Induction of an interleukin-1 receptor (IL-1R) on monocytic cells. Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA

Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.     

Presence of high affinity receptor for interleukin-1 (IL-1) on cultured porcine thyroid cells

Using 125I-interleukin-1 beta (125I-IL-1 beta) as a ligand, a specific receptor of high affinity dissociation constant (1.1 +/- 0.2 x 10(-10) M) with binding sites (350 +/- 40/cell) for interleukin-1 beta (IL-1 beta) has been demonstrated on cultured porcine thyroid cells. IL-1 alpha almost equally cross-reacted with the receptor (Kd = 1.2 +/- 0.3 x 10(-10) M and 350 +/- 50 binding sites/cell). TSH, IL-2 and other peptide hormones did not inhibit the binding of 125I-IL-1 beta to thyroid cells. Crosslinking study revealed a major band (approximately 95 kD) with a corrected molecular mass of approximately 78 kD. Moreover, both IL-1 beta and IL-1 alpha stimulated prostaglandin E2 production of cultured porcine thyroid cells, although the potency of IL-1 alpha was slightly greater than that of IL-1 beta. These results suggest that IL-1 may be involved in the regulation of thyroid cell function.     

Soluble interleukin-2 receptor (S IL-2R) in renal and pancreatic transplantation.

The monitoring of plasma soluble interleukin-2 receptor (S IL-2R) concentrations has been proposed in organ transplantation, especially to detect early manifestations of rejection. In organ transplantation, immune activation occurs in various circumstances such as rejections and infections. We performed S IL-2R determination 3 times a week in the sera of 106 patients undergoing kidney and/or pancreas transplantation. In kidney transplantation, S IL-2R was increased before the transplant. It also increased under prophylactic and especially under curative anti-rejection OKT3 or ATG therapy. In 90% cases, S IL-2R increased 2 to 4 days before creatininemia rise. In the other 10% cases, no correlation could be found with any clinical status modification. S IL-2R concentrations never increased in isolated acute tubular necrosis or in cyclosporine A (CsA) nephrotoxicity. In pancreas transplantation, the correlation between S IL-2R concentrations and possible pancreas rejection, was very poor. During cytomegalovirus (CMV) infection, only 50% patients with clinical CMV manifestations had high concentrations of S IL-2R. During Dihydroxy Propoxy Methyl Guanine (DPHG = Ganciclovir) treatment, S IL-2R still increased at the beginning, then it decreased progressively when therapy was efficient on CMV infection. The monitoring of S IL-2R concentrations may be useful in the weeks following organ transplantation provided that results are interpreted in the context of clinical and other laboratory findings, particularly with the renal function status and creatininemia.     

Imbalance production between interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1Ra) in bronchial asthma

Genes of the IL-1 family encode three different peptides, IL-1alpha, IL-1beta, and IL-1Ra, respectively. IL-1 operates through IL-1RI, and is involved in airway inflammation in asthmatic subjects, whereas IL-1Ra appears to be a specific competitive inhibitor of IL-1. All genes are on chromosome 2q12-21 where genomewide searches have identified linkage for asthma. To test whether variants of IL-1 relate to asthma, we conducted a genetic association study in a Japanese population. We show that the A2 allele of IL1RN (encoding IL-1Ra) associates with nonatopic asthma [OR = 5.71, 95% CI: 1.63-19. 8, Pc = 0.007]. Both atopic and nonatopic asthmatics with the A2 allele had significantly lower serum IL-1Ra levels in both types of asthmatics. Peripheral blood cells from asthmatics with A2 alleles, however, produced as much IL-1 as did those with A1 homozygotes. Since Th1 and Th2 cytokines differentially regulate the ratio between IL-1beta and IL-1Ra, these findings suggest that dysregulation of IL-1beta/IL-1Ra, probably due to interaction between epithelium and immuno-competent cells in the airway, is important in asthma inflammation.     

