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Summary: The interleukin-1 (IL-1), IL-18, and IL-33 families of cytokines are related by mechanism of origin, receptor structure, and signal transduction pathways utilized. All three cytokines are synthesized as precursor molecules and cleaved by the enzyme caspase-1 before or during release from the cell. The NALP-3 inflammasome is of crucial importance in generating active caspase-1. The IL-1 family contains two agonists, IL-1alpha and IL-1beta, a specific inhibitor, IL-1 receptor antagonist (IL-1Ra), and two receptors, the biologically active type IL-1R and inactive type II IL-1R. Both IL-1RI and IL-33R utilize the same interacting accessory protein (IL-1RAcP). The balance between IL-1 and IL-1Ra is important in preventing disease in various organs, and excess production of IL-1 has been implicated in many human diseases. The IL-18 family also contains a specific inhibitor, the IL-18-binding protein (IL-18BP), which binds IL-18 in the fluid phase. The IL-18 receptor is similar to the IL-1 receptor complex, including a single ligand-binding chain and a different interacting accessory protein. IL-18 provides an important link between the innate and adaptive immune responses. Newly described IL-33 binds to the orphan IL-1 family receptor T1/ST2 and stimulates T-helper 2 responses as well as mast cells.

Interleukin-1 (IL-1) plays a pivotal role in the pathogenesis of inflammatory bowel disease (IBD). IL-1 action is regulated in part by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1Ra). Four splice variants of IL-1Ra gene product have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Although sIL-1Ra and icIL-1Ra1 bind to type I IL-1 receptor with equal affinity, icIL-1Ra1 may carry out unique functions inside cells. The goal of this study was to determine the role of icIL-1Ra1 in regulation of cytokine-induced IL-6 and IL-8 production in Caco-2 intestinal epithelial cells. icIL-1Ra1 inhibited IL-1-induced IL-6 and IL-8 production. IL-1 activated all three mitogen-activated protein (MAP) kinase family members: p38 MAP kinase, extracellular-regulated kinases (ERK), and c-Jun amino-terminal kinases (JNK). Specific inhibitors of each MAP kinase pathway decreased IL-1-induced IL-6 and IL-8 production. Overexpression of icIL-1Ra1 inhibited p38 MAP kinase phosphorylation, but had no effect on ERK and JNK phosphorylation. In addition, icIL-1Ra1 inhibited nuclear translocation of NF-kappaB after IL-1 stimulation. In conclusion, these data indicate that icIL-1Ra1, acting in the cytoplasm of Caco-2 cells, decreased IL-1-induced IL-6 and IL-8 production. This intracellular anti-inflammatory activity of icIL-1Ra1 was mediated through inhibition of p38 MAP kinase and NF-kappaB signal transduction pathways.

IL-1 is an important mediator of inflammation and tissue damage in multiple organs, both in experimental animal models of disease and in human diseases. The IL-1 family consists of two agonists, IL-1alpha and IL-1beta, two receptors, biologically active IL-1RI and inert IL-1RII, and a specific receptor antagonist, IL-1Ra. The balance between IL-1 and IL-1Ra in local tissues plays an important role in the susceptibility to and severity of many diseases. An allelic polymorphism in the IL-1Ra gene has been associated with a variety of human diseases primarily of epithelial and endothelial cell origin. This association may be secondary to an imbalance in the IL-1 system with enhanced production of IL-1beta and reduced production of the major intracellular isoform of IL-1Ra. Treatment of RA with daily subcutaneous injections of recombinant IL-1Ra protein has been shown to be efficacious. Gene therapy approaches with IL-1Ra are being evaluated for the treatment of RA and other human diseases.

Objectives: To summarize the role of cytokine imbalance in the pathogenesis of rheumatoid arthritis., Methods: The literature on cytokines in rheumatoid arthritis from American and European medical journals was reviewed., Results: An important role of interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha in the mediation of tissue damage in the rheumatoid joint has been well established over the past 10 years. The IL-1 family consists of 2 agonists, IL-1alpha and IL-1beta, and a specific naturally occurring receptor antagonist, IL-1Ra. Both forms of IL-1 induce intracellular responses through binding to the type 1 IL-1 receptor (IL-1R) on target cells. IL-1Ra binds to IL-1R with an avidity equal to that of IL-1 but fails to stimulate the cells, thus functioning as an inhibitor of IL-1 binding. Endogenous production of IL-1Ra is an important anti-inflammatory mechanism both in animal models of disease and in human disease. In the rheumatoid synovium, an imbalance exists in this system, because the relative levels of production of IL-1Ra are not adequate to effectively block the proinflammatory effects of IL-1. Studies in different animal models of inflammatory arthritis indicate that a deficiency of IL-1Ra relative to IL-1 leads to more severe disease and even to the spontaneous development of arthritis as observed in IL-1Ra knockout mice. A restoration of the balance between IL-1Ra and IL-1 in human disease can theoretically be achieved through the administration of recombinant IL-1ra protein, gene therapy with the IL-1Ra complementary DNA, or stimulation of production of endogenous IL-1Ra., Conclusions: An imbalance between proinflammatory cytokines and cytokine antagonists or inhibitors may be one factor predisposing to initiation or perpetuation of rheumatoid synovitis., (Copyright 2001 by W.B. Saunders Company.)

