDO. These findings identify CD28 and CD80/CD86 as important molecular components of the interaction between myeloma cells and the bone marrow microenvironment, point to similar interaction for normal plasma cells, and suggest novel therapeutic strategies to target malignant and pathogenic (e.g., in allergy and autoimmunity) plasma cells.     

Serum soluble interleukin 2 receptor alpha in human cancer of adults and children: a review.

Cancer growth and development is associated with the stimulation of the innate immune system, including enhanced interleukin 2 receptor (IL-2R) expression in immune cells and its shedding into the circulation in a soluble form of sIL-2Ralpha. In most haematological malignancies, including different types of leukaemias and lymphomas, sIL-2Ralpha has been found to be released directly from the surface of neoplastic cells thus reflecting the tumour bulk, turnover and activity. Several studies have proved that not only lymphoid cancer cells, but also some non-lymphoid cancer cells, express IL-2R on their surface. They include malignant melanoma and carcinomas of the kidney, head and neck, oesophagus and lung. It is suggested that in most malignant solid tumours, elevated levels of sIL-2Ralpha are likely to be the product of normal peripheral mononuclear cells activated in response to the neoplasm's growth or that they are released from activated lymphoid cells infiltrating neoplastic tissues. This latter hypothesis has been proved by discovering the high expression of CD25 on the cell surface of most of these cells. Although the precise source and biological role of sIL-2Ralpha has not been clarified definitively, pretreatment serum levels of sIL-2Ralpha have been shown to reflect the activity, advancement and biological aggressiveness of many types of cancer in adults and children as well as to correlate with prognosis and overall survival. The possibility of enriching the diagnostic tools of oncologists with a new biochemical marker of activity of neoplasms resulted in numerous studies and reports concerning the clinical usefulness of sIL-2Ralpha measurements in adult and, less frequently, in paediatric malignancies. This article presents the actual knowledge concerning the structure, source and biological function of sIL-2Ralpha in patients with haematological and non-haematological malignancies. The authors review the published data on clinical applicability of soluble IL-2Ralpha determination in terms of diagnostics, prognosis and treatment monitoring of particular types of malignant disorders both in adults and in children. They also provide an insight into the clinical usefulness of sLL-2Ralpha-blocking antibodies in patients with cancer, and in those who reject organ transplants, develop graft-versus-host disease after allogeneic bone marrow transplantation and are affected with autoimmune disorders.     

Interleukin-2 receptor positive cells and circulating soluble interleukin-2 receptors in patients with solid tumors are not correlated

Activated lymphocytes can release a soluble form of IL-2 receptor (sIL-2R), which retains the capacity to bind IL-2. Abnormally high values of sIL-2R have been observed in patients with advanced solid tumors. In an attempt to further understand the biological significance of sIL-2R in solid tumors, this study investigated the relation between sIL-2R and Tac-positive cells. sIL-2R serum levels and Tac-positive cells were determined in 18 patients with solid tumors metastatic, 108 non-metastatic. Tumor types were: breast 7; lung 6; colon 2; stomach 1; testis 1; larynx 1. No correlation was found between circulating sIL-2R values and Tac-positive cells, and there was no difference between Tac-positive cell mean number in patients with high and normal sIL-2R levels. These preliminary results suggest that different mechanisms are responsible for sIL-2R release in the blood and IL-2 receptor expression on the immune cell surface.     

Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.     

Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3) increased concentrations of sgp130 should produce an efficient buffering effect on the IL-6/sIL-6R complex without incidence on the level of free IL-6. According to this model, the IL-6/sIL-6R complex appears to be an important support of IL-6 signaling in the most commonly encountered in vivo situations. The concentration of this complex is directly under the control of the concentration of sIL-6R; its bio-availability should be efficiently buffered by increased sgp130 concentrations.     

The interleukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145 confers increased proteolytic conversion rates by ADAM proteases

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant.     

Serum levels of interleukin-6 type cytokines and soluble interleukin-6 receptor in patients with rheumatoid arthritis.

