Soluble IL-15Rα Research Articles

Serum and muscle interleukin-15 levels decrease in aging mice: Correlation with declines in soluble interleukin-15 receptor alpha expression

Interleukin-15 (IL-15) is a skeletal muscle-derived cytokine with favorable effects on muscle mass and body composition. Modulation of IL-15 levels has been suggested as a treatment for sarcopenia and age-associated increases in adiposity. However, it is unclear whether IL-15 levels change during aging, as measurement of IL-15 at physiological concentrations in mice has been technically difficult, and translational regulation of IL-15 is complex. Moreover, the IL-15 receptor alpha (IL-15Rα) can comprise part of a membrane-associated receptor complex, or appear as a soluble form which stabilizes IL-15 and facilitates IL-15 secretion. Here, we report measurement of physiological levels of murine IL-15, and determine that muscle and serum IL-15 levels decline progressively with age. However, expression of IL-15 mRNA and membrane-associated subunits of the IL-15 receptor did not change with age in muscle. Expression of soluble IL-15Rα (sIL-15Rα) mRNA declined 5-fold with age, and serum IL-15 levels correlated highly with muscle sIL-15 mRNA expression, suggesting declines in sIL-15Rα expression lead to decreased circulating IL-15 levels during aging. These findings complement studies which described several single-nucleotide polymorphisms in the human IL-15Rα gene which impact muscularity and adiposity, and provide a technical basis for further investigation of IL-15 and the sIL-15Rα in determining body composition in aging mice, as a model for humans.

A Programmed Switch from IL-15- to IL-2-Dependent Activation in Human NK Cells

IL-2 and IL-15 differentially control the development, activation and proliferation of human NK cells, although they share common signal-transducing receptor chains CD122 and common gamma. To explore this issue, we analyzed in detail the kinetics of cytokine receptor expression, cytokine binding, and signaling responses in human NK cells treated with common gamma-chain family cytokines. We provide evidence for the sequential expression of IL-15Ralpha and IL-2Ralpha at the surface of cytokine-stimulated human NK cells, independent of the cytokine used for stimulation (IL-2, IL-15, or IL-7). Binding experiments confirmed the switch of high-affinity receptor from IL-15R to IL-2R between 18 and 48 h after stimulation. Consequently, phospho-STAT5 signaling responses to IL-15 were efficient in human NK cells pretreated with cytokines for 18 h, but were abolished at 48 h. Functional NK cell responses to IL-15, including IFN-gamma secretion and CD107a expression, followed a similar pattern, indicating the physiological relevance of the cytokine receptor switch. Importantly, IL-15 complexed to soluble IL-15Ralpha preserved the capacity to activate cytokine-stimulated human NK cells at 48 h, suggesting that human NK cells remained competent for IL-15 trans-presentation, while they had become refractory to free diffusible IL-15. These findings define a common cytokine receptor expression program, which increases human NK cell sensitivity to free IL-15 in early activation and redirects responses toward IL-2 and trans-presented IL-15 at later stages. Such a program may prevent excessive human NK cell activation by effectors of innate immunity and regulate the transition between the innate and adaptive stages of immune responses.

The Exon-3-Encoded Domain of IL-15Rα Contributes to IL-15 High-Affinity Binding and Is Crucial for the IL-15 Antagonistic Effect of Soluble IL-15Rα

