Characterization of a low molecular weight isoform of IL-1 receptor antagonist.

IL-1R antagonist (IL-1Ra) exists in two well-characterized forms, 17-kDa secretory IL-1Ra (sIL-1Ra) and 18-kDa intracellular IL-1Ra (icIL-1Ra), that arise by alternative transcription of the same IL-1Ra gene. A third, lower molecular mass form (approximately 16 kDa) was detected by immunoblot within lysates of a variety of cells, including human monocytes and myelomonocytic cell lines. The 16-kDa isoform was designated icIL-1RaII, and the previously established 18-kDa form was designated icIL-1RaI. Intracellular IL-1RaII bound type I IL-1R up to fivefold less avidly than did sIL-1Ra and icIL-1RaI. Microsequencing of cyanogen bromide fragments of purified icIL-1RaII provided evidence consistent with initiation of protein translation at the second start site in either IL-1Ra mRNA. The results of site-directed mutation experiments established that icIL-1RaII could be derived by alternative translation initiation. In vitro transcription and translation of intact sIL-1Ra cDNA in rabbit reticulocyte lysates led to both pro-sIL-1Ra and icIL-1RaII proteins, whereas transcription and translation of icIL-1RaI cDNA produced both icIL-1RaI and icIL-1RaII proteins. Mutation of the first 5' ATG in sIL-1Ra cDNA led to translation of only icIL-1RaII, while only sIL-1Ra was observed after mutation of the second ATG. These results indicate that icIL-1RaII is a third member of the IL-1Ra family and is a 16-kDa, 143-amino acid intracellular protein derived by alternative translation initiation from either sIL-1Ra mRNA or icIL-1Ra mRNA. The role in biology of either intracellular form of IL-1Ra remains unknown.

The differential production of three forms of IL-1 receptor antagonist by human neutrophils and monocytes.

IL-1R antagonist (IL-1Ra) exists as three well-characterized isoforms. The 17-kDa secretory IL-1Ra (sIL-1Ra) and 18-kDa intracellular IL-1Ra (icIL-1RaI) arise by alternative transcription of the same IL-1Ra gene. The recently described 16-kDa intracellular IL-1Ra (icIL-1RaII) is formed by alternative translation initiation of sIL-1Ra mRNA. Transcription and translation of IL-1Ra isoforms were examined in LPS-stimulated human neutrophils and PBMC using RT-PCR, ELISA, and Western blot analysis. LPS stimulation of neutrophils resulted in elevated sIL-1Ra mRNA levels by 1 h, whereas icIL-1RaI mRNA remained undetectable through 22 h of culture. Extracellular glycosylated sIL-1Ra protein and intracellular icIL-1RaII were observed in LPS-stimulated neutrophils by 3 h of culture; no icIL-1RaI protein was detected by immunoblot. LPS stimulation of PBMC resulted in elevated sIL-1Ra mRNA levels by 1 h and detectable icIL-1RaI mRNA at 8 h of culture. LPS-stimulated PBMC demonstrated extracellular glycosylated sIL-1Ra protein and intracellular icIL-1RaII within 3 h of stimulation, whereas detection of icIL-1RaI protein was delayed until 15 h of culture. Subcellular localization experiments established that both icIL-1RaI and icIL-1RaII were present primarily within the cytoplasmic compartment, as expected by their lack of a signal peptide. These results demonstrate that although both LPS-stimulated neutrophils and PBMC synthesize sIL-1Ra and icIL-1RaII, only PBMC transcribe and translate icIL-1RaI. Furthermore, sIL-Ra transcription and translation (and translation of icIL-1RaII) are early events, whereas icIL-1RaI transcription in PBMC is delayed.

Interleukin-1 receptor antagonist: role in biology.

