Soluble interleukin-1 receptor accessory protein ameliorates collagen-induced arthritis by a different mode of action from that of interleukin-1 receptor antagonist.

OBJECTIVE: To discern the mode of interleukin-1 (IL-1) inhibition of soluble IL-1 receptor accessory protein (sIL-1RAcP) by comparison with IL-1 receptor antagonist (IL-1Ra) in arthritis. METHODS: Adenoviral vectors encoding either sIL-1RAcP or IL-1Ra were administered systemically before onset of collagen-induced arthritis in DBA/1 mice. Anti-bovine type II collagen IgG and IL-6 were quantified in serum. Proliferative response of splenic T cells was determined in the presence of sIL-1RAcP or IL-1Ra. The effect on IL-1 inhibition of recombinant sIL-1RAcP and IL-1Ra was further examined in vitro, using NF-kappaB luciferase reporter cell lines. Quantitative polymerase chain reaction was used to determine the relative messenger RNA expression of the IL-1 receptors. RESULTS: Adenoviral overexpression of both sIL-1RAcP and IL-1Ra resulted in amelioration of the collagen-induced arthritis. Both IL-1 antagonists reduced the circulating levels of antigen-specific IgG2a antibodies, but only IL-1Ra was able to inhibit lymphocyte proliferation. By using purified lymphocyte populations derived from NF-kappaB reporter mice, we showed that sIL-1RAcP inhibits IL-1-induced NF-kappaB activity in B cells but not T cells, whereas IL-1Ra inhibited IL-1 on both cell types. A study in a panel of NF-kappaB luciferase reporter cells showed that the sIL-1RAcP inhibits IL-1 signaling on cells expressing either low levels of membrane IL-1RAcP or high levels of IL-1RII. CONCLUSION: We show that the sIL-1RAcP ameliorated experimental arthritis without affecting T cell immunity, in contrast to IL-1Ra. Our results provide data in support of receptor competition by sIL-1RAcP as an explanation for the different mode of IL-1 antagonism in comparison with IL-1Ra.

Effectiveness of the soluble form of the interleukin-1 receptor accessory protein as an inhibitor of interleukin-1 in collagen-induced arthritis.

OBJECTIVE: To investigate whether the soluble form of interleukin-1 (IL-1) receptor accessory protein (sIL-1RAcP), whose physiologic function remains to be established, can serve as a specific inhibitor of IL-1 signaling in vitro, and to evaluate its applicability in collagen-induced arthritis (CIA). METHODS: Soluble IL-1RAcP was cloned from murine liver complementary DNA and expressed by the use of either an adenoviral vector (AdRGD) for sIL-1RAcP or a stable-transfected NIH3T3 fibroblast cell line. The ability of affinity-purified sIL-1RAcP to inhibit IL-1 signaling was tested on NF-kappaB luciferase reporter fibroblasts and quantified by luminometer. To investigate therapeutic efficacy, sIL-1RAcP was both locally (knee joint) and systemically overexpressed in collagen-immunized male DBA/1 mice. Severity of arthritis was monitored visually, and the pathologic process in the joint was examined histologically. Serum was obtained from mice to quantify IL-6 and anti-bovine type II collagen (BCII) antibody levels. RESULTS: Incubation of the NF-kappaB reporter fibroblast with purified sIL-1RAcP protein showed a marked reduction of IL-1-induced, but not tumor necrosis factor-induced, NF-kappaB activation. This showed a novel role for sIL-1RAcP as a specific inhibitor of IL-1 signaling. Local transplantation of sIL-1RAcP-producing NIH3T3 fibroblasts into the knee before onset of CIA had little or no effect on general disease severity in these mice. Histologic evaluation of the knee joints receiving sIL-1RAcP cell transplantation showed a marked reduction in both joint inflammation and bone and cartilage erosion. Local treatment with sIL-1RAcP had no profound effect on serum levels of IL-6 and anti-BCII antibodies, which is indicative of the ongoing presence of arthritis in distal joints. In contrast to local treatment, systemic treatment with the AdRGD for sIL-1RAcP markedly ameliorated CIA in all joints. CONCLUSION: In this study we demonstrated that sIL-1RAcP is a biologically active and innovative inhibitor of IL-1, and treatment of mice with sIL-1RAcP had a profound prophylactic effect on collagen-induced arthritis.

Interleukin-12 upregulates the IL-18Rbeta chain in BALB/c thymocytes.