We previously showed that a natural soluble form of interleukin-15 (IL-15) Rα corresponding to the full-length ectodomain of IL-15Rα behaved as a potent antagonist of IL-15 action through IL-15Rα/β/γ, whereas a recombinant soluble IL-15Rα sushi domain did not, but instead acted as an agonist of IL-15 action through IL-15Rβ/γ. In order to determine precisely the molecular basis governing these antagonistic versus agonistic actions, we compared the binding properties and biological effects of recombinant soluble IL-15Rα (sIL-15Rα) species containing the sushi domain and different remaining parts of the ectodomain. We first demonstrate that the exon-3-encoded domain and, more particularly, its N-terminal 13-amino-acid (aa) peptide are important, in addition to the adjacent exon-2-encoded sushi domain, for the stabilization of the high-affinity IL-15·IL-15Rα complex by slowing down its dissociation rate and by contributing to about 10–20% of the free energy of interaction. We next show that all sushi-containing sIL-15Rα are agonists on IL-15Rβ/γ, coordinately increasing IL-15 binding and IL-15-induced proliferation. Their agonistic potencies are proportional to their respective affinities for IL-15. We then show that the antagonistic effect of sIL-15Rα in the context of IL-15Rα/β/γ is due to the 13-aa peptide that creates a sterical constraint impeding the binding of the sIL-15Rα·IL-15 complex to the membrane-anchored IL-15Rα/β/γ. In the frame of the soluble IL-15Rα sushi domain–IL-15 fusion protein that contains the 13-aa peptide, this constraint is alleviated as a result of a conformational effect due to the covalent linking of the 13-aa peptide to the N-terminus of IL-15. The soluble IL-15Rα sushi domain–IL-15 fusion protein is therefore able to bind and activate both the IL-15Rβ/γ and the IL-15Rα/β/γ receptors.

CD8 T cell response to IL‐15/IL‐15Rα complex is MHC class I dependent

Recent findings illustrate that IL‐15 operates via a unique mechanism termed transpresentation. In this system, IL‐15 produced by one cell type is bound to IL‐15Rα expressed by the same cell and presented to apposing cells expressing the IL‐15Rβ/γC complex. We have recently shown that administering soluble IL‐15Rα complexed with IL‐15 can greatly enhance IL‐15 activity by increasing IL‐15 half‐life and forcing transpresentation of IL‐15. Furthermore, IL‐15/IL‐15Rα complex can cause the proliferation and maturation of naïve CD8 T cells. We now show that the CD8 T cell response to exogenous IL‐15/IL‐15Rα complex is MHC class I dependent. In the absence of MHC class I, naïve CD8 T cells scarcely proliferate in response to IL‐15/IL‐15Rα complex. In addition, providing specific peptide along with IL‐15/IL‐15Rα complex can enhance the proliferation of antigen‐specific CD8 T cells. The responding antigen‐specific CD8 T cells undergo an expansion and contraction phase, resulting in a larger frequency of remaining memory‐phenotype cells. These data suggest that IL‐15/IL‐15Rα complex has selective effects on antigen activated CD8 T cells. Our findings have important implications for directing IL‐15/IL‐15Rα complex based therapy to specific antigen targets, and illustrate the possible adjuvant uses of IL‐15/IL‐15Rα complex.NIH Grant AI‐051583 (to L.L.) and NCC Predoctoral Fellowship (to T.A.S.).

Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor alpha (IL-15Ralpha) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Ralpha, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Ralpha expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Ralpha also increased the number of CD44(high) memory cells with effector phenotype (CD44(high)CD62L-). Thus, mutual stabilization of IL-15 and IL-15Ralpha leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor alpha either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Ralpha in the same cell and suggest that the IL-15Ralpha is part of the active IL-15 cytokine rather than part of the receptor.

11 Response to Infliximab Treatment in Crohn’s Disease is Correlated with Increased Soluble IL-15Rα Serum Levels

11 Response to Infliximab Treatment in Crohn’s Disease is Correlated with Increased Soluble IL-15Rα Serum Levels

Membrane-Bound Interleukin (IL)-15 on Renal Tumor Cells Rescues Natural Killer Cells from IL-2 Starvation-Induced Apoptosis