Recent studies have described the spontaneous development of arthritis or vasculitis in IL-1 receptor antagonist (IL-1Ra) knockout mice bred on specific and different genetic backgrounds. The levels of both secreted and intracellular isoforms of IL-1Ra produced in the rheumatoid joint or in the arterial wall may not be adequate to effectively inhibit the excess amounts of locally produced IL-1. Thus, an imbalance between IL-1 and IL-1Ra may predispose to local inflammatory disease in particular tissues in the presence of other as yet unknown genetically influenced factors.

Interleukin-1 receptor antagonist (IL-1Ra) is produced by hepatocytes with characteristics of an acute-phase protein. To examine the role of IL-4 and IL-13 in production of IL-1Ra, human primary hepatocytes and HepG2 human hepatoma cells were cultured in the presence of IL-4 or IL-13 in combination with IL-1beta and/or IL-6. The results indicated that both IL-4 and IL-13 amplified the stimulatory effect of IL-1beta on production of IL-1Ra protein and messenger RNA (mRNA) by both human primary hepatocytes and HepG2 cells. IL-1Ra refers to three different peptides, one secreted (sIL-1Ra) and two intracellular (icIL-1RaI and icIL-1RaII), derived from the same gene. sIL-1Ra and icIL-1RaI are the products of two different mRNA, whereas icIL-1RaII is synthesized by alternative translation initiation mainly from sIL-1Ra mRNA. Our results show that both sIL-1Ra and icIL-1RaII, but not icIL-1RaI, are produced by HepG2 cells and human hepatocytes. Transient transfection experiments as well as mRNA stability studies indicated that IL-4 stimulated sIL-1Ra production primarly at the level of transcription. Gel retardation assays showed that IL-4 induced the formation of a STAT6-DNA complex with a STAT6 binding element within the sIL-1Ra promoter, but had no effect on IL-1-induced NF-kappaB binding activity. In contrast to IL-1Ra, production of C-reactive protein by human primary hepatocytes was stimulated by IL-6 and decreased by the addition of IL-4.

Extensive evidence from both in vivo and in vitro experiments indicate that IL-1, a prototypic proinflammatory cytokine, is involved in the mechanisms that lead to progressive joint destruction in RA. IL-1Ra, a member of the IL-1 family, binds IL-1 receptors but does not induce any cellular responses. IL-1Ra competitively inhibits the binding of IL-1 to its cell surface receptors and thus, acts as an endogenous antiinflammatory mediator. However, the results of several studies suggest that a relatively deficient production in IL-1Ra as compared to that of IL-1 in RA synovium may predispose to the perpetuation of chronic inflammation. Systemic administration of IL-1Ra, or local delivery into the joint by gene therapy, in different experimental animal models of arthritis attenuated the severity of the inflammatory response and reduced articular destruction. In addition, treatment of rheumatoid patients with IL-1Ra led to an improvement in different clinical and biological parameters and to a reduction in the radiological signs of joint erosions. Encouraging results also have been reported in both in vitro and in vivo experimental animal models of arthritis through using other strategies designed to block the effects of IL-1 at the level of production, prevent the binding of IL-1 to its cell surface receptors, or interfere with the effects of IL-1 at the post-receptor level.

