In vitro effects of diacerhein and rhein on interleukin 1 and tumor necrosis factor-alpha systems in human osteoarthritic synovium and chondrocytes

OBJECTIVE: To evaluate the in vitro effects of diacerhein, a new drug for the treatment of osteoarthritis (OA), and its active metabolite, rhein, on interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) synthesis and expression in human OA synovial membrane, and on the IL-1beta and TNF-alpha receptors on human OA chondrocytes. METHODS: Levels of IL-1beta and TNF-alpha were determined using specific ELISA in culture medium of human synovial membrane explants incubated in the presence of 1 microg/ml of lipopolysaccharide with or without therapeutic concentrations of diacerhein (1.4, 2.7, 5.4 x 10(-5) M) and rhein (1.7, 3.5, 7.0 x 10(-5) M). IL-1beta mRNA level was quantitated by Northern blotting. Using radioligand binding experiments, we determined the effects of these agents on the density and affinity of chondrocyte IL-1 and TNF receptors. RESULTS: IL-1beta synthesis was significantly inhibited by diacerhein and rhein, with maximum inhibition at 5.4 x 10(-5) M for diacerhein (p < 0.02) and 3.5 x 10(-5) M for rhein (p < 0.05). The effect of both agents on IL-1beta was found to be translational and/or post-translational, judging by the absence of effect on gene expression level. Both agents produced dose and time dependent decreases in the number of IL-1 receptors (IL-1R) on OA chondrocytes. This effect was mediated through a reduction in the level of the type I IL-1R as shown by experiments using a blocking monoclonal antibody against this receptor type. Both agents also markedly reduced the IL-1 induced synthesis and expression of stromelysin 1. Neither diacerhein nor rhein significantly affected the level of synthesis of TNF-alpha or the level of TNF-R. CONCLUSION: Diacerhein and rhein can effectively inhibit the synthesis of IL-1beta on human OA synovium, as well as the action of this cytokine at the cartilage level, by reducing the number of chondrocyte IL-1R. The effects of these agents seemed "selective" to the IL-1 system.     

Interleukin-1 in multiple myeloma: producer cells and their role in the control of IL-6 production

We studied the role of interleukin (IL)-1beta in patients with multiple myeloma. By in situ hybridization and immunochemistry, myeloid and megakaryocytic cells expressed high levels of the IL-1beta gene and produced IL-1beta. Myeloma cells less potently expressed the IL-1beta gene and IL-1beta protein. IL-1beta gene expression was not constitutive since it was detected in the bone marrow myeloma cells of two patients, unlike circulating tumoural cells. In addition, nine myeloma cell lines failed to express the IL-1beta gene and this expression could not be induced by 12 different cytokines. We demonstrated that IL-1 was mainly responsible for IL-6 production in the tumoural environment through a PGE2 loop. In fact, an IL-1 receptor antagonist (IL-1RA) blocked PGE2 synthesis and IL-6 production by 80%; this blockage could be reversed by adding synthetic PGE2. Similar findings were found with indomethacin, an inhibitor of cyclooxygenase that blocks PGE2 synthesis. Taken together, these data emphasize the possibility of blocking IL-1 by using IL-1RA or other antagonists in order to block IL-6 production, which is a major tumoural survival and proliferation factor.     

Coexistence of CD14-dependent and independent pathways for stimulation of human monocytes by gram-positive bacteria

The cell wall is a key inflammatory agent of gram-positive bacteria. Possible receptors mediating cell wall-induced inflammation include CD14 and platelet-activating factor (PAF) receptor. To delineate the conditions under which these various receptors might be used, human monocytic THP-1 cells and heparinized whole human blood were stimulated with lipopolysaccharide (LPS), intact Streptococcus pneumoniae bacteria, or purified pneumococcal cell wall. THP-1 culture supernatant or cell-free plasma was analyzed for the presence of tumor necrosis factor, interleukin-1beta (IL-1beta), and IL-6. For the cultured monocytes, anti-CD14 inhibited induction of the inflammatory cytokines by the cell wall and LPS but not by intact pneumococcal bacteria. Despite the difference in CD-14 usage, the intracellular pathways induced by the three agents demonstrated similarities, as revealed in the presence of specific signal transduction inhibitors such as cholera toxin, pertussis toxin, and genistein. Cytokine production in whole human blood indicated that anti-CD14 failed to block responses to cell wall and intact pneumococci, whereas while LPS-induced responses were inhibited. PAF receptor antagonist had no effect under any conditions in both assays. These results indicate that although cell walls bind to both CD14 and PAF receptor, only CD14 appears to engender a cytokine response under restricted conditions. Furthermore, host cell responses to intact pneumococci are consistently independent of CD14 and PAF receptor.     