Mechanical stretch induced interleukin-18 (IL-18) expression through Angiotensin subtype 1 receptor (AT1R) and endothelin-1 in cardiomyocytes

Interleukin-18 (IL-18) is a proinflammatory cytokine with multiple biological functions. We and others have demonstrated that an increased level of circulating IL-18 is one of the risk factors for cardiovascular diseases. Endothelin-1 (ET-1) has been reported to be a potent hypertrophy-promoting factor through RhoA and Rho-Kinase. Mechanical stretch induces a hypertrophic response, partly through the production of ET-1 through Endothelin A receptor (ETAR). Moreover, it has also been reported that mechanical stretch induces cardiac hypertrophy through Angiotensin subtype 1 receptor (AT1R). However, the mechanism by which the IL-18 gene expression is regulated in cardiomyocytes has not yet been fully understood. This study was designed to elucidate the functional significance of IL-18 gene expression in response to mechanical stretch. Neonatal rat cardiomyocytes cultured on silicone dishes were subjected to stretch. The moderate 20% mechanical stretch resulted in the elevation of IL-18 expression in a time-dependent manner with the maximal level achieved 36 hours after the stretch. Olmesartan, AT1R antagonist inhibited stretch-induced IL-18 expression. ETAR blockade BQ123 inhibited stretch-induced IL-18 expression. However, the Endothelin B receptor (ETBR) receptor blockade BQ788 did not inhibit this reaction. ET-1 induced IL-18 expression, with a peak induction after 4 hours of incubation. These results might suggest that stretch stimulation of cardiomyocytes induced ET-1 and, subsequently, ET-1 up-regulated the IL-18 expression. Furthermore, Fasudil, a Rho-Kinase inhibitor, and Simvastatin, a HMG-CoA reductase inhibitor, led to a significant reduction in mechanical stretch-induced IL-18 expression. These results indicated, for the first time, that IL-18 expression is induced by mechanical stretch in cardiomyocytes via the ETAR, AT1R, and the Rho/Rho-K pathways. The induction of IL-18 from cardiomyocytes by mechanical stress might cause the deterioration of cardiac functions in autocrine and paracrine fashion. The inhibition of IL-18 expression induced by mechanical stress might be one of the mechanisms that account for the beneficial cardiovascular effects of AT1R antagonist, ETAR blockade, Statin, and Rho-Kinase inhibitor.     

Expression of interleukin-2 receptor (IL-2R) in feline peripheral blood lymphocytes

A monoclonal anti-interleukin-2 receptor (IL-2R) antibody has been identified as a putative antibody against the human IL-2R. In the present study, anti-Tac antibody CD-25 was used to determine cell expressing IL-2 receptor in feline peripheral blood lymphocytes by means of direct immunofluorescence tests and complement-mediated lymphocytotoxicity tests. With complement-mediated lymphocytotoxicity, approximately 18% of feline peripheral blood lymphocytes expressed the receptors. By the direct immunofluorescence test, we found approximately 22% of IL-2R positive cells in lymphocytes of feline peripheral-blood.     

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.     

Amino acids conserved in interleukin-1 receptors (IL-1Rs) and the Drosophila toll protein are essential for IL-1R signal transduction.

The cytoplasmic domain of the human T cell-type interleukin-1 receptor (hIL-1R) is not involved in the binding, internalization, or nuclear localization of interleukin-1 (IL-1), but is essential for signal transduction. We have previously localized a 50-amino acid region (residues 477-527) critical for IL-1-mediated activation of the interleukin-2 promoter in T cells. This region displays a striking degree of amino acid conservation in human, murine, and chicken IL-1Rs. Here we report the results of a site-directed mutational analysis of the cytoplasmic domain of the hIL-1R. We have introduced single-amino acid substitutions at positions conserved in all three receptors and at nonconserved positions and identified key amino acids for IL-1R function in signal transduction. Three basic (Arg431, Lys515, and Arg518) and 3 aromatic (Phe513, Trp514, and Tyr519) amino acids that are conserved in human, murine, and chicken IL-1Rs could not be replaced without abolishing IL-1R-mediated signal transduction. A substitution at another conserved position (Pro521) reduces significantly the ability of the IL-1R to transmit the IL-1 signal. Nonconserved residues could be replaced without affecting signal transduction. The cytoplasmic domain of the IL-1R is related to that of the Drosophila Toll protein, with a 26% identity and a 43% similarity in amino acid sequence. The amino acids shown to be essential for IL-1R function are conserved in the Toll protein. Our experimental data indicate that the amino acid sequence similarity between the IL-1R and the Drosophila toll protein reflects a functional homology between the two proteins.     

