Dexamethasone up-regulates type II IL-1 receptor in mouse primary activated astrocytes

Brain astrocytes play a pivotal role in the brain response to inflammation. They express IL-1 receptors including the type I IL-1 receptor (IL-1RI) that transduces IL-1 signals in cooperation with the IL-1 receptor accessory protein (IL-1RAcP) and the type II IL-1 receptor (IL-1RII) that functions as a decoy receptor. As glucocorticoid receptors are expressed on astrocytes, we hypothesized that glucocorticoids regulate IL-1 receptors expression. IL-1beta-activated mouse primary astrocytes were treated with 10(-6) M dexamethasone, and IL-1 receptors were studied at the mRNA and protein levels. Using RT-PCR, IL-1RI and IL-1RII but not IL-1RAcP mRNAs were found to be up-regulated by dexamethasone in a time-dependent manner. Dexamethasone (Dex), but not progesterone, had no effect on IL-1RI but strongly increased IL-1RII mRNA expression. Binding studies revealed an increase in the number of IL-1RII binding sites under the effect of Dex, but no change in affinity. These findings support the concept that glucocorticoids have important regulatory effect on the response of astrocytes to IL-1.     

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.     

Reversal of autocrine and paracrine effects of interleukin 1 (IL-1) in human arthritis by type II IL-1 decoy receptor. Potential for pharmacological intervention

Interleukin 1 (IL-1), produced by both synovial cells and chondrocytes, plays a pivotal role in the pathogenesis of cartilage destruction in osteoarthritis (OA). We examined the specific expression and function of IL-1 receptor family-related genes in human joint tissues. Gene array analysis of human normal and OA-affected cartilage showed mRNA expression of IL-1 receptor accessory protein (IL-1RAcp) and IL-1 type I receptor (IL-1RI), but not IL-1 antagonist (IL-1ra) and IL-1 type II decoy receptor (IL-1RII). Similarly, human synovial and epithelial cells showed an absence of IL-1RII mRNA. Functional genomic analyses showed that soluble (s) IL-1RII, at picomolar concentrations, but not soluble TNF receptor:Fc, significantly inhibited IL-1beta-induced nitric oxide (NO) and/or prostaglandin E(2) production in chondrocytes, synovial and epithelial cells. In OA-affected cartilage, the IC(50) for inhibition of NO production by sIL-1RII was 2 log orders lower than that for sIL-1RI. Human chondrocytes that overexpressed IL-1RII were resistant to IL-1-induced IL-1beta mRNA accumulation and inhibition of proteoglycan synthesis. In osteoarthritis, deficient expression by chondrocytes of innate regulators or antagonists of IL-1 such as IL-1ra and IL-1RII (soluble or membrane form) may allow the catabolic effects of IL-1 to proceed unopposed. The sensitivity of IL-1 action to inhibition by sIL-1RII has therapeutic implications that could be directed toward correcting this unfavorable tissue(s) dependent imbalance.     

Selectivity release of the type II decoy IL-1 receptor

The type II IL-1 receptor (IL-1RII) is a non-signalling molecule which acts as a decoy target for IL-1. Various signals (e.g. chemoattractants and phorbol ester) induce metalloprotease-mediated rapid shedding of the IL-1RII. The present study was designed to compare the susceptibility to shedding of the decoy IL-1RII vs the signalling IL-1RI. HEK 293 cells and COS cells were transfected with cDNAs encoding the human IL-1RI, IL-1RII or both. Slow spontaneous release and rapid phorbol ester-induced shedding were only observed for the decoy IL-1RII. Similarly, OVCAR-3 cells, which express substantial amounts of both IL-1RI and IL-1RII, only released the IL-1RII. These results indicate that the IL-RII, but not the IL-1RI, is susceptible to proteolytic shedding, a finding consistent with the decoy function of this molecule.     

Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding

Proteolytic cleavage of the extracellular domain of the type II IL-1 decoy receptor (IL-1RII) generates soluble IL-1-binding proteins that prevent excessive bioactivity by binding free IL-1. In this study we report that an aminopeptidase, aminopeptidase regulator of TNFR1 shedding (ARTS-1), is required for IL-1RII shedding. Coimmunoprecipitation experiments demonstrate an association between endogenous membrane-associated ARTS-1 and a 47-kDa IL-1RII, consistent with ectodomain cleavage of the membrane-bound receptor. A direct correlation exists between ARTS-1 protein expression and IL-1RII shedding, as cell lines overexpressing ARTS-1 have increased IL-1RII shedding and decreased membrane-associated IL-1RII. Basal IL-1RII shedding is absent from ARTS-1 knockout cell lines, demonstrating that ARTS-1 is required for constitutive IL-1RII shedding. Similarly, PMA-mediated IL-1RII shedding is almost entirely ARTS-1-dependent. ARTS-1 expression also enhances ionomycin-induced IL-1RII shedding. ARTS-1 did not alter levels of membrane-associated IL-1RI or IL-1R antagonist release from ARTS-1 cell lines, which suggests that the ability of ARTS-1 to promote shedding of IL-1R family members may be specific for IL-1RII. Further, increased IL-1RII shedding by ARTS-1-overexpressing cells attenuates the biological activity of IL-1beta. We conclude that the ability of ARTS-1 to enhance IL-1RII shedding represents a new mechanism by which IL-1-induced cellular events can be modulated. As ARTS-1 also promotes the shedding of the structurally unrelated 55-kDa, type I TNF receptor and the IL-6R, we propose that ARTS-1 may play an important role in regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.     

IL-10 and IL-4 regulate type-I and type-II IL-1 receptors expression on IL-1β-activated mouse primary astrocytes

When activated by its ligand, the interleukin receptor type I (IL-1RI) transduces signals in cooperation with the IL-1 receptor accessory protein (IL-1RacP). In contrast, IL-1RII functions as a decoy receptor without participating in IL-1 signalling. Brain astrocytes are cellular targets of IL-1 and play a pivotal role in brain responses to inflammation. The regulation of IL-1 receptors on astrocytes by anti-inflammatory cytokines such as IL-4 and IL-10 has not been studied, despite its importance for understanding the way these cells respond to IL-1. Using RT-PCR, we first showed that the expression of IL-1RI and IL-1RII, but not IL-1RacP, mRNAs are up-regulated by IL-1 beta in a time-dependent manner. Using a radioligand binding technique, we then showed that astrocytes display an equivalent number of IL-1RI and IL-1RII. IL-1 beta decreases the number of IL-1RI binding sites, whereas it increases those of IL-1RII. IL-4 and IL-10 both up-regulate IL-1RII IL-1 beta-induced, but only IL-4 does so for IL-1RI. At the protein level, IL-4 and IL-10 dramatically reverse the ability of IL-1 beta to inhibit expression of IL-1RI but neither affects the ability of IL-1 beta to enhance the number of IL-1RII. Collectively, these results establish the existence of receptor cross-talk between pro- and anti-inflammatory cytokines on a critical type of cell that regulates inflammatory events in the brain.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

IL-1 beta scavenging by the type II IL-1 decoy receptor in human neutrophils

IL-1 elicits its cellular effects by binding a heterodimeric receptor consisting of IL-1RI and the accessory protein, IL-1RAcPr. In addition, it binds to IL-1RII, which lacking signaling function has been ascribed a decoy role. The fate of the ligand following interaction with the decoy receptor was examined in human polymorphonuclear cells (PMN), which express predominantly (>90%) IL-1RII. Incubation of PMN with IL-1beta results in a rapid decrease in cell surface-associated ligand accompanied by a concomitant increase in internalized IL-1 with 50-60% of IL-1beta located intracellularly within 1 h at 37 degrees C. The use of blocking Abs revealed that IL-1 internalization is mediated exclusively by the decoy receptor. The results of inhibitor analysis demonstrate that internalization requires ATP synthesis and involves clathrin-mediated endocytosis. Following removal of the ligand, the receptor was rapidly re-expressed on the cell surface. Cyclohexamide, a protein synthesis inhibitor, had no effect upon the process, suggesting that the re-expressed receptor was recycled. In addition, human keratinocytes stably transfected with IL-1RII (HaCAT 811) also internalized the IL-1RII with 43% cell surface receptor internalized after 90 min. Immunofluorescence microscopy revealed colocalization of the internalized receptor with wheat germ agglutinin-labeled internalized glycoproteins and early endosome Ag-1, a protein associated with the early endosome compartments, indicative of cellular uptake of IL-1RII by endocytosis. In contrast, little or no internalization was observed in other cells of immune origin. These results suggest that the decoy receptor IL-1RII can act as a scavenger of IL-1, representing a novel autoregulatory mechanism of the IL-1 system.     