Impaired IL-1beta-induced neutrophil accumulation in tachykinin NK1 receptor knockout mice

Tachykinin NK1 receptors play an important role in the development of neurogenic inflammatory responses. We have used the murine air-pouch model to investigate whether the neurogenic component of the cellular inflammatory response to interleukin-1beta (IL-1beta, 10 ng into the air-pouch) is altered in NK1 receptor knockout mice compared to wild type controls. Air-pouches were washed following a 4 h IL-1beta treatment, the wash collected and neutrophil number estimated using a Neubauer haemocytometer. The response to IL-1beta was significantly attenuated in NK1 receptor +/- (40% reduction) and -/- mice (62% reduction) compared to wild type controls (+/+), whilst the response to cytokine-induced neutrophil chemoattractant (CINC, 0.3 microg) was unaffected. The response to substance P (7.5 nmol) was attenuated by approximately 50% in both NK1 receptor +/- and -/- mice compared to wild type controls. In conclusion NK1 receptors play a significant role in the cellular response to IL-1beta in a model of inflammation.     

A neuromodulatory role of interleukin-1beta in the hippocampus

It is widely accepted that interleukin-1beta (IL-1beta), a cytokine produced not only by immune cells but also by glial cells and certain neurons influences brain functions during infectious and inflammatory processes. It is still unclear, however, whether IL-1 production is triggered under nonpathological conditions during activation of a discrete neuronal population and whether this production has functional implications. Here, we show in vivo and in vitro that IL-1beta gene expression is substantially increased during long-term potentiation of synaptic transmission, a process considered to underlie certain forms of learning and memory. The increase in gene expression was long lasting, specific to potentiation, and could be prevented by blockade of potentiation with the N-methyl-D-aspartate (NMDA) receptor antagonist, (+/-)-2-amino-5-phosphonopentanoic acid (AP-5). Furthermore, blockade of IL-1 receptors by the specific interleukin-1 receptor antagonist (IL-1ra) resulted in a reversible impairment of long-term potentiation maintenance without affecting its induction. These results show for the first time that the production of biologically significant amounts of IL-1beta in the brain can be induced by a sustained increase in the activity of a discrete population of neurons and suggest a physiological involvement of this cytokine in synaptic plasticity.     

Activation of vagal afferents after intravenous injection of interleukin-1beta: role of endogenous prostaglandins

Intravenous administration of interleukin-1 (IL-1) activates central autonomic neuronal circuitries originating in the nucleus of the solitary tract (NTS). The mechanism(s) by which blood-borne IL-1 regulates brain functions, whether by operating across the blood-brain barrier and/or by activating peripheral sensory afferents, remains to be characterized. It has been proposed that vagal afferents originating in the periphery may monitor circulating IL-1 levels, because neurons within the NTS are primary recipients of sensory information from the vagus nerve and also exhibit exquisite sensitivity to blood-borne IL-1. In this study, we present evidence that viscerosensory afferents of the vagus nerve respond to intravenously administered IL-1beta. Specific labeling for mRNAs encoding the type 1 IL-1 receptor and the EP3 subtype of the prostaglandin E2 receptor was detected in situ over neuronal cell bodies in the rat nodose ganglion. Moreover, intravenously applied IL-1 increased the number of sensory neurons in the nodose ganglion that express the cellular activation marker c-Fos, which was matched by an increase in discharge activity of vagal afferents arising from gastric compartments. This response to IL-1 administration was attenuated in animals pretreated with the cyclooxygenase inhibitor indomethacin, suggesting partial mediation by prostaglandins. In conclusion, these results demonstrate that somata and/or fibers of sensory neurons of the vagus nerve express receptors to IL-1 and prostaglandin E2 and that circulating IL-1 stimulates vagal sensory activity via both prostaglandin-dependent and -independent mechanisms.     