The interleukin-1 receptor in Raji human B-lymphoma cells. Molecular characterization and evidence for receptor-mediated activation of gene expression.

In a previous paper [Horuk, Huang, Covington & Newton (1987) J. Biol. Chem. 262, 16275-16278] we reported that there were fundamental differences in the biochemical properties of the interleukin-1 (IL-1) receptor between Raji and EL4 cell lines. In the present study we have investigated the basis for these differences. Kinetic studies measuring the on and off rates of IL-1 receptor binding revealed that the low-affinity IL-1-binding sites observed in Raji cells, compared with EL4 cells, result from a combination of a lower association rate and a higher dissociation rate in the Raji cells. The turnover of the Raji IL-1 receptor, measured by inhibiting protein synthesis with cycloheximide, was much faster than that of the EL4 IL-1 receptor, with a half-time of 2 h as against 5 h. Treatment of 125I-IL-1-labelled IL-1 receptors in Raji and EL4 cells with neuraminidase decreased their molecular mass by approx. 2-5 kDa as assessed by SDS/polyacrylamide-gel electrophoresis (PAGE). The covalently labelled IL-1 receptors in both cell types were sensitive to treatment with endoglycosidase F, which decreased their molecular mass on SDS/PAGE by 12-13 kDa. Incubation of Raji cells with maximally stimulating doses of IL-1 resulted in an increase in the nascent RNA levels of several genes, including the IL-2 receptor and the proto-oncogenes c-Ha-ras and c-myc.     

A novel splice variant of interleukin-1 receptor (IL-1R)-associated kinase 1 plays a negative regulatory role in Toll/IL-1R-induced inflammatory signaling

The interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) is a member of the IRAK kinase family that plays a pivotal role in the Toll/IL-1 receptor (TIR) family signaling cascade. We have identified a novel splice variant, IRAK1c, which lacks a region encoded by exon 11 of the IRAK1 gene. IRAK1c expression was confirmed by both RNA and protein detection. Although both IRAK1 and IRAK1c are expressed in most tissues tested, IRAK1c is the predominant form of IRAK1 expressed in the brain. Unlike IRAK1, IRAK1c lacks kinase activity and cannot be phosphorylated by IRAK4. However, IRAK1c retains the ability to strongly interact with IRAK2, MyD88, Tollip, and TRAF6. Overexpression of IRAK1c suppressed NF-kappaB activation and blocked IL-1beta-induced IL-6 as well as lipopolysaccharide- and CpG-induced tumor necrosis factor alpha production in multiple cellular systems. Mechanistically, we provide evidence that IRAK1c functions as a dominant negative by failing to be phosphorylated by IRAK4, thus remaining associated with Tollip and blocking NF-kappaB activation. The presence of a regulated, alternative splice variant of IRAK1 that functions as a kinase-dead, dominant-negative protein adds further complexity to the variety of mechanisms that regulate TIR signaling and the subsequent inflammatory response.     

Constitutive and interleukin-1 (IL-1)-inducible factors interact with the IL-1-responsive element in the IL-6 gene.

The interleukin-6 (IL-6) promoter is rapidly and transiently activated with other cytokines, including IL-1, tumor necrosis factor, and platelet-derived growth factor, as well as phorbol esters and agents that increase intracellular cyclic AMP. In this study, we have investigated cis-acting regulatory elements and trans-acting factors responsible for IL-1-induced IL-6 gene expression. Studies on the 5' deletion mutants of the human IL-6 gene suggested that the IL-1-responsive element was mapped within the IL-6 promoter region (-180 to -123) which was homologous to the c-fos serum-responsive enhancer element. Gel retardation assay identified two types of nuclear factors that bound to this region, one constitutive and the other inducible. These two factors recognized a 14-base-pair (bp) palindromic sequence, ACATTGCACAATCT. Furthermore, three copies of this 14-bp palindrome conferred IL-1 responsiveness to the basal enhancerless IL-6 promoter, indicating that a 14-bp-dyad symmetry sequence was an IL-1-responsive element in the IL-6 gene.     