The epistatic interrelationships of IL-1, IL-1 receptor antagonist,and the type I IL-1 receptor

Mice lacking the gene for the IL-1R antagonist (IL-1ra) show abnormal development and homeostasis as well as altered responses to infectious and inflammatory stimuli. A reduction in the level of IL-1 signaling, either by deletion of the receptor or increased expression of IL-1ra, does not affect development or homeostasis, but does alter immune responses. In this study we use genetic epistasis to investigate the interdependence of selected genes in the IL-1 family in the regulation of these developmental and immunological processes. Deletion of the gene encoding the type I IL-1R (IL-1RI) is epistatic to deletion of the IL-1ra gene. Therefore, all functions of IL-1ra depend upon the presence of a functional receptor; there is no other target. Similarly, overexpression of the mRNA encoding the secreted form of IL-1ra is epistatic to deletion of the receptor antagonist, leaving the role of the intracellular splice variants of IL-1ra unknown. The abnormal development of IL-1ra-deficient mice is probably due to chronic overstimulation of the proinflammatory pathway via IL-1, but a clear single pathological defect is not apparent. These results support the model that the only essential function of IL-1ra in both health and disease is competitive inhibition of the IL-1RI.     

Proteolysis induces increased binding affinity of the monocyte type II FcR for human IgG

Human monocytes express two types of IgG FcR, Fc gamma RI and Fc gamma RII. These can be assayed by using indicator E sensitized by human IgG (EA-human IgG) or mouse IgG1, (EA-mouse IgG1), respectively. On mouse macrophages, Fc gamma RI is sensitive to trypsin, whereas Fc gamma RII is trypsin resistant. We studied the effects of the proteolytic enzymes pronase and trypsin on human monocyte Fc gamma R. Neither enzyme caused a decrease in rosetting mediated by monocyte Fc gamma RI. Human Fc gamma RII is polymorphic, and monocytes interact either strongly or weakly with mouse IgG1. The interaction of low responder monocytes with mouse IgG1 was dramatically increased (to the level exhibited by high responder monocytes) by protease treatment. The effects of proteases on Fc gamma RII were investigated in more detail by using monocytes from which Fc gamma RI was selectively modulated by using immobilized immune complexes. Proteolysis of such modulated monocytes induced an increased interaction with EA-human IgG. Fc gamma RII appears to mediate this interaction. This conclusion is supported by the observation that after proteolysis, the Fc gamma RII-mediated binding of EA-mouse IgG1 becomes susceptible to inhibition by (monomeric) human IgG. To quantify the effect of proteolytic enzymes on Fc gamma RII, we performed binding studies with cell line K562, that expresses only Fc gamma RII. A significant increase in Ka of Fc gamma RII for dimeric human IgG complexes was observed when K562 cells were treated with protease. To elucidate the mechanism of this enhancement of Ka by proteolysis, we performed immunoprecipitation studies. Neither m.w., nor IEF pattern of Fc gamma RII were influenced by proteolysis. Moreover, the expression of Fc gamma RII was not affected by proteolysis as evidenced by immunofluorescence studies and Scatchard analysis, and neither were Fc gamma RI or Fc gamma RIII induced. We conclude that proteolysis increases the affinity of Fc gamma RII for human IgG, and speculate that such a proteolysis-induced change may also occur in vivo, e.g., at inflammatory sites.     

Augmentation of type I IL-1 receptor expression and IL-1 signaling by IL-6 and glucocorticoid in murine hepatocytes