Renal cell carcinoma primary tumors and lung metastases are infiltrated by activated natural killer (NK) cells. Interleukin (IL)-15, a major cytokine involved in cross-talk between accessory cells (dendritic cells and macrophages) and NK cells, is produced by epithelial renal cells. We show that renal cell carcinoma cells and normal renal cells express IL-15 mRNA and membrane-bound IL-15 (MbIL-15). These cells also express IL-15 receptor alpha (IL-15Ralpha). Silencing of IL-15Ralpha by specific small interfering RNA in renal cell carcinoma had no effect on MbIL-15 production, indicating that the cytokine is not cross-presented by IL-15Ralpha in renal cell carcinoma cells but anchored to the membrane. Furthermore, we show that MbIL-15 from renal cell carcinoma cells is functional and involved in rapid nuclear translocation of phosphorylated signal transducers and activators of transcription 3 in IL-2-starved NK cells. MbIL-15 on the target did not interfere with resting NK cell activation and target cell cytolysis but rescued NK cells from IL-2 starvation-induced apoptosis through contact-dependent interaction. Masking of MbIL-15 with soluble IL-15Ralpha molecules restored NK cell apoptosis. These findings suggest that IL-15 produced by renal tumor cells is involved in the maintenance of active NK cells at the tumor site.

Soluble Interleukin (IL)-15Rα Is Generated by Alternative Splicing or Proteolytic Cleavage and Forms Functional Complexes with IL-15

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.

Augmenting the biological activity of IL-15 with soluble IL-15Ralpha (95.21)

Abstract IL-15 is thought to be normally presented in vivo as a cell-associated cytokine bound to IL-15Rα. We show here that the biological activity of soluble IL-15 is much improved after interaction with recombinant soluble IL-15Rα; after injection, soluble IL-15/IL-15Rα complexes rapidly induce strong and selective expansion of memory CD8+ cells and natural killer cells. Surprisingly, we also find that sIL-15/IL-15Rα complexes can drive conversion of naive T cells to memory T cells in vivo, without administration of antigen. This conversion is associated with an acquisition of effector function. The enhancing effect of IL-15Rα binding may explain why IL-15 normally functions as a cell-associated cytokine. Our results suggest the biological activity of IL-15 as a therapeutic reagent could be considerably enhanced by administration of preformed soluble IL-15/IL-15Rα complexes.

Exchange Protein Activated by Cyclic AMP (Epac)-Mediated Induction of Suppressor of Cytokine Signaling 3 (SOCS-3) in Vascular Endothelial Cells

Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs) by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling complex (soluble IL-6Ralpha/IL-6). This was associated with the induction of suppressor of cytokine signaling 3 (SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3 induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP may mediate its potent anti-inflammatory effects.

Converting IL-15 to a superagonist by binding to soluble IL-15Rα

IL-15 is normally presented in vivo as a cell-associated cytokine bound to IL-15Ralpha. We show here that the biological activity of soluble IL-15 is much improved after interaction with recombinant soluble IL-15Ralpha; after injection, soluble IL-15/IL-15Ralpha complexes rapidly induce strong and selective expansion of memory-phenotype CD8(+) cells and natural killer cells. These findings imply that binding of IL-15Ralpha to IL-15 may create a conformational change that potentiates IL-15 recognition by the betagamma(c) receptor on T cells. The enhancing effect of IL-15Ralpha binding may explain why IL-15 normally functions as a cell-associated cytokine. Significantly, the results with IL-2, a soluble cytokine, are quite different; thus, IL-2 function is markedly inhibited by binding to soluble IL-2Ralpha.

High-dose glucocorticoids increase serum levels of soluble IL-6 receptor α and its ratio to soluble gp130: an additional mechanism for early increased bone resorption