This review has summarized information published over the last 5 years on the presence and pathophysiologic role of IL-1 and TNF alpha in RA. The evidence to date shows that 5 of 6 criteria for identifying mediators of tissue damage in human autoimmune diseases are satisfied (Table 1). The last criterion, prevention of clinical progression in patients with RA, is currently being evaluated. Many new therapeutic approaches are currently being developed, including the use of soluble receptors to IL-1 or TNF, monoclonal antibodies to TNF alpha, a specific IL-1 receptor antagonist, and gene therapy with the latter molecule. It should be emphasized that both IL-1 and TNF alpha play important roles in normal host defense; the possible complications of blocking their production or effects need to be carefully evaluated in long-term studies. A recent review has emphasized that although IL-1 and TNF alpha have many overlapping biologic properties, each may exhibit distinct effects in joint disease (99). Anti-TNF treatment may be primarily antiinflammatory but blocking IL-1 may be more effective in preventing cartilage destruction (100). The possibility exists that simultaneous inhibition of TNF alpha and IL-1 may be more therapeutically efficacious than blockade of either agent alone, as was recently demonstrated with IL-1ra and soluble TNF receptors in bacterial cell wall-induced arthritis in rats (101). The next level of clinical studies in rheumatoid arthritis should include the use of two biologic response modifiers together, or one agent combined with a more traditional form of therapy.

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.

Objective: To quantify interleukin-1 receptor antagonist (IL-1ra) and IL-1 production and gene expression by rheumatoid arthritis (RA) synovial tissue (ST) cells., Methods: IL-1 alpha, IL-1 beta, and IL-1ra protein levels were measured by enzyme-linked immunosorbent assay in fresh and cultured ST cells, purified synovial macrophages, and fibroblast-like synoviocytes (FLS). The relative expression of the secreted form of IL-1ra (sIL-1ra) and the alternatively spliced intracellular form (icIL-1ra) was determined by reverse transcription polymerase chain reaction (RT-PCR) techniques., Results: IL-1 alpha, IL-1 beta, and IL-1ra were present in fresh and cultured ST cell samples of synovium from RA and osteoarthritis patients. IL-1ra:IL-1 ratios ranged from 1.2 to 3.6, which is below the 10-100-fold excess of IL-1ra needed to inhibit IL-1 bioactivity. Isolated CD14+ synovial macrophages secreted IL-1ra, but the amount was much less than that of alveolar or in vitro-derived macrophages. Cultured FLS contained intracellular IL-1ra but secreted little IL-1ra into the culture supernatants. RT-PCR showed that icIL-1ra mRNA was more abundant than sIL-1ra mRNA in FLS and unfractionated ST cells., Conclusion: IL-1ra production by RA ST cells is deficient relative to total production of IL-1.

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.

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.

Interleukin 1 receptor antagonist (IL-1ra) is induced in monocytes stimulated with lipopolysaccharide (LPS) or cultured on adherent immunoglobulin G (IgG). We examined the effects of various cytokines on monocyte IL-1ra protein production and compared it to IL-1 beta, which is regulated differently. IL-3 and GM-CSF induced near equivalent amounts of IL-1ra protein as does LPS. IL-1 alpha and IL-4 were weaker inducers. IL-3 and GM-CSF did not affect LPS or IgG induction of IL-1ra or LPS-induced IL-1 beta. However, our data confirmed that IL-4 up-regulated LPS-induced IL-1ra and down-regulated LPS-induced IL-1 beta. The kinetics of IL-1ra production by monocytes varied between stimulation with adherent IgG and cytokines or LPS. Cells cultured on adherent IgG exhibited a higher level of and more prolonged IL-1ra production. Relative IL-1ra mRNA levels after 8 h were in the order: adherent IgG > LPS or GM-CSF > IL-1 alpha, IL-3 or IL-4. The following cytokines failed to induce IL-1ra production: IL-2, IL-6, G-CSF, M-CSF, IFN-gamma, TGF beta 1, TGF beta 2, TNF alpha, acidic and basic FGF, PDGF and EGF. These results suggest that IL-1 alpha, IL-3, IL-4 and GM-CSF may play important roles in regulating monocyte IL-1ra production and that different mechanisms may be involved in induction of IL-1ra by adherent IgG in comparison to LPS or other cytokines.