The "interleukin 1 receptor antagonist" is a partial agonist of prostaglandin synthesis by human decidual cells

In many systems the interleukin-1 receptor antagonist opposes the effects of interleukin-1 beta. We considered that it might block interleukin-1 beta-stimulated prostaglandin production from human decidual cells. Very high levels of interleukin-1 receptor antagonist (> 1000 pg/ml) had limited inhibitory effects on IL-1 beta-stimulated PGE2 synthesis, and lower levels of antagonist (< 1000 pg/ml) increased the effects of IL-1 beta. Low concentrations of the antagonist alone (1-100 pg/ml) increased basal PGE2 production, whereas higher levels (10-100 ng/ml) had less effect. It seems, therefore, that in human decidua the "antagonist" is more accurately described as a partial agonist. It has been suggested that the IL-1 receptor antagonist could be used to inhibit decidual prostaglandin synthesis and thereby prevent preterm labor, but this report shows that caution should be exercised before using the receptor antagonist.     

p38 mitogen-activated protein kinase down-regulates nitric oxide and up-regulates prostaglandin E2 biosynthesis stimulated by interleukin-1beta

The inflammatory cytokine interleukin 1beta (IL-1beta) induces both cyclooxygenase-2 (Cox-2) and the inducible nitric-oxide synthase (iNOS) with increases in the release of prostaglandins (PGs) and nitric oxide (NO) from glomerular mesangial cells. However, the intracellular signaling mechanisms by which IL-1beta induces iNOS and Cox-2 expression is obscure. Our current studies demonstrate that IL-1beta produces a rapid increase in p38 mitogen-activated protein kinase (MAPK) phosphorylation and activation. Serum starvation and SC68376, a drug which selectively inhibits p38 MAPK in mesangial cells, were used to investigate whether p38 MAPK contributes to the signaling mechanism of IL-1beta induction of NO and PG synthesis. Serum starvation and SC68376 selectively inhibited IL-1beta-induced activation of p38 MAPK. Both SC68376 and serum starvation enhanced NO biosynthesis by increasing iNOS mRNA expression, protein expression, and nitrite production. In contrast, both SC68376 and serum starvation suppressed PG release by inhibiting Cox-2 mRNA, protein expression, and PGE2 synthesis. These data demonstrate that IL-1beta phosphorylates and activates p38 MAPK in mesangial cells. The activation of p38 MAPK may provide a crucial signaling mechanism, which mediates the up-regulation of PG synthesis and the down-regulation of NO biosynthesis induced by IL-1beta.     

Prediction of sleep disorders induced by beta-adrenergic receptor blocking agents based on receptor occupancy

beta-adrenergic receptor blocking agents (beta-blocking agents) have been widely used clinically for the treatment of various cardiovascular conditions. However, beta-blocking agents are liable to cause sleep disturbance, such as vivid dreams, nightmares, increased waking, and insomnia. The mechanisms of the sleep disorders are not known, but several may conceivably be responsible for these CNS-related side effects. In the present study, we hypothesized that the sleep disorders are induced by the blockade of central or peripheral beta 2 receptors and/or central serotonin (5-HT) receptors. To verify the hypothesis, we retrospectively analyzed the relationships between the extent of the sleep disorders and the beta 1, beta 2, or 5-HT receptor occupancies for four beta-blocking agents (atenolol, metoprolol, pindolol, and propranolol). No significant correlations were observed among pharmacokinetic/physicochemical parameters (therapeutic dose, plasma concentration, plasma unbound concentration, cerebrospinal fluid concentration, and lipid solubility) and pharmacodynamic parameters (the scores of the sleep disorders such as the number of dreams). Furthermore, no significant relationship (correlation coefficient: r < 0.3) was observed between beta 1 receptor occupancies of the drugs and the number of dreams. On the other hand, good relationships (r > 0.95) were observed between central and peripheral beta 2 or central 5-HT receptor occupancies and the number of dreams. These findings suggest that beta 2 and/or 5-HT receptor occupancy is superior to beta 1 receptor occupancy as an index for the sleep disorders.     