The interleukin-1 receptor antagonist (IL-1Ra) is a member of the IL-1 family that binds to IL-1 receptors but does not induce any intracellular response. Two structural variants of IL-1Ra have previously been described: a 17-kDa form that is secreted from monocytes, macrophages, neutrophils, and other cells (sIL-1Ra) and an 18-kDa form that remains in the cytoplasm of keratinocytes and other epithelial cells, monocytes, and fibroblasts (icIL-1Ra). An additional 16-kDa intracellular isoform of IL-1Ra has recently been described in neutrophils, monocytes, and hepatic cells. Both of the major isoforms of IL-1Ra are transcribed from the same gene through the use of alternative first exons. The two promoters regulating transcription of the secreted and intracellular forms have been cloned, and some of the functional cis-acting DNA regions have been characterized. The production of IL-1Ra is stimulated by many substances including adherent IgG, other cytokines, and bacterial or viral components. The tissue distribution of IL-1Ra in mice indicates that sIL-1Ra is found predominantly in peripheral blood cells, lungs, spleen, and liver, while icIL-1Ra is found in large amounts in skin. Studies in transgenic and knockout mice indicate that IL-1Ra is important in host defense against endotoxin-induced injury. IL-1Ra is produced by hepatic cells with the characteristics of an acute phase protein. Endogenous IL-1Ra is produced in numerous experimental animal models of disease as well as in human autoimmune and chronic inflammatory diseases. The use of neutralizing anti-IL-1Ra antibodies has demonstrated that endogenous IL-1Ra is an important natural antiinflammatory protein in arthritis, colitis, and granulomatous pulmonary disease. Treatment of human diseases with recombinant human IL-1Ra showed an absence of benefit in sepsis syndrome. However, patients with rheumatoid arthritis treated with IL-1Ra for six months exhibited improvements in clinical parameters and in radiographic evidence of joint damage.

Binding of IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist by soluble IL-1 receptors and levels of soluble IL-1 receptors in synovial fluids.

These studies have examined the binding of the three IL-1 ligands, IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1 ra), to soluble forms of types I and II IL-1Rs (sIL-1RI and sIL-1RII). This interaction was measured in direct binding experiments, in which the ligands bound to immobilized sIL-1R, and in inhibition experiments, in which sIL-1R in solution inhibited the binding of IL-1 ligands to immobilized sIL-1R. In addition, the effects of sIL-1R on the detection of IL-1 ligands by ELISA were examined. Finally, levels of sIL-1R in synovial fluid samples were determined, and their effects on measurement of IL-1 in these samples were estimated. IL-1 beta bound more avidly to sIL-1RII than IL-1 alpha or IL-1ra, primarily because of a slow dissociation rate. In contrast, IL-1 ra bound more avidly than IL-1 alpha or IL-1 beta to sIL-1RI, again because of a slow dissociation rate. sIL-1RII and sIL-1RI inhibited the detection of IL-1 beta and IL-1ra, respectively, by ELISA. Low levels of sIL-1RI (approximately 1.0-2.5 ng/ml) were present in all synovial fluids, irrespective of the degree of inflammation, and were correlated inversely with the levels of measured IL-1ra. In contrast, higher levels of sIL-1RII (approximately 10-20 ng/ml) were found in inflammatory synovial fluids and were not correlated with IL-1ra levels. IL-1 beta could not be detected in any synovial fluid. These results suggest that some IL-1 beta and IL-1ra may be bound in vivo to sIL-1RII and sIL-1RI, respectively, leading to underestimations of cytokine concentrations in body fluids when measured by ELISA.

The effects of interleukin-10 on interleukin-1 receptor antagonist and interleukin-1 beta production in human monocytes and neutrophils.

LPS stimulates human monocytes and neutrophils to produce IL-1 beta and IL-1 receptor antagonist (IL-1ra). IL-10 has been shown to inhibit LPS-induced IL-1 beta production in human monocytes. The objective of these studies was to examine the effects of IL-10 on human monocyte and neutrophil IL-1ra protein and mRNA production and compare it to IL-1 beta. IL-10 markedly inhibited LPS-induced IL-1 beta mRNA and protein production in both cell types. IL-10 alone induced IL-1ra mRNA production in monocytes with a low level of protein production. Furthermore, IL-10 led to enhanced levels of IL-1ra mRNA in LPS-induced monocytes at 2 to 6 h and of IL-1ra protein at 4-16 h, but there was no increase in levels of IL-1ra protein at 24 h or later. In neutrophils IL-10 alone did not stimulate IL-1ra protein or mRNA production and did not augment LPS-induced IL-1ra mRNA. These net effects of IL-10 resulted in an increase in the ratio of IL-1ra to IL-1 beta in both neutrophils and monocytes. The net effects of IL-10 on inflammatory cells may be important in modulation of biologic responses to IL-1.

Peripheral blood neutrophil production of interleukin-1 receptor antagonist and interleukin-1 beta.