We investigated the serum concentrations of interleukin-6 (IL-6) and two IL-6 family of cytokines (leukaemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) as well as IL-6 soluble receptor (sIL-6R) using an enzyme-linked immunosorbent assay (ELISA) in 66 patients with rheumatoid arthritis (RA) and 24 healthy controls. We examined a possible association between the serum levels of these peptides and RA activity according to the Mallya and Mace scoring system and Ritchie''s index. We also evaluated the correlation between the serum levels of IL-6, LIF, CNTF and sIL-6R and duration of the disease and calculated sIL-6R/IL-6 ratio in RA patients and in the control group. IL-6 and sIL-6R were detectable in all 66 patients with RA and 24 normal individuals. LIF was also found in the serum of all patients with RA and in 16 (66.7%) normal individuals. In contrast CNTF was measurable only in 15 (22.7%) patients with RA and 24 (33.3%) normal individuals. The highest IL-6 and sIL-6R levels were found in the patients with Stages 3 and 4 of RA activity and the lowest in the control group. In contrast there were no statistically significant differences between the LIF and CNTF levels in RA patients and normal individuals. We found positive correlation between IL-6 and sIL-6R concentrations and Ritchie''s index and a lack of such correlation with LIF and CNTF. IL-6 serum level correlated positively with the disease duration, but sIL-6R, LIF and CNTF did not. Serum sIL-6R/IL-6 ratio was significantly lower in RA patients than in healthy controls. In conclusion, an increase in the serum levels of IL-6 and sIL-6R, but not LIF and CNTF concentrations, may be useful markers for RA activity.     

Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor.

Soluble forms of the interleukin (IL)-2, IL-4 and IL-7 receptors which lack the transmembrane domain have been described. IL-6 is a growth factor important in the final differentiation of B-cells into plasma cells and in the pathogenesis of multiple myeloma. To determine whether the receptor for IL-6 may exist as a soluble molecule, RNA was analysed from the transformed B-cell lines U266, CESS and Daudi, from bone marrow from two myeloma patients, and from normal leukocytes. Using polymerase chain reaction, oligonucleotide primers which flank the transmembrane domain were selected to generate a 339 bp fragment. All samples produced equivalent amounts of the expected 339 bp fragment plus a smaller 245 bp fragment except Daudi which exhibited virtual absence of both. Sequence analysis of the smaller fragments from each of the five samples demonstrated the deletion of the entire transmembrane region from codons 356 (G-TG) to 387 (AG-G). The boundaries of this deletion were identical in all cases. Partial sequence analysis of the ligand-binding domain for U266 demonstrated identical sequences for the membrane-bound and soluble forms of the IL-6 receptor cDNAs. In summary, an mRNA which encodes a soluble form of the IL-6 receptor is expressed in both normal and myeloma cells.     

Relationship between circulating interleukin-10 (IL-10) with interleukin-6 (IL-6) type cytokines (IL-6, interleukin-11 (IL-11), oncostatin M (OSM)) and soluble interleukin-6 (IL-6) receptor (sIL-6R) in patients with multiple myeloma