The morphogenic/cytotoxic and prostaglandin-stimulating activities of interleukin-1 beta in the rat ovary are nitric oxide independent

Nitric oxide (NO) has been implicated as a mediator of physiologic and pathologic cellular injury. Since the cytokine interleukin-1 beta (IL-1 beta) induces nitric oxide synthase (NOS) activity as well as effects morphogenic/cytotoxic changes and increased prostaglandin (PGE2) levels in cultured whole ovarian dispersates, we set out to determine whether these actions are interrelated. Treatment with IL-1 beta resulted in a marked increase in media nitrite and nitrate accumulation, morphological alterations, and increased release of lactate dehydrogenase (LDH) into media. Addition of IL-1 receptor antagonist (RA) eliminated these IL-1 beta effects. In contrast, specific inhibitors of NOS failed to reverse IL-1 beta-induced morphogenic changes or LDH release in spite of complete reduction of media nitrite to control levels. Similarly, treatment with transforming growth factor beta 1, inhibited IL-1 beta-induced nitrite accumulation, but had no effect on the morphologic or cytotoxic endpoints. Moreover, the addition of sodium nitroprusside, an NO generator, resulted in progressive increments in media nitrite content without a corresponding increase in the IL-1 beta-associated morphogenic changes or media LDH content. Furthermore, IL-1-induced PGE2 accumulation remained unaffected by specific NOS inhibition. These observations support the view that NO does not mediate the morphogenic/cytotoxic or inflammatory-like (e.g., PGE2 inducing) properties of IL-1 beta in cultured whole ovarian dispersates. Although the precise role of NO in ovarian physiology remains unknown, it is possible that NO participates in the periovulatory modulation of ovarian blood flow by virtue of its potent vasodilatory activity.     

Bispecific humanized anti-IL-2 receptor alpha beta antibodies inhibitory for both IL-2- and IL-15-mediated proliferation

Humanized anti-Tac (HAT) and Mik beta1 (HuMik beta 1) Abs directed at IL-2R alpha and IL-2R beta, respectively, inhibit IL-2 binding and biological activity and together act synergistically in vitro. The Abs have been used successfully in primate models of allograft rejection, graft-vs-host disease, and autoimmunity. We produced bifunctional humanized anti-IL-2R alpha beta Abs (BF-IgG) to combine the specificity of the two Abs into one entity by fusing HAT-producing NSO cells and HuMik beta 1-producing Sp2/0 cells. BF-IgG was purified using protein G-Sepharose affinity chromatography, followed by IL-2R alpha and IL-2R beta affinity chromatography and hydrophobic interaction chromatography. BF-IgG exhibited both anti-IL-2R alpha and anti-IL-2R beta specificities in binding assays. While the Ab binds the IL-2R with intermediate affinity (Kd = 2.82 nM), it does not inhibit IL-15 binding to its high affinity IL-15R. In Kit225/K6 (IL-2R alpha beta gamma+) cells, BF-IgG was 10-fold more potent than a HAT/HuMik beta 1 equimolar mixture in blocking IL-2-induced proliferation and, unexpectedly, was at least 65-fold more active than the mixture in blocking IL-15-induced proliferation. This dual inhibitory activity may be due to cross-linking of the IL-2R alpha and IL-2R beta, thus blocking IL-2 binding and possibly impeding the association of IL-2R beta with IL-15R. BF-IgG has potent immunosuppressant activities against both IL-2- and IL-15-mediated responses, and this antagonist could be more efficacious than HAT and/or HuMik beta 1 for the treatment of autoimmunity and the prevention of allograft rejection.     

Interleukin-1 beta induced transient diabetes mellitus in rats. A model of the initial events in the pathogenesis of insulin-dependent diabetes mellitus?