Conformational changes mediate interleukin-10 receptor 2 (IL-10R2) binding to IL-10 and assembly of the signaling complex

Interleukin-10 receptor 2 (IL-10R2) is a critical component of the IL-10.IL-10R1.IL-10R2 complex which regulates IL-10-mediated immunomodulatory responses. The ternary IL-10 signaling complex is assembled in a sequential order with the IL-10.IL-10R1 interaction occurring first followed by engagement of the IL-10R2 chain. In this study we map the IL-10R2 binding site on IL-10 using surface plasmon resonance and cell-based assays. Critical IL-10R2 binding residues are located in helix A adjacent to the previously identified IL-10R1 recognition surface. Interestingly, IL-10R2 binding residues located in the N-terminal end of helix A exhibit large structural differences between unbound cIL-10 and cIL-10.IL-10R1 crystal structures. This suggests IL-10R1-induced conformational changes regulate IL-10R2 binding and assembly of the ternary IL-10.IL-10R1.IL-10R2 complex. The basic mechanistic features of the assembly process are likely shared by six additional class-2 cytokines (viral IL-10s, IL-22, IL-26, IL-28A, IL28B, and IL-29) to promote IL-10R2 binding to six additional receptor complexes. These studies highlight the importance of structure in regulating low affinity protein-protein interactions and IL-10 signal transduction.     

Effect of interleukin-1 receptor antagonist (IL-1RA) on histamine and serotonin release by rat basophilic leukemia cells (RBL-2H3) and peritoneal mast cells

Recently, it has been appreciated that cultured mast cells are significant sources of cytokines. However, the role of interkeukin-1 (IL-1) on mast cells and/or basophil degranulation is still unclear. In this report we provide evidence that rat basophilic leukemia cells (RBLC) cultured with a natural inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1RA) (500 ng/ml) for 48 h, strongly inhibited the spontaneous release of serotonin (5HT) and histamine (from 22.50 to 43.49%), compared to untreated cells (control). When IL-1RA-treated and untreated RBLC were stimulated with a secretagogue (anti-IgE), no difference was found in the percent of 5HT and histamine release. Moreover, in another set of experiments using rat peritoneal mast cells (RPMC) treated and untreated with IL-1RA, we found that IL-1RA did not affect the release of 5HT or histamine, even when the secretagogue anti-IgE or compound 48/80 (C48/80) were used. The present studies describe an additional biological activity of IL-1RA, inhibiting histamine and 5HT release from RBLC cultures.Abbreviations IL-1 interleukin-1 - RA receptor antagonist - 5HT serotonin - RBLC rat basophilic leukemia cells - RPMC rat peritoneal mast cells - IgE immunoglobulin E - Fc immunoglobulin E receptor - CPM counts per minute - BSA bovine serum albumin - C48/80 compound 48/80 - TNF tumor necrosis factor     

Cloning and characterization of the rainbow trout (Oncorhynchus mykiss) type II interleukin-1 receptor cDNA.

A homologue of mammalian type II interleukin-1 receptor (IL-1RII) was isolated from a rainbow trout cDNA library by differential hybridization using a suppression subtractive hybridization generated probe enriched for sequences upregulated after immune stimulation. The trout cDNA has an ORF encoding 441 amino acids, and represents the first piscine IL-1 receptor described. The predicted amino-acid sequence has 29 and 26% identity with human and mouse IL-1RII, respectively. The trout IL-1 receptor has a domain organization similar to that of mammalian type II receptor, with a short cytoplasmic tail of 24 amino acids. These results suggest that type II receptor is also present in lower vertebrates, and therefore the duplication of an ancestral gene that generated type I and type II IL-1 receptors occurred prior to the time mammals emerged.