Interleukin-18 (IL-18) is a proinflammatory cytokine that plays an important role in innate immunity and specific Th1 immune responses. During infection, IL-18 is produced by activated macrophages and primarily induces interferon-gamma (IFN-gamma) production in synergy with IL-12 in primed T cells and natural killer (NK) cells. IL-18 exerts its function by binding to a specific receptor complex (IL-18R) consisting of two subunits, the IL-18Ralpha chain and the IL-18Rbeta chain. Two individual mechanisms for synergism between IL-12 and IL-18 have been reported. First, IL-12-induced expression of the IL-18 receptor complex was described. Second, IL-18 and IL-12 together induced expression of target genes on a transcriptional level. In this study, we show that freshly isolated thymocytes from BALB/c mice constitutively expressed mRNA for the IL-18Ralpha chain but not for the IL-18Rbeta chain. These thymocytes were unresponsive to IL-18. Treatment with a combination of concanavalinA (ConA) plus IL-12 did not affect expression of the IL-18Ralpha chain but strongly induced expression for IL-18Rbeta mRNA, rendering these thymocytes highly responsive to IL-18 treatment. Thus, the synergistic effects of IL-12 and IL-18 on BALB/c mouse thymocytes resulted from the induction and regulation of the IL-18Rbeta chain and not the IL-18Ralpha chain.

Model of interaction of the IL-1 receptor accessory protein IL-1RAcP with the IL-1beta/IL-1R(I) complex.

A preliminary model has been calculated for the activating interaction of the interleukin 1 receptor (IL-1R) accessory protein IL-1RAcP with the ligand/receptor complex IL-1beta/IL-1R(I). First, IL-1RAcP was modeled on the crystal structure of IL-1R(I) bound to IL-1beta. Then, the IL-1RAcP model was docked using specific programs to the crystal structure of the IL-1beta/IL-1R(I) complex. Two types of models were predicted, with comparable probability. Experimental data obtained with the use of IL-1beta peptides and antibodies, and with mutated IL-1beta proteins, support the BACK model, in which IL-1RAcP establishes contacts with the back of IL-1R(I) wrapped around IL-1beta.

The membrane form of the type II IL-1 receptor accounts for inhibitory function.

IL-1 signaling is mediated by the type I IL-1R (IL-1RI). The nonsignaling type II receptor has a regulatory function, since it reduces IL-1 effects by scavenging free IL-1 molecules. This regulatory function has been demonstrated only for the soluble form, released from the membrane receptor by action of specific proteases, but is still ill-defined for the membrane receptor itself. To assess the function of membrane IL-1RII, a modified IL-1RII cDNA was constructed, in which the cleavable domain was replaced with the corresponding uncleavable sequence of the epidermal growth factor receptor. The human keratinocyte line HaCaT, which does not express wild-type IL-1RII (wtIL-1RII), was stably transfected with this modified cDNA (unconventionally cleavable IL-1RII (uIL-1RII)). Cells transfected with uIL-1RII expressed the membrane form of IL-1RII, but were unable to produce the 60-kDa soluble receptor. Upon analysis of IL-1 responsiveness, parental HaCaT and vector-transfected cells (E27), expressing IL-1RI and the accessory chain IL-1R accessory protein, were responsive to IL-1. Conversely, cells overexpressing wtIL-1RII (811) or uIL-1RII (9D4) showed comparable reduction in responsiveness to both IL-1alpha (bound by membrane and soluble receptors) and IL-1beta (recognized by the membrane receptor only), suggesting that the membrane form of the IL-1RII is mainly responsible for IL-1 inhibition. In contrast with wtIL-1RII, uIL-1RII did not interact with IL-1R accessory protein. Thus, the membrane form of IL-1RII possesses strong IL-1-inhibitory activity, independent of sequestration of the accessory protein and circumscribed to its ligand sink function.

The first two N-terminal immunoglobulin-like domains of soluble human IL-1 receptor type II are sufficient to bind and neutralize IL-1beta.

Two forms of soluble human type II interleukin (IL)-1 receptor (shIL-1RII) were generated, one consisting of the complete extracellular three immunoglobulin (Ig)-like domains and one containing only the first two N-terminal Ig-like domains. Both forms bound IL-1beta with a dissociation constant (K(d)) of 200 pM and neutralized IL-1beta in a bioassay. They did not bind or neutralize IL-1alpha. This demonstrates that the two Ig-like domains of shIL-1RII are sufficient to bind IL-1beta with an affinity comparable to full length shIL-1RII. This suggests that this short form of shIL-1RII contributes to the anti-inflammatory effect of soluble IL-1 receptors in vivo.

Endogenous serum levels of thrombopoietic cytokines in healthy whole-blood and platelet donors: implications for plateletpheresis.