When aiming at preventing IDDM in man, knowledge of the molecular mechanisms leading to beta cell destruction may facilitate identification of new possible intervention modalities. A model of IDDM pathogenesis in man suggests that cytokines, and IL-1 in particular, are of major importance in the initial events (Nerup et al 1994) (Fig. 1). In vitro rat experiments demonstrated that rhIL-1 beta inhibits beta cell function and induces beta cell death both in isolated islets of Langerhans and in the isolated perfused pancreatic gland. With the long term goal of identifying new modalities capable of preventing IDDM in man, the aim af this review was to investigate the effects of rhIL-1 beta on beta-cell function and viability in normal rats. This review discussed 1) the pharmacokinetics of IL-1 beta in rats as the basis for choice of route of administration and dose of rhIL-1 beta, 2) the effects and molecular mechanisms of IL-1 beta on temperature and food intake used as control parameters for successful injection of rhIL-1 beta in rats, 3) the effects of one or more injection of IL-1 beta on rat beta cell function, 4) the molecular mechanisms leading to IL-1 beta induced beta cell inhibition in vivo, and some possible intervention modalities based on the molecular mechanisms, 5) the effects of IL-1 beta on spontaneous diabetes mellitus in DP BB rats, and 6) the effects and molecular mechanisms of IL-1 beta induced inhibition of thyroid epithelial cell function and aggravated thyroiditis in DP BB rats, compared to the effects of IL-1 beta on rat beta cell function. Finally, this review discussed the effects of IL-1 beta on human beta cells in vitro, and the clinical relevance of these experiments, with special reference to a clinical trial with the aim of preventing IDDM in man. The pharmacokinetic studies suggested that IL-1 beta is distributed according to a two-compartment model with a first-order elimination. Interleukin-1 beta reached all the investigated organs in the rats, was accumulated in kidneys and was excreted in the urine. The data suggested that IL-1 beta also accumulated in the islets of Langerhans. After injection of 4.0 micrograms/kg pathophysiologically relevant concentrations of rhIL-1 beta were reached and intact rhIL-1 beta persisted for up to 5 hrs in plasma. Peripheral injections of IL-1 beta dose-dependently induced fever and anorexia in rats, probably via induction of PGE2 in the brain or in peripheral tissues thereafter passing the blood-brain barrier. Nitric oxide produced by cNOS seems to be a molecular mediator of IL-1 beta induced fever but not of anorexia. Fever and anorexia are well described effects of IL-1 beta in rats, and are as such usefull control parameters of the absorption and biological activity of IL-1 beta after peripheral injection. Injections of rhIL-1 beta to normal, non-diabetes prone rats induced initial beta cell stimulation followed by inhibition, in accordance with in vitro data. Furthermore, induction of peripheral insulin resistance coincided with beta cell inhibition after one daily injection for 5 days, leading to a transient diabetes mellitus-like state, characterized by hyperglycemia and hypoinsulinemia. At this time point, electron-microscopy did not demonstrate beta cell destruction. However, IL-1 beta induced intercellularly edema and microvillous processes on the beta cells, which might be early evidence of apoptosis. The diabetes mellitus-like state was not aggravated if the daily injections were continued beyond 5 days. Daily injections of rhIL-1 beta for 2 to 4 weeks induced formation of blocking IL-1 beta-antibodies in normal rats. Hence, injections exceeding 2 weeks should only be performed using species homologous IL-1 beta. The molecular mechanism of IL-1 beta induced beta cell inhibition in rats in vivo as in vitro, are likely to involve binding of IL-1 beta to the IL-1RtI, since the IL-1RtII is considered to be a decoy receptor. (ABSTRACT TRUNCATED)     

Induction of biologically active IL-1 beta-converting enzyme and mature IL-1 beta in human keratinocytes by inflammatory and immunologic stimuli