Glucocorticoids (GCs) at pharmacological doses stimulate bone resorption. Mechanisms of this action are unclear. The osteoclastogenic cytokine interleukin (IL)-6 acts through an oligomeric receptor consisting of two subunits, gp80 (or IL-6 receptor alpha, IL-6Ralpha) and gp130; both exist in membrane and soluble forms. Soluble IL-6Ralpha (sIL-6Ralpha) enhances, while sgp130 inhibits IL-6 signalling. In vitro, GCs enhance many effects of IL-6 by up-regulation of IL-6Ralpha. The aim of the present study was to assess acute changes of IL-6 system in the peripheral blood of patients given high-dose GCs. Serum levels of IL-6, sIL-6Ralpha, sgp130 and bone turnover markers were assessed before and each day during treatment in 24 multiple sclerosis (MS) patients undergoing high-dose (prednisolone, 15 mg/kg per day), short-term (3 to 5 days) intravenous GC therapy for relapse at the Regional Multiple Sclerosis Centre. An immediate and marked fall of osteocalcin and an early increase of C-terminal telopeptide of type I collagen were already noticed at day 2 (P < 0.001 and P < 0.02, respectively); both became more apparent in the subsequent days. IL-6 was always below or near the detection limit of our ELISA. sgp130 showed a slight increase. sIL-6Ralpha significantly increased, peaking at day 4 (P < 0.01). However, inter-individual variability of response was noticed. Four patients showed a slight decrease, while no change was observed in one patient and an increase was noticed in the remaining nineteen (maximum change ranging from +10% to +67% with respect to baseline). In these patients, a significant increase of sIL-6Ralpha/sgp130 ratio was apparent. No correlation was found between bone turnover markers and any measured component of the IL-6 system. sIL-6Ralpha and sIL-6Ralpha/sgp130 ratio are precociously increased in the peripheral blood of the vast majority of patients given high-dose, intravenous GCs. The increase of systemically available sIL-6Ralpha conceivably results in the enhancement of IL-6-dependent osteoclastogenesis. The role of such a mechanism in the bone loss observed in inflammatory and immune-mediated diseases (where abundancy of IL-6 in the bone microenvironment is expected) requires further investigation.

The Structure of the Interleukin-15α Receptor and Its Implications for Ligand Binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.

Membrane-bound and soluble IL-15/IL-15Rα complexes display differential signaling and functions on human hematopoietic progenitors

AbstractMembrane-bound and soluble interleukin-15 (IL-15)/IL-15 receptor α (Rα) complexes trigger differential transcription factor activation and functions on human hematopoietic progenitors. Indeed, human spleen myofibroblasts (SMFs) are characterized by a novel mechanism of IL-15 trans-presentation (SMFmb [membrane-bound]-IL-15), based on the association of an endogenous IL-15/IL-15Rα complex with the IL-15Rβγc chains. SMFmb-IL-15 (1) induces lineage-specific signaling pathways that differ from those controlled by soluble IL-15 in unprimed and committed normal progenitors; (2) triggers survival and proliferation of leukemic progenitors expressing low-affinity IL-15R (M07Sb cells); (3) causes only an antiapoptotic effect on leukemic cells expressing high-affinity receptors (TF1β cells). This behavior is likely due to the IL-15Rα chain present on these cells that interact with the SMFmb-IL-15, inhibiting signal transducer and transcriptional activator 5 (STAT5) activation. On the other hand, the soluble IL-15/IL-15Rα complex (hyper IL-15) displays a dominant pattern of action, activating only those cells expressing low-affinity IL-15R (IL-15Rβγc). Thus, hyper IL-15 induces antiapoptotic effects on M075b cells and the up-regulation of STAT6 activation on adult peripheral blood (PB) pre-natural killer (NK) committed progenitors. The latter effect using 100-fold concentrations of recombinant (r)-IL-15. In conclusion, SMFmb-IL-15 and soluble IL-15Rα/IL-15 complexes seem to play a pivotal role in the control of the survival, proliferation and differentiation of both normal and leukemic circulating progenitors, highlighting new functions of IL-15 and of IL-15Rα.

Bacterial Induction of TNF-α Converting Enzyme Expression and IL-6 Receptor α Shedding Regulates Airway Inflammatory Signaling

Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-alpha converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Ralpha on the apical surface of airway cells, and by 24 h, soluble IL-6Ralpha accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Ralpha were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.