Interleukin-1 (IL-1) may have a significant pro-inflammatory effect in the skin; an imbalance in its production has been linked to cutaneous disease processes. IL-1 receptor antagonist (IL-1ra) is a recently described competitive inhibitor of IL-1 alpha and IL-1 beta that binds to human types I and II IL-1 receptors without apparent cell activation. Human keratinocytes synthesize IL-1ra, IL-1 alpha, and IL-1 beta but fail to secrete these cytokines. This study investigated IL-1ra and IL-1 alpha accumulation by cultured keratinocytes stimulated by tumor necrosis factor-alpha (TNF-alpha), IL-3, IL-4, IL-6, IL-10, interferon-gamma, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and macrophage colony-stimulating factor and by various extracellular matrix proteins, conditions that these cells may encounter in normal or inflamed skin in vivo. IL-1ra and IL-1 alpha proteins were measured by specific enzyme-linked immunosorbent assay in keratinocyte supernatants and lysates. Only TNF-alpha induced IL-1ra and IL-1 alpha production. TNF-alpha added to culture in amounts of 10 ng/ml or higher, induced a twofold increase in intracellular levels of both IL-ra and IL-1 alpha without secretion at 48 h. The IL-1ra concentration in keratinocyte lysates increased from 9.6 to 17.6 ng/ml after TNF-alpha stimulation, and the IL-1 alpha concentration increased from 1.0 to 3.3 ng/ml. Keratinocytes also exhibited comparable increases in IL-1 alpha and IL-1ra mRNA levels after 12 h in culture with TNF-alpha, as determined by in vitro hybridization to specific cDNA probes. The IL-1 alpha and IL-1ra response to TNF-alpha stimulation showed a varied pattern among different keratinocyte strains over 72 h of culture on plain plastic. In contrast, extracellular matrix proteins (laminin, fibronectin, collagen I and IV, and vitronectin) did not stimulate keratinocyte accumulation of IL-1 alpha or IL-1ra proteins after 72 h in culture. When TNF-alpha was added to cells cultured on these matrices, no change in IL-1 alpha or IL-1ra production was observed above that which could be attributed to TNF-alpha alone. In conclusion, TNF-alpha, but not the extracellular matrix proteins tested, stimulated production of intracellular IL-1 alpha and IL-1ra by keratinocytes. The ratio of IL-1ra to IL-1 alpha after TNF-alpha stimulation of keratinocytes may influence the inflammatory profile in the epidermis.

IL-1ra is the first described naturally occurring receptor antagonist of any cytokine or hormone-like molecule. IL-1ra is a member of the IL-1 family by three criteria: amino acid sequence homology of 26 to 30% to IL-1 beta and 19% to IL-1 alpha; similarities in gene structure; and common gene localization to human chromosome 2q14. Two structural variants of IL-1ra exist: sIL-1ra, a secretory molecule produced by monocytes, macrophages, neutrophils, fibroblasts, and other cells; and icIL-1ra, an intracellular molecule produced by keratinocytes and other epithelial cells, macrophages, and fibroblasts. IL-1ra production by monocytes, macrophages, and neutrophils may be regulated in a differential fashion with IL-1 beta. Human IL-1ra binds to both human IL-1RIs and IL-1RIIs on cell surfaces, although with 100-fold greater avidity to IL-1RIs. IL-1ra may bind preferentially to soluble IL-1RIs and not at all to soluble IL-1RIIs. IL-1ra competitively inhibits binding of both IL-1 alpha and IL-1 beta to cell surface receptors without inducing any discernible intracellular responses. All three forms of IL-1 may bind to IL-1 receptors in a similar fashion but IL-1ra may lack the secondary interactions necessary to trigger cell responses. A 100-fold or greater excess of IL-1ra over IL-1 may be necessary to inhibit biological responses to IL-1 both in vitro and in vivo. The roles of sIL-1ra and icIL-1ra in normal physiology or in host defense mechanisms remain unclear. The administration of IL-1ra blocks the effects of IL-1 in some animal models of septic shock, inflammatory arthritis, graft-versus-host disease, and inflammatory bowel disease. The preliminary results of clinical trials in humans indicate possible efficacy of IL-1ra in sepsis syndrome, rheumatoid arthritis, and GVHD.

The objective of these studies was to characterize the IL-1 inhibitory activity present in normal and psoriatic epidermis from clinically stable lesions. Fractionation of normal epidermal cytosol on a molecular sizing column failed to reveal the presence of IL-1 inhibitory bioactivity. However, specific ELISAs indicated that both the IL-1 receptor antagonist (IL-1ra) and IL-1 alpha were present in overlapping peaks. Further fractionation of the normal epidermal cytosol by anion exchange chromatography separated these two molecules, revealing the IL-1 inhibitory bioactivity of the IL-1ra molecule. Similar studies on psoriatic epidermal cytosol indicated the presence of IL-1 inhibitory bioactivity and IL-1ra protein. The IL-1 inhibitory bioactivity of both normal and psoriatic cytosol was neutralized by a mAb specific for IL-1ra. The ratio of IL-1ra to IL-1 alpha proteins was significantly increased in involved psoriatic skin compared with normal skin. By Western blot analysis this IL-1ra was approximately 20 kD, slightly larger than monocyte-derived IL-1ra and equivalent to an intracellular variant of IL-1ra expressed by keratinocytes. Polymerase chain reaction indicated the presence of mRNA for both forms of IL-1ra in normal epidermis, with both forms increased in psoriatic-involved skin. Immunofluorescence studies revealed the IL-1ra protein to be concentrated in the stratum granulosum of normal skin and in the basal-midbasal layers of psoriatic epidermis. These results suggest that the balance between intracellular IL-1ra and IL-1 alpha may be an important influence on keratinocyte growth and/or differentiation, as well as on the inflammatory potential of IL-1 in injured skin.