IL-1beta, a major mediator of inflammatory and immunologic skin disease, undergoes post-translational site-specific cleavage by a novel cysteine protease termed IL-1beta-converting enzyme (ICE). Although in human skin keratinocytes produce significant amounts of the 31-kDa IL-1beta precursor protein, they fail under nonpathologic conditions to convert it to the 17.5-kDa bioactive form. In this study, we examined whether haptens and inflammatory agents might serve as stimuli for ICE activity in human keratinocytes, and, if so, whether ICE activity might precipitate enzymatic processing of IL-1beta to its 17.5-kDa form. Baseline levels of ICE mRNA were detected in keratinocyte cultures devoid of Langerhans cells and were up-regulated by nontoxic concentrations of the reactive hapten urushiol and by the irritant chemicals sodium lauryl sulfate and PMA. Although untreated keratinocytes expressed the 31-kDa form of the protein, 17.5-kDa IL-1beta was easily detected in keratinocytes and keratinocyte supernatants treated with either urushiol or the irritant chemicals. Enzymatic conversion from the 31-kDa to the 17.5-kDa form of IL-1beta was blocked by addition of a highly specific aldehyde inhibitor that contained a tetrapeptide recognition sequence specific for ICE, but not by an aldehyde inhibitor of a related ICE-like cysteine protease. Induction of IL-1beta-converting enzyme by immunologic and inflammatory stimuli may be one of the key regulatory elements in the pathogenesis of allergic and irritant contact hypersensitivity.     

Interleukin-1beta-induced cyclooxygenase-2 expression requires activation of both c-Jun NH2-terminal kinase and p38 MAPK signal pathways in rat renal mesangial cells

The inflammatory cytokine interleukin-1beta (IL-1beta) induces cyclooxygenase-2 (Cox-2) expression with a concomitant release of prostaglandins from glomerular mesangial cells. We reported previously that IL-1beta rapidly activates the c-Jun NH2-terminal/stress-activated protein kinases (JNK/SAPK) and p38 mitogen-activated protein kinase (MAPK) and also induces Cox-2 expression and prostaglandin E2 (PGE2) production. The current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 JNK2/SAPKbeta reduces Cox-2 expression and PGE2 production stimulated by IL-1beta. Similarly, overexpression of the kinase-dead form of p38 MAPK also inhibits IL-1beta-induced Cox-2 expression and PGE2 production. These results suggest that activation of both JNK/SAPK and p38 MAPK is required for Cox-2 expression after IL-1beta activation. Furthermore, our experiments confirm that IL-1beta activates MAP kinase kinase-4 (MKK4)/SEK1, MKK3, and MKK6 in renal mesangial cells. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta- induced Cox-2 expression with inhibition of both JNK/SAPK and p38 MAPK phosphorylation. Overexpression of the kinase-dead form of MKK3 or MKK6 demonstrated that either of these two mutant kinases inhibited IL-1beta-induced p38 MAPK phosphorylation and Cox-2 expression but not JNK/SAPK phosphorylation and activation. This study suggests that the activation of both JNK/SAPK and p38 MAPK signaling cascades is required for IL-1beta-induced Cox-2 expression and PGE2 synthesis.     

Differential effect of dexamethasone on interleukin 1beta- and cyclic AMP-triggered expression of GTP cyclohydrolase I in rat renal mesangial cells

1. Endogenous synthesis of tetrahydrobiopterin (BH4) is an essential requirement for cytokine-stimulated nitric oxide (NO) synthesis in rat mesangial cells. GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, is expressed in renal mesangial cells in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin (IL)-1beta and agents that elevate cellular levels of cyclic AMP. 2. We examined the action of the potent anti-inflammatory drug dexamethasone on GTP cyclohydrolase I induction in response to IL-1beta and a membrane-permeable cyclic AMP analogue, N6, O-2'-dibutyryladenosine 3'-5'-phosphate (Bt2cyclic AMP). 3. Nanomolar concentrations of dexamethasone markedly attenuated IL-1beta-induced GTP cyclohydrolase I mRNA steady state level as well as IL-1beta-induced GTP cyclohydrolase I protein expression and enzyme activity. In contrast, dexamethasone did not inhibit Bt2cyclic AMP-triggered increase in GTP cyclohydrolase I mRNA level and protein expression, and low (1 nM) or high (1 and 10 microM) doses of dexamethasone consistently increased Bt2cyclic AMP-induced GTP cyclohydrolase activity. 4. In summary, these results suggest that glucocorticoids act at several levels, critically dependent on the stimulus used, to control GTP cyclohydrolase I expression.