Serum concentrations of the thrombopoiesis-enhancing cytokines thrombopoietin (TPO), erythropoietin (EPO), interleukin (IL)-6 and IL-11 were determined in 119 healthy whole-blood (WBD) and 101 platelet donors (PD) prior to donation. The 90% TPO reference interval in WBD of 64-867 pg/ml (median 163, 100% range 45-7572) was significantly higher than in PD of 56-524 (median 122, range 44-801, P = 0.004), whereas their platelet counts were lower (P < 0.001). EPO levels were not different (WBD 7.7 +/- 3.8, PD 8.0 +/- 4.9 IU/l), IL-6 and IL-11 were below the detection limit in >/=90% of cases (IL-6 < 3.2 pg/ml, IL-11 < 31.2 pg/ml). None of the cytokines correlated with platelet counts, other blood parameters, or in the PD group with the frequency of platelet donations within the last 6 months. We conclude that plateletpheresis does not lead to a lasting increase of thrombopoietic cytokines and provide reference data for potential platelet mobilization strategies with recombinant growth factors.

Effects of IL-1 receptor-associated kinase (IRAK) expression on IL-1 signaling are independent of its kinase activity.

Interleukin-1 (IL-1) stimulates the association of the IL-1 receptor-associated protein kinase (IRAK) with the heterodimer of IL-IRI and IL-IRAcP via the adapter protein MyD88. In the receptor complex IRAK becomes heavily phosphorylated and concomitantly activated. Here we show that overexpression of a kinase-inactive mutant of IRAK (K239S) inhibits neither IL-1-stimulated activation of the transcription factor NF-kappaB, nor that of the c-Jun N-terminal kinase nor IL-2 production in murine EL-4 cells, but enhances these effects in a manner comparable to wild type IRAK. This strongly suggests that the intrinsic kinase activity is not required for downstream signaling via IRAK.

The type II IL-1 receptor interacts with the IL-1 receptor accessory protein: a novel mechanism of regulation of IL-1 responsiveness.

IL-1 binds to two types of receptors on the cell membrane, of which only type I (IL-1RI) transduces signals in concert with the coreceptor IL-1 receptor accessory protein (IL-1RAcP) while type II (IL-1RII) allegedly functions solely as ligand sink and decoy receptor without participating in IL-1 signaling. To investigate the regulatory role of IL-1RII on IL-1 responsiveness, a chimeric receptor encompassing the extracellular and transmembrane portions of IL-1RII and the cytoplasmic signal-transducing domain of IL-1RI was transfected into two murine EL-4-derived sublines that do or do not express IL-1RAcP, respectively. The chimeric receptor was able to transduce the IL-1 signal and induce IL-2 production only in the cell line which expressed IL-1RAcP, suggesting effective interaction between the extracellular domains of IL-1RII and IL-1RAcP in the presence of IL-1. The physical association of ligated IL-1RII with IL-1RAcP was proven by crosslinking experiments with radio-iodinated IL-1 and subsequent immunoprecipitations in normal human B cells and in EL-4 D6/76 cells transiently cotransfected with IL-1RII and IL-1RAcP, respectively. Based on these findings, it is proposed that upon IL-1 binding IL-1RII can recruit IL-1RAcP into a nonfunctional trimeric complex and thus modulate IL-1 signaling by subtracting the coreceptor molecule from the signaling IL-1RI. In this novel mechanism of coreceptor competition, the ratio between IL-1RII and IL-1RI becomes the central factor in determining the IL-1 responsiveness of a cell and the availability of IL-1RAcP becomes limiting for effective IL-1 signaling.

Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4.

The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms.

Withdrawal of 2-mercaptoethanol induces apoptosis in a B-cell line via Fas upregulation.

Mouse lymphoid cell cultures are dependent on reducing agents in their culture medium to allow proliferation and survival of the cells. In the case of the mouse CD5+-pre-B cell line SPGM-1, withdrawal of 2-mercaptoethanol (2-ME) resulted in rapid inhibition of proliferation and subsequent cell death by apoptosis. The pathways leading to cell death by withdrawal of 2-ME or by incubation with ionomycin, a known inducer of apoptosis, were compared. Both kinds of stimulation resulted in apoptosis of the whole population, but cell death occurred with different kinetics. Only apoptosis induced by ionomycin was inhibited by coincubation with the phorbol ester PMA, while apoptosis induced by withdrawal of 2-ME was not. Overexpression of the human bcl-2 proto-oncogene in these cells delayed the death process induced by either method. SPGM-1xbcl-2 cells accumulated in the G0/G1 and G2/M cell cycle phases after removal of 2-ME from the medium, whereas treatment with ionomycin resulted in an arrest only in the G0/G1 transition. Interestingly, both stimuli induced the expression of the Fas receptor, but with different kinetics, while the Fas ligand (FasL) was expressed constitutively in SPGM-1 cells. These data demonstrate that withdrawal of 2-ME and incubation with ionomycin both induce rapid cell death by apoptosis, possibly mediated by an autocrine Fas/FasL loop. Although the initial pathways activated by the two forms of treatment must be different, they converge on a common level controlled by the anti-apoptotic gene product Bcl-2.