The objective of this study was to characterize interleukin-1 receptor antagonist (IL-1ra) and interleukin-1 beta (IL-1 beta) production by human peripheral blood neutrophils (polymorphonuclear leukocytes; PMN). Unstimulated PMN contained IL-1ra protein in the absence of IL-1ra mRNA; IL-1 beta mRNA and protein were undetectable. Lipopolysaccharide (LPS), granulocyte/macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), individually, transiently increased IL-1ra steady-state mRNA levels in PMN, with associated increases in IL-1ra protein synthesis. LPS, GM-CSF, and TNF-alpha generated similar increases in IL-1 beta mRNA, yet only LPS resulted in detectable synthesis of IL-1 beta protein. IL-4 enhanced LPS-induced IL-1ra production by PMN and inhibited LPS-induced IL-1 beta production. by PMN and inhibited LPS-induced IL-1 beta production. IL-1ra protein present within stimulated PMN supernatants existed in the 22- to 25-kD glycosylated form. Polymerase chain reaction amplification determined that only sIL-1ra mRNA was present within stimulated PMN; icIL-1ra mRNA was undetectable. These results indicate that freshly isolated PMN possess a small amount of IL-1ra protein and that these cells can respond to stimuli with a low level of sIL-1ra transcription and translation. PMN may be a major source of IL-1ra in inflammatory exudates where these cells predominate.

Levels of synovial fluid interleukin-1 receptor antagonist in rheumatoid arthritis and other arthropathies. Potential contribution from synovial fluid neutrophils.

OBJECTIVE: To measure synovial fluid (SF) levels of interleukin-1 receptor antagonist (IL-1ra) and to determine the capacity of SF neutrophils (PMN) to synthesize and release IL-1ra. METHODS: A sensitive and specific enzyme-linked immunosorbent assay was used to measure SF IL-1ra protein concentrations and IL-1ra production by isolated SF PMN: RESULTS: SF IL-1ra levels were elevated in 13 of 16 samples from patients with rheumatoid arthritis (RA) (mean 17.1 ng/ml), in 6 of 18 samples from patients with infectious or inflammatory, non-RA arthropathies (mean 10.6 ng/ml), and in none of 11 noninflammatory SF samples. SF IL-1ra levels correlated with SF PMN concentrations (r = 0.680, P < 0.00001). Isolated SF PMN contained preexisting IL-1ra protein in the absence of messenger RNA (mRNA). In addition, both lipopolysaccharide and granulocyte-macrophage colony-stimulating factor induced modest increases in IL-1ra mRNA by cultured SF-PMN. CONCLUSION: IL-1ra levels are increased in > 80% of RA SF samples. SF PMN produce IL-1ra, possibly contributing to the levels of IL-1ra present within the SF.

IL-1ra ELISA: reduction and alkylation of synovial fluid eliminates interference by IgM rheumatoid factors.

IL-1 and a specific receptor antagonist of IL-1, IL-1ra, may play important roles in the pathophysiology of rheumatoid arthritis and in other types of inflammatory synovitis. Measurement of IL-1ra in synovial fluids and in other body fluids may lead to a greater understanding of its possible activity as a modulator of the immune and inflammatory systems in vivo. Therefore, a modified sandwich ELISA was developed to measure IL-1ra protein concentration in synovial fluids. The antibodies used in this ELISA were polyclonal and derived from rabbits hyperimmunized with human recombinant IL-1ra. IgM rheumatoid factors within synovial fluid resulted in false elevation of determined IL-1ra by the sandwich ELISA through binding of the primary and secondary antibodies. Reduction and alkylation of synovial fluid samples before application to the ELISA plate eliminated the interference caused by greater than or equal to 2000 micrograms/ml IgM rheumatoid factor (latex agglutination titer of 1/5.120). This ELISA was specific for IL-1ra; there was no detection of IL-1 alpha, IL-1 beta, or lysozyme. The sensitivity of this ELISA was less than 200 pg/ml, making it a useful assay for the accurate measurement of synovial fluid IL-1ra protein concentration.

The biological role of naturally-occurring cytokine inhibitors.

Four different mechanisms of cytokine inhibition might be involved in regulation of cytokine effects in vivo. Different cytokines may exhibit opposing biological effects on a specific target cell or in a particular disease. Autoantibodies to cytokines may block cytokine effects in vivo or may function as carriers to deliver cytokines to tissues. Soluble receptors of cytokines, particularly for IL-2 and TNF alpha, may be released by cell activation. Lastly, a specific receptor antagonist of IL-1, IL-1ra, is synthesized by human monocytes and macrophages, particularly under the influence of GM-CSF. IL-1ra is the first described naturally-occurring receptor antagonist of any cytokine or hormone-like molecule. It is not yet known whether IL-1ra is produced in tissues in human diseases as an endogenous anti-inflammatory factor. Whether any of these potential mechanisms to regulate cytokine effects will be of value in the treatment of human diseases remains to be determined.