mRNA expression and protein production of interleukin-1 alpha, interleukin-1 beta and intracellular and secreted forms of an interleukin-1 receptor antagonist were measured in visually confluent monolayers of unstimulated cultured human retinal pigment epithelial cells and after cells were stimulated with recombinant cytokines. Using reverse transcription polymerase chain reaction, transcripts for interleukin-1 alpha and interleukin-1 beta were not detected in unstimulated cells from any of six donors whereas mRNA expression for both interleukin-1 alpha and interleukin-1 beta was readily induced in all six cell lines after cells were stimulated with recombinant IL-1 (alpha or beta), tumor necrosis factor alpha, or lipopolysaccharide. The combination of cycloheximide and recombinant interleukin-1 caused a 14-fold enhancement of interleukin-1 alpha and interleukin-1 beta mRNA expression above that observed after cells were stimulated with interleukin-1 alone. After stimulation by interleukin-1, cells produced intracellular interleukin-1 alpha protein, but did not secrete it into medium. In contrast, interleukin-1 beta protein was not detected in cell lysates or conditioned-medium after stimulation with interleukin-1. An intracellular interleukin-1 receptor antagonist was expressed constitutively by human retinal pigment epithelial cells; mRNA transcripts were enhanced in a dose and time dependent manner after cells were exposed to recombinant interleukin-1 or tumor necrosis factor alpha. In contrast, mRNA for a secreted form of the interleukin-1 receptor antagonist was not detected under basal conditions or after cells were stimulated by recombinant cytokines. Interleukin-1 receptor antagonist protein was found primarily in cell lysates; little interleukin-1 receptor antagonist protein was secreted by the cells. The presence of cell-associated interleukin-1 receptor antagonist was confirmed by immunocytochemistry. Levels of cell-associated IL-1 receptor antagonist protein were not significantly influenced by recombinant interleukin-1 or tumor necrosis factor alpha. Endogenous expression of interleukin-1 receptor antagonist may attenuate the effect of exogenous or endogenous interleukin-1, thus providing the RPE cell a means of maintaining interleukin-1 homeostasis in ocular inflammatory disease.

Human monocytes may synthesize simultaneously both the agonist IL-1 beta and a specific receptor antagonist of IL-1 (IL-1ra). These studies examined whether monocyte production of these two structurally related cytokines was regulated differently. IL-1ra and IL-1 beta protein levels in cell supernatants and lysates were measured with specific ELISA. Relative steady-state mRNA levels, relative transcriptional rates as determined by the nuclear run-on technique, and mRNA stability were all assessed using specific cDNA probes. Monocytes were stimulated with LPS alone, adherent IgG, or both LPS and adherent IgG. Monocytes stimulated with LPS produced near equivalent amounts of IL-1ra and IL-1 beta proteins over 22 h; relative steady-state mRNA levels paralleled the protein levels. In addition, LPS-induced monocytes exhibited enhanced rates of transcription for both IL-1ra and IL-1 beta, in comparison to adherent control cells without LPS. mRNA half-lives in LPS-induced monocytes also were similar for IL-1ra and IL-1 beta. Monocytes cultured on adherent IgG exhibited a low level of IL-1 beta transcription with an absence of protein production. In contrast, adherent IgG led to a high and prolonged rate of IL-1ra protein production. Furthermore, monocytes cultured on adherent IgG exhibited a specific induction of IL-1ra transcription and a marked prolongation in IL-1ra mRNA stability. However, LPS in a high concentration, 1 microgram/ml, reversed the IgG induction of IL-1ra production by decreasing both transcriptional rate and mRNA stability. These results indicate that production of IL-1ra by human monocytes is characterized by different patterns of regulation in comparison with IL-1 beta. LPS induces production of both proteins whereas adherent IgG stimulates only IL-1ra production. The effects of IgG and LPS on induction of IL-1ra production in human monocytes are mediated at both transcriptional and post-transcriptional levels.