Blocking IL-15 Prevents the Induction of Allergen-Specific T Cells and Allergic Inflammation In Vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.

Regulating innate immune response cytokines in synovitis

Several studies have provided powerful proof of concept for the notion that single cytokine targeting can manifest effective immune suppression even in the context of complex networks of cytokine activities. We have studied the biology and functional expression of the innate response cytokine IL-15 and, more recently, members of the IL-1/IL-1 receptor superfamily in inflammatory synovitis. These cytokines are characterised by early upregulation during inflammatory responses and characteristic functional synergy and cross-regulation in the intact immune response. IL-15 is an IL-2-like cytokine of 15 kDa that possesses broad proinflammatory activities via its heterotrimeric receptor (IL-15Rα/IL-15/2Rβ/common γ), including promoting T-cell maturation, activation, and cytokine production, natural killer cell maturation and effector function, neutrophil activation, dendritic cell activation, endothelial cell rescue from apoptosis, macrophage activation and B-cell activation and isotype switching. IL-15 is expressed at mRNA and protein levels in several inflammatory arthritides including rheumatoid arthritis. When targeted in rodent models of inflammation using either soluble IL-15Rα or modified IL-15 mutant fusion proteins, amelioration of onset and of existing arthritis is observed. A recent phase I study in which a fully human IgG1 monoclonal antibody (HuMax-IL15; Genmab, Copenhagen, Denmark) was administered to 30 patients with active rheumatoid arthritis indicated that IL-15 blockade was well tolerated up to 8 weeks, with early indications of efficacy detected. We have now observed close interactions between IL-15 and IL-1 receptor superfamily signalling in promoting T-cell activation. Specifically, IL-15 synergises with the TLR2 ligand BLP to promote memory T-cell activation (co-stimulatory) and also via a cell membrane-dependent pathway to promote adjacent macrophage activation and tumour necrosis factor release. We have also observed that a further member of the IL-1 receptor superfamily, namely ST2, is expressed in synovial fibroblasts. Membrane-bound ST2 negatively regulates type I IL-1 receptor and TLR4 signalling by sequestrating the adaptors MyD88 and Mal. Intriguingly, soluble ST2 suppresses development of collagen-induced arthritis in DBA/1 mice if given prophylactically, and also established collagen-induced arthritis if administered after disease onset. Together these data suggest that complex interactions between the common γ-receptor chain and IL-1 receptor superfamily signalling cytokines are of importance in developing synovial inflammatory responses and that elucidation of these pathways offers therapeutic utility.

On-column refolding and characterization of soluble human interleukin-15 receptor α-chain produced in Escherichia coli

Interleukin-15 receptor α-chain (IL-15Rα) is a member of the new cytokine receptor family, which possesses the sushi domain. To investigate the biochemical and biophysical characteristics of soluble human IL-15Rα (shIL-15Rα), shIL-15Rα was recombinantly expressed in Escherichia coli. The shIL-15Rα containing a six histidine-tag was expressed as inclusion bodies, which were solubilized with urea, immobilized on a Ni–nitrilotriacetic acid column, and refolded by a decreasing gradient of urea concentration. The refolded shIL-15Rα exhibited a highly flexible structure, neutralized human interleukin-15-induced cell proliferation effectively, and bound to its ligand with the same affinity as human IL-15Rα on the cell surface, as demonstrated by circular dichroism, a cell proliferation assay, and surface plasmon resonance, respectively. Thus, we succeeded in refolding shIL-15Rα to an active form on an affinity column.

The IL-6-soluble IL-6Ralpha autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation: an experimental model involving thrombin.

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1alphabeta and TNF-alpha. Addition of physiological concentrations of exogenous soluble IL-6Ralpha (sIL-6Ralpha) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Ralpha blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Ralpha/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Ralpha because IL-8 induced IL-6Ralpha shedding from the neutrophil membranes and increased in parallel sIL-6Ralpha concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Ralpha from neutrophil membranes. sIL-6Ralpha may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.