We investigated the serum concentration of the interleukin-10 (IL-10), along with cytokines of interleukin-6 (IL-6) family (IL-6, IL-11 and oncostatin M - OSM), as well as soluble receptor for IL-6 (sIL-6R), in 121 patients with multiple myeloma (MM) and 28 healthy subjects. We studied the interactions between IL-10 and other cytokines, and the receptor. The correlation between IL-10 and some clinical and laboratory parameters associated with the disease activity were also analysed. The IL-10 was detectable in all patients with multiple myeloma and in all controls. The IL-10 concentration was significantly increased in myeloma patients compared with healthy persons (mean - 7.09 and 2.1 pg/ml, respectively) (p = 0.008). The level of IL-10 correlated positively with the advanced stage of disease estimated according to the Salmon and Durie classification (I versus III stage - p = 0.03). Higher values of IL-10 were found in patients with the light chain disease, hypercalcaemia, and correlated with the elevated concentrations of C-reactive protein (CRP). IL-6 was detected in 117 of the 121 patients and in all controls. The concentration of IL-6 was statistically increased in MM patients compared with control group (mean - 16.06 and 4.49 pg/ml, respectively) (p = 0.01). We found a positive correlation between IL-10 and IL-6 serum levels in MM patients. The relationship, expressed as Spearman's rank sum coefficient (rho = 0.249, p = 0.006) was significant. IL-11 was detected in 26 of the 121 MM patients and in 3 of the 28 healthy subjects at the mean concentration of 1.2 and 0.6 pg/ml respectively (p > 0.05). OSM was at detectable levels in 51 of the 121 patients and in only 4 of the 28 controls (mean - 3.84 and 0.1 pg/ml, p = 0. 002). The correlation between IL-10 and IL-11 levels in MM patients was not significant, but there was a strong statistical correlation between IL-10 and OSM concentrations (rho= 0.327, p = 0.0002). The serum concentration of sIL-6R was measurable in all patients and all controls (mean - 66.00 and 39.57 ng/ml respectively), but the difference between these groups was not significant. We found significant, positive correlation between the levels of IL-10 and sIL-6R (rho= 0.233, p = 0.01). In conclusion, we state that the serum concentrations of IL-10, IL-6, OSM and sIL-6R in MM patients may be a useful markers for the evaluation of the disease activity.     

Construction and characterization of a replication-deficient adenovirus expressing rat-soluble interleukin-6 receptor.

BACKGROUND: The pleiotropic cytokine interleukin-6 mediates its multiple effects at the cell level through a multimeric receptor consisting of a binding protein (gp80) and a signal transducer (gp130). A soluble form of gp80 (sIL-6R or gp55) is found released from the surface of cells and appears to possess interleukin-6 (IL-6) agonist activity. Increases in circulating levels of sIL-6R have been reported in different pathological conditions but the precise role of this protein in vivo remains unknown. MATERIALS AND METHODS: The cDNA encoding the extracellular domain of the rat IL-6R (sIL-6R) with an appropriate leader sequence has been cloned into the E1 region of an adenovirus vector under the control of the hCMV promoter (Ad5.sIL-6R). RESULTS: Infection of different human or rodent cell lines with Ad5.sIL-6R leads to extended production of recombinant sIL-6R protein into the culture media. The kinetics of transgene expression depends both on the cell type and the species. sIL-6R produced in this manner is biologically active as it confers responsiveness of human hepatoma cells (HepG2) to rat IL-6 stimulation. Adenovirus vectors have been shown to be highly effective for transient delivery of cytokines in vivo. Antibodies against recombinant rat soluble IL-6R were generated and an ELISA developed that allowed us to quantify sIL-6R concentrations. The sIL-6R expressing adenovirus vector has been instilled intratracheally into rats and induced an increase in lung sIL-6R concentration from Day 1 up to Day 10. We demonstrate the potency of our system to deliver in vivo or in vitro soluble cytokine receptors in a prolonged but transient manner.     

Purification and characterization of soluble human IL-6 receptor expressed in CHO cells

An immunosorbent assay system to detect genetically engineered IL-6 receptor (IL-6R) was established, whereby soluble IL-6 receptor (sIL-6R) was detected in the culture medium when sIL-6R cDNA was transfected into COS1 cells. A stably transformed Chinese hamster ovary (CHO) cell line constitutively expressing sIL-6R has been established. The recombinant sIL-6R was purified to homogeneity by sequential filtration and chromatography of the culture medium. The recombinant sIL-6R augmented the sensitivity of M1 cells to IL-6 in growth inhibition assay in a dose-dependent manner. Furthermore, a radioisotope immunosorbent assay (RIA) utilizing recombinant sIL-6R was established which could detect IL-6 in a quantity as low as 10 ng/ml.