Human monocytes aged in medium exhibit a decrease in LPS-induced IL-1 beta production in comparison with fresh cells. The objective of these experiments was to evaluate the effects of differentiation for 1 or 6 days in IFN-gamma, 1,25-(OH)2-vitamin D3, granulocyte-macrophage CSF, or in various combinations of these agents on both steady state IL-1 beta mRNA levels and protein production in LPS-stimulated monocytes. Monocytes preincubated in IFN-gamma for 1 day, then cultured for 24 h with LPS, exhibited similar kinetics of IL-1 beta mRNA production, but a higher peak mRNA level at 8 h after LPS stimulation, in comparison with cells cultured in medium before LPS stimulation. An increase in IL-1 beta protein production with variable secretion was noted in monocytes preincubated in IFN-gamma before stimulation with LPS. Monocytes preincubated for 1 day in 1,25-(OH)2-D3 alone or in both IFN-gamma and 1,25-(OH)2-D3 exhibited similar kinetics and peak expression of LPS-induced IL-1 beta mRNA levels as cells cultured in medium. However, monocytes preincubated for 1 day in both agents displayed a 50% or greater restoration in LPS-induced IL-1 beta synthesis and secretion in comparison with fresh cells. Granulocyte-macrophage-CSF did not augment the effects of the other differentiating agents on IL-1 beta production. Monocytes cultured over 6 days in differentiating agents failed to exhibit any restoration in LPS-induced IL-1 beta production. These in vitro-derived macrophages exhibited very low levels of both IL-1 beta mRNA and protein production under any culture condition. These results suggest that the effects of differentiating agents on LPS-induced IL-1 beta production may in part be related to the state of maturation of the monocyte. Furthermore, a repression in IL-1 beta transcription that is not reversed by exposure to differentiating agents may be acquired by monocytes as they mature into macrophages in vitro.

Three interleukin-1 inhibitors have been purified to homogeneity from medium conditioned by human monocytes. Partial sequence analysis and digestion with N-glycanase indicate that these are glycosylation forms of a single protein. The protein binds to the interleukin-1 receptor but has no interleukin-1-like activity, even at very high concentrations, and is therefore a pure receptor antagonist.

The objective of these studies was to examine the ability of phorbol myristic acetate (PMA), Fc fragments, and various forms of immune complexes to induce the production by human monocytes of factors stimulatory to chondrocytes or thymocytes. All of these materials were prepared free of detectable contamination with bacterial lipopolysaccharides (LPS) at the level of less than 0.1 ng/ml. Supernatants and lysates from stimulated human monocytes were assayed for their ability to induce collagenase production in cultured rabbit articular chondrocytes or to augment mitogen-induced proliferation of murine thymocytes. The activity detected by these assays exhibited an m.w. of approximately 15,000, and electrophoretic heterogeneity in the pH ranges of 5 to 5.5 and 6.5 to 7.0, characteristics of human interleukin 1 (IL 1) or IL 1-like factors. Monocytes cultured with 2 ng/ml LPS produced chondrocyte and thymocyte stimulatory factors. PMA, Fc fragments, and soluble, precipitated, particulate, or adherent immune complexes were inactive in stimulating the monocytes. However, complement fixation by precipitated immune complexes did generate activity capable of inducing monocytes to synthesize and secrete chondrocyte and thymocyte stimulatory factors. Adherent immune complexes and PMA were biologically active, as evidenced by induction of superoxide generation in the human monocytes. Supernatants from monocytes cultured on adherent immune complexes contained a factor inhibitory to chondrocyte and thymocyte responsiveness. This factor had a m.w. approximately 22,000 and appeared to inhibit specifically IL 1 stimulation, not interleukin 2 stimulation or cell proliferation. It was concluded that PMA, Fc fragments, and various forms of immune complexes in the absence of complement do not induce IL 1 production in human monocytes. However, complement fixation by immune complexes does lead to activation of monocytes to produce IL 1. Monocytes cultured on adherent immune complexes produce an IL 1 inhibitor.