Effects of IL-1 receptor accessory protein on IL-1 binding.

Interleukin-1 (IL-1) is an important pro-inflammatory cytokine that exerts its diverse biological functions by binding to a receptor complex consisting of two transmembrane proteins, type I IL-1 receptor (IL-1RI) and IL-1 receptor accessory protein (IL-1RAcP). Both receptor chains are indispensable for IL-1 signaling, but only IL-1RI is able to bind the cytokine. The effects of IL-1RAcP on IL-1 binding are unclear. This study shows that although expression of IL-1RAcP does not alter the equilibrium dissociation constant of IL-1, it has an effect on the non-equilibrium binding modus, most likely due to changes in on/off rates. This defines an additional function of IL-1RAcP: it stabilizes the active IL-1 receptor complex.

The interleukin-1 receptor complex and interleukin-1 signal transduction.

The aim of this mini-review is to give an update on the current developments concerning the IL-1 plasma membrane receptor complex and the modes of signal transduction through the plasma membrane. The mechanisms of signal generation at the cytosolic side of the plasma membrane will be summarized. Possible coupling mechanisms to down-stream signalling elements will be discussed, which result in the complex activation of transcription factors responsible for the cellular responses to IL-1. It is not the aim of this review to extensively summarize IL-1 induced cytosolic kinase cascades and activation of transcription factors.

The interleukin-1 receptor accessory protein (IL-1RAcP) is essential for IL-1-induced activation of interleukin-1 receptor-associated kinase (IRAK) and stress-activated protein kinases (SAP kinases).

Interleukin-1 (IL-1) is a central mediator of the immune system involved in acute and chronic inflammatory responses. Although the sequences of two types of IL-1 receptors are known, the exact molecular events resulting in signal transduction and coupling to downstream signaling elements remain unclear. The recently cloned IL-1 receptor accessory protein (IL-1RAcP) has been suggested as a co-receptor molecule for IL-1RI, supported by the observation that its expression correlates to IL-1 responsiveness. We transfected the EL-4 subline D6/76 with IL-1RAcP cDNA. This cell line is an IL-1 non-responder expressing IL-1RI but lacking constitutive IL-1RAcP expression. The expression of IL-1RAcP in EL-4 D6/76 was sufficient to restore IL-1-induced activation of interleukin-1 receptor-associated kinase and of stress-activated protein kinases, translocation of the transcription factors NFkappaB and IL-1 NF to the nucleus, and induction of IL-2 mRNA synthesis. These results proved that IL-1RAcP is an indispensible molecule in the IL-1 receptor signal transduction complex, necessary to link events on the plasma membrane level to downstream signaling pathways, allowing IL-1-dependent activation of transcription factors and gene expression.

Overexpression of the bcl-2 oncogene in the mouse pre-B cell line SPGM-1 protects from apoptosis, but does not affect blocked B-lineage differentiation and lineage switch towards macrophage like cells.

The CD5(+) mouse pre-B cell line SPGM-1 is able to undergo lineage conversion towards a macrophage like cell type. Although one of the kappa-light chain alleles is rearranged in V-Jkappa4 configuration, no protein is expressed nor could the expression be induced. Infection with and expression of the human bcl-2 gene protected SPGM-1 cells from apoptosis, allowing the rearrangement of their second kappa-allele in V-Jkappa5 configuration. mRNA transcripts of the V-Jkappa regions were detected only in SPGM-1 x bcl-2 cells, but not in SPGM-1, while kappa-light chain protein was not found in any of the cell lines. The myeloid differentiation potential of SPGM-1 cells was not affected by overexpression of the human bcl-2 gene. Upon appropriate stimulation, SPGM-1xbcl-2 cells became enlarged, adhered to the plastic surfaces and lost their immunoglobulin mu heavy chain expression.

Co-expression of mRNA for type I and type II interleukin-1 receptors and the IL-1 receptor accessory protein correlates to IL-1 responsiveness.

Three cell surface molecules participate in Interleukin-1 (IL-1) binding and signal generation, the two distinct types of receptors (type I IL-1R and type II IL-1R) and the IL-1 receptor accessory protein (IL-1RAcP). Low surface expression hampers the detection of all three components on a protein level in most cell types, thus the highly sensitive RT-PCR was used to analyse the mRNA expression in a panel of 18 murine cell types of different hemopoietic lineages and fibroblasts. The transcription of both types of IL-1 receptors was detected in all cell lines tested. In most cell lines the IL-1RAcP was co-expressed with the IL-1 receptors, and only these lines responded to IL-1. However, in three cell lines no mRNA for the IL-1RAcP could be detected, and these cells did not respond to IL-1. These results suggest that the expression of the IL-1RAcP correlates with IL-1 responsiveness and they point to a pivotal role for the IL-1RAcP in IL-1 signal generation.