IL-1 signal is transduced through type I receptor (IL-1RI). We have recently reported that LPS augments IL-1RI mRNA expression in the hepatocytes of mice in vivo, and the augmentation is mediated by the interaction of IL-1, IL-6, and glucocorticoid (GC). In this study, we examined whether IL-1RI mRNA expression level in the hepatocytes reflects those of cell surface molecule and IL-1 signaling. When primary cultured murine hepatocytes were treated with dexamethasone (Dex) or IL-6, these two reagents synergistically up-regulated IL-1RI mRNA expression in the cells. 125I-labeled IL-1 binding experiment showed that the level of binding was also up-regulated by the treatment with Dex and IL-6. Scatchard analysis revealed that the number of IL-1R increased. The increased binding of IL-1 was completely inhibited by an Ab against murine IL-1RI, indicating that Dex and IL-6 augmented the expression of cell surface IL-1RI molecule. When hepatocytes were pretreated with Dex and IL-6, the activation of IL-1R-associated kinase was augmented in response to IL-1, indicating that IL-1 signaling was also augmented. In addition, IL-1 treatment following administration of the combination of Dex and IL-6 into mice markedly increased the serum level of serum amyloid A. These results indicate that GC and IL-6 augment the expression of cell surface IL-1RI in hepatocytes, as well as IL-1 signaling and IL-1R-associated kinase activation, through up-regulation of IL-1RI mRNA level, which represents a novel regulatory network between IL-1, GC, and IL-6.     

Development of a novel noncompetitive antagonist of IL-1 receptor

IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed "101.10"), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC(50) approximately 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.     

Isolation, characterization, and expression of cDNA clones encoding the mouse Fc receptor for IgE (Fc epsilon RII)1

We have isolated cDNA clones encoding a mouse low affinity receptor for IgE (Fc epsilon RII) from a cDNA library of BALB/c splenic B cells activated with LPS and IL-4. The 2.2-kb cDNA clone encodes a 331 amino acid membrane glycoprotein that is homologous to human Fc epsilon RII (CD23) and a family of carbohydrate-binding proteins. COS7 cells transfected with the cDNA clones expressed a 45,000 m.w. protein that bound IgE and the anti-Fc epsilon RII mAb, B3B4. Fc epsilon RII mRNA was up-regulated in mouse B cells by culture with IL-4, but not in B cells cultured with IgE. Fc epsilon RII mRNA was detected in IgM+/IgD+ B cell lines, but not in pre-B cell lines or in B cell lines which have undergone differentiation to secrete Ig. The monocyte line P388D1, mast cell lines MC/9 and PT18, and peritoneal macrophages stimulated with IL-4 lacked detectable Fc epsilon RII mRNA, as did Thy-1.2+, CD4+, and CD8+ normal T cells and Thy-1.2+ T cells from Nippostrongylus brasiliensis-infected mice.     

IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4, and IL-10.

T cell-derived cytokines IFN-gamma and IL-4 have different regulatory effects on two functionally important molecules on human monocytes: MHC class II Ag and the Fc receptor for monomeric IgG, Fc gamma RI (CD64). MHC class II Ag, and Fc gamma RI are both upregulated in the presence of IFN-gamma. IL-4 induces MHC class II Ag expression but reduces Fc gamma RI expression. Recently, we showed that the cytokine IL-10 also affects MHC class II Ag expression. Here, we demonstrate that in contrast to the down-regulation of MHC class II Ag expression, IL-10 stimulates Fc gamma RI expression on human monocytes comparable to the levels of Fc gamma RI expression induced by IFN-gamma. The IL-10-induced Fc gamma RI expression is specific because anti-IL-10 antibodies completely reverse the IL-10-induced surface expression of Fc gamma RI and correlate with an enhanced capacity to lyse anti-D-coated human rhesus-positive erythrocytes. IL-10 fails to induce the expression of Fc gamma RII (CD32) and Fc gamma RIII (CD16). Furthermore, we demonstrate that IL-10 is able to prevent down-regulation in surface membrane expression of all three Fc gamma R that can be found when monocytes are cultured in the presence of IL-4. In contrast to IFN-gamma, IL-10 does not restore the reduced antibody-dependent cellular cytotoxicity (ADCC) activity of IL-4-cultured monocytes. Together, these results show that, similar to IFN-gamma, IL-10 is capable of enhancing Fc gamma R expression and ADCC activity, and that IFN-gamma, IL-4, and IL-10 have different regulatory effects on both monocyte Ag-presenting capacity and ADCC activity.     

Optimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and II

The biological effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the number of receptors on the cell. We determined the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labelled antibodies to the IL-1Rs, and the mean absolute number of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different numbers of molecules of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the number of IL-1RI expressed, and a significant decrease in the mean number of IL-1RII molecules per cell. Determination of the mean number of membrane-bound IL-1R molecules using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.