Cultured human monocytes and tissue macrophages exhibit a marked decrease in LPS-stimulated IL-1 production. Preincubation of monocytes in 100 U/ml IFN-gamma or in 0.25 microgram/ml cycloheximide (Cx) leads to a partial maintenance of the ability to produce IL-1 in response to subsequent stimulation with LPS, whereas preincubation in both reagents leads to a near complete maintenance of this response. These studies were performed to determine the mechanisms of these alterations in regulation of IL-1 production in cultured monocytes. In comparison to LPS-induced fresh monocytes, cells preincubated in medium for 1 day, then stimulated for 24 h with 100 ng/ml LPS, exhibited a 50% or more decrease in steady-state IL-1 beta mRNA levels. This reduction was accompanied by a four- to fivefold decrease in relative transcriptional rate, as determined by the nuclear run-on technique. The medium-aged cells also displayed greatly decreased IL-1 beta production with deficient secretion. Monocytes preincubated in IFN-gamma for 1 day before LPS stimulation exhibited a variable maintenance in IL-1 beta production, secondary both to prolonged duration of transcription and to increased IL-1 beta mRNA stability. Monocytes were preincubated for 1 day in Cx alone or in both IFN-gamma and cycloheximide, then these substances were washed out before a subsequent 24-h culture in LPS. Cx preincubation led to increases in both the peak and duration of transcription as well as to enhanced secretion of IL-1 beta protein. Monocytes preincubated in both IFN-gamma and Cx exhibited increased IL-1 beta mRNA levels due to both enhanced transcription and mRNA stability; more IL-1 beta protein secretion also was present in these cells. In summary, IFN-gamma and Cx may maintain LPS-induced IL-1 beta production in cultured human monocytes through multiple mechanisms. These alterations in regulation of IL-1 beta production during monocyte differentiation may be relevant to tissue macrophages where impaired IL-1 production may be present.

The objectives of these studies were to study the effects of bacterial lipopolysaccharide (LPS) on interferon-gamma (IFN-gamma)-induced Fc receptor expression on human monocytes and to examine whether these effects were mediated through stimulation of interleukin 1 (IL-1) production. Fc receptor expression was determined by binding of monomeric monoclonal murine immunoglobulin (Ig)G2a and cytofluorographic analysis. IL-1 activity in monocyte supernatants and lysates was assayed by augmentation of mitogen-induced murine thymocyte proliferation. IFN-gamma induced the expression of Fc receptors on human monocytes that were specific for murine IgG2a. This induction was inhibited by the addition of LPS in amounts as low as 2 to 8 pg/ml. LPS inhibition of IFN-gamma-induced Fc receptor expression was paralleled by the appearance of IL-1 in monocyte lysates and supernatants. The addition of purified human or recombinant IL-1 beta at the initiation of culture similarly inhibited the expression of IFN-gamma-induced Fc receptors on the monocytes. LPS also inhibited Fc receptor expression on the human myelomonocytic cell line THP-1 after induction with IFN-gamma or phorbol myristate acetate alone or with both agents together. This inhibition also was paralleled by the production of IL-1 but the addition of exogenous IL-1 to the THP-1 cells had no effect on IFN-gamma-induced Fc receptor expression. Tumor necrosis factor (TNF) inhibited IFN-gamma-induced Fc receptor expression on human monocytes but was much less potent than comparable amounts of IL-1. TNF also did not inhibit Fc receptor expression on THP-1 cells. In fact, IL-1 or TNF led to an enhancement in IFN-gamma-induced Fc receptor expression on THP-1 cells. These results indicate that LPS can inhibit IFN-gamma-induced Fc receptor expression on human monocytes and that IL-1 and TNF may mediate these effects of LPS. Thus, an autocrine or paracrine role is suggested for these cytokines. The possibility exists that intracellular IL-1 resulting from LPS stimulation may be at least in part responsible for inhibition of Fc receptor expression.

The objective of these studies was to characterize some aspects of interleukin 1 (IL-1) synthesis and secretion by human monocytes after stimulation with bacterial lipopolysaccharides (LPS). Various molecular species of LPS were incubated with adherent monocytes for 24 h. IL-1 activity in monocyte supernatants (secretion) and lysates (synthesis) was determined by stimulation of collagenase production in rabbit articular chondrocytes and augmentation of mitogen-induced proliferation of murine thymocytes. The presence of cytochalasin B enhanced LPS-induced IL-1 secretion without altering IL-1 synthesis. Monocytes preincubated in dexamethasone or hydrocortisone failed to exhibit any IL-1 activity in supernatants after LPS stimulation but the cell lysates still possessed 50% of control IL-1 activity. Studies with different LPS preparations indicated that the presence of diphosphoryl groups in lipid A enhanced the IL-1-inducing activities. Butanol-extracted LPS preparations, containing associated proteins, were not completely inhibited by 5 micrograms/ml polymyxin B in induction of IL-1 production at LPS concentrations of 10 or 100 ng/ml. These results indicate that the failure of polymyxin B to inhibit stimulation of IL-1 production by tests materials cannot be assumed to mean an absence of contaminating LPS.

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.

The objective of these studies was to characterize some aspects of collagenase production by rabbit articular chondrocytes cultured with stimulated monocyte supernatants. Supernatants from human monocytes stimulated with 20 ng/ml bacterial lipopolysaccharide (LPS) induced the synthesis and secretion of latent collagenase by the chondrocytes beginning at 6 hr. The time course and dose response of collagenase production by the chondrocytes were identical using crude monocyte supernatants or semipurified interleukin 1 (IL-1). Recombinant or purified human interleukin 2 failed to induce collagenase production in the cultured chondrocytes. The response of the chondrocytes was inhibited by actinomycin D or cycloheximide and not by corticosteroids. Phorbol myristate acetate (PMA) alone failed to directly stimulate the chondrocytes. However, PMA led to enhanced collagenase production by chondrocytes when incubated with submaximal amounts of LPS-stimulated monocyte supernatant or semipurified IL-1. LPS alone in amounts between 0.1 and 10.0 micrograms/ml directly stimulated collagenase production in chondrocytes between 4 and 11 days in culture. These data confirm those of other laboratories that IL-1 may be the active factor in monocyte supernatants responsible for inducing collagenase production in cultured chondrocytes. Further characterization of this response indicates that the collagenase is not preformed in the cells and stimulation of its production is not inhibited by corticosteroids. Cell supernatants or IL-1 preparations containing PMA as low as 1.0 ng/ml or LPS as low as 1.0 microgram/ml may give falsely high values for IL-1 activity when assayed by stimulation of collagenase production in cultured chondrocytes.

The objective of these studies was to investigate mechanisms of regulation of interleukin 1 (IL-1) production by human monocytes. IL-1 production was measured by augmentation of phytohemagglutinin-induced proliferation of murine thymocytes. Adherent human monocytes incubated in medium for one day exhibited a marked decrease in lipopolysaccharide (LPS)-induced IL-1 production over a second day. Cells pre-incubated in 100 U/ml gamma interferon (gamma-IFN) for 24 h displayed a partial to complete maintenance of LPS-induced IL-1 production. Studies with inhibitors of cyclo-oxygenase or lipoxygenase indicated that prostaglandins or leukotrienes were not responsible for the alterations in IL-1 production observed with cultured cells or for the effects of gamma-IFN. Monocytes were pre-incubated in cycloheximide for 24 h and the drug was washed out. These cells exhibited an enhancement in IL-1 production over a second 24 h culture in the presence of 2 ng/ml LPS. Furthermore, the partial maintenance of LPS-induced IL-1 production seen after cells were pre-incubated in gamma-IFN was markedly increased by the inclusion of 0.25 microgram/ml cycloheximide during the 24 h pre-incubation. These results indicate that IL-1 production may be inhibited by newly-synthesized proteins during maturation in vitro or differentiation of monocytes into macrophages. Pre-incubation in gamma-IFN and cycloheximide leads to separate but synergistic effects on the maintenance of LPS-induced IL-1 production in cultured monocytes.

Previous studies have described a 22 kD IL-1 inhibitor in the supernatant of human monocytes cultured on adherent immune complexes (J. Immunol. 134:3868, 1985). The studies reported herein further detail the conditions of production and biological properties of this IL-1 inhibitor. The inhibitor was produced by human monocytes cultured on adherent human IgG with maximal production between 8 and 24 hr. The IL-1 inhibitor was not performed in the cells but required transcription and new protein synthesis. The inhibitor blocked IL-1 augmentation of PHA-induced murine thymocyte proliferation but not IL-2-induced stimulation of CTLL or HT-2 cell lines. In addition, the inhibitor blocked IL-1-stimulated collagenase production from rabbit articular chondrocytes and IL-1-induced PGE2 production from human fibroblasts and synovial cells. The IL-1 inhibitor was not transforming growth factor beta (TGF beta) as determined by: the failure of anti-TGF beta antibodies to reduce IL-1 inhibitory activity, the separation of TGF beta from the IL-1 inhibitor by ion exchange chromatography, and the failure of TGF beta to inhibit IL-1-induced PGE2 production from synovial cells. IL-1 and the inhibitor showed no immunological cross-reactivity by Western blot analysis. The inhibitor specifically blocked binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1. These results indicate that a specific inhibitor of IL-1-induced immune and inflammatory cell responses is produced by monocytes cultured on adherent immune complexes or adherent IgG. This IL-1 inhibitor may be of importance in modulating the effects of IL-1 in the monocyte microenvironment.