Antiinflammatory properties of hepatic acute phase proteins: preferential induction of interleukin 1 (IL-1) receptor antagonist over IL-1 beta synthesis by human peripheral blood mononuclear cells

This study was undertaken to determine whether acute phase proteins (APP) induce the synthesis of interleukin 1 beta (IL-1 beta) and its specific antagonist, IL-1 receptor antagonist (IL-1Ra), in human peripheral blood mononuclear cells (PBMC). PBMC from healthy volunteers were incubated with C-reactive protein (CRP), alpha 1-antitrypsin (alpha 1-AT), or alpha 1-acid glycoprotein (AGP), and the levels of IL-1 beta and IL-1Ra produced were measured by specific radioimmunoassay. To evaluate the effects of alpha 1-AT further, a synthetic pentapeptide FVYLI corresponding to the minimal binding sequence for the serpine-enzyme complex receptor was also evaluated. PBMC incubated for 24 h with CRP, alpha 1-AT, or the pentapeptide FVYLI synthesized large quantities of IL-1Ra, 5-10-fold greater than the amount of IL-1 beta produced by these cells. AGP induced significantly less IL-1Ra than the other APP tested. These effects were shown to be specific, in that polyclonal antibodies against CRP, alpha 1-AT, and AGP eliminated the cytokine production induced by these respective proteins. CRP, alpha 1-AT, FVYLI, and AGP were synergistic with low concentrations of endotoxin in the induction of both IL-1Ra and IL-1 beta synthesis. We suggest that the preferential induction of IL-1Ra by APP may contribute to their antiinflammatory effects and provide an important regulatory signal for the acute phase response.     

Sustained exposure to a glycine receptor partial agonist differentially alters NMDA receptor agonist and antagonist potencies in cultured spinal cord neurons

The present study examined the effects of K+ channel inhibitors on the basal tone and on KCl- or methacholine-induced contraction of the mouse-isolated trachea. Glibenclamide and iberiotoxin, procaine, quinine and tetraethylammonium did not induce any contraction of the indomethacin-treated mouse trachea. 4-Aminopyridine induced concentration-dependent contraction. This action of 4-aminopyridine was abolished by atropine and reduced by tetrodotoxin and nifedipine. Glibenclamide failed to modify KCl- or methacholine-induced contraction. Iberiotoxin and 4-aminopyridine potentiated KCl- and methacholine-induced contractions. Nifedipine, procaine, quinine and tetraethylammonium inhibited KCl- and methacholine-induced contractions. These data suggest that the closure of large Ca2+-dependent K+ channels can potentiate KCI- and methacholine-induced contraction. The effects of 4-aminopyridine on the mouse trachea reflect chiefly activation of muscarinic receptors. Procaine, quinine and tetraethylammonium inhibit depolarization-induced and receptor-mediated contractions of the mouse-isolated trachea.     

Interleukin-1 receptor antagonist synthesis by peripheral blood mononuclear cells in hemodialysis patients

BACKGROUND: Pro-inflammatory cytokines like interleukin (IL)-1 beta and tumor necrosis factor-alpha (TANF-alpha) are believed to play a significant role in dialysis-related morbidity. It has been previously demonstrated that the endogenous synthesis of interleukin-1 receptor antagonist (IL-1Ra) is a reliable marker of the level of IL-1 beta synthesis in hemodialysis (HD) patients. In this study, we assessed the impact of clinical and laboratory variables on IL-1Ra synthesis by peripheral blood mononuclear cells (PBMC) in patients on HD with unsubstituted cellulose dialyzers. METHODS: IL-1Ra by PBMC was measured by a specific non-cross-reactive radioimmunoassay. Day to day variation in cytokine synthesis, the correlation between cytokine synthesis under different in vitro stimulatory conditions, and the influence of clinical and laboratory variables on cytokine synthesis were studied. RESULTS: Although there was a trend towards greater IL-1Ra synthesis by unstimulated, endotoxin-stimulated and IgG-stimulated PBMC drawn before the second and third dialysis sessions of the week when compared to the first dialysis treatment, this was not statistically significant. There was a strong correlation between IL-1Ra synthesis by PBMC cultured under different stimulatory conditions that was best observed between IL-1Ra cell content and from endotoxin-stimulated PBMC (r = 0.51, P = 0.0001), and endotoxin- and IgG-stimulated PBMC (r = 0.44, P = 0.0001). In addition, there was a close correlation between total synthesis (cell associated and secreted) and secreted levels of IL-1Ra in unstimulated (r = 0.59, P = 0.0001) and endotoxin-stimulated PBMC (r = 0.69, P = 0.0001). Interestingly, there was an inverse correlation between IL-1Ra synthesis and duration of dialysis that was strongest for secreted IL-1Ra from unstimulated (r = -0.50, P = 0.002) and endotoxin-stimulated PBMC (r = -0.34, P = 0.04). There was no significant correlation between IL-1Ra synthesis by PBMC and other clinical and laboratory indices. CONCLUSIONS: The observations from this study indicate that: (1) in HD patients, there were no significant differences in cytokine synthesis by PBMC drawn before the three different dialysis treatments during the week; (2) there is a close relationship between IL-1Ra synthesis from PBMC cultured under different stimulatory conditions; (3) the secreted levels of IL-1Ra correlate directly with total synthesis (cell-associated and secreted); (4) with the exception of duration of dialysis, none of the other clinical or laboratory parameters correlated with cytokine synthesis; and (5) the diminished endotoxin- or IgG-stimulated IL-1Ra synthesis with increasing time on dialysis is possibly another sign of the impaired host-defense system in patients on long-term hemodialysis.     

GR89,696 is a kappa-2 opioid receptor agonist and a kappa-1 opioid receptor antagonist in the guinea pig hippocampus

Receptor binding studies and electrophysiological studies demonstrated the existence of at least two kappa opioid receptors, which have been designated kappa-1 and kappa-2. Several agonists and antagonists are selective for the kappa-1 receptor whereas no known ligands are selective for the kappa-2 receptor. In this study, the kappa opioid GR89,696 was tested in the guinea pig hippocampal slice preparation for kappa-1 versus kappa-2 activity. The perforant path-evoked population spike in the dentate was use to evaluate activity at the kappa-1 receptor, and the Schaffer collateral-evoked N-methyl-D-aspartate (NMDA) receptor-mediated synaptic current in CA3 pyramidal cells was used to measure kappa-2 receptor activation. GR89,696 had no effect on the perforant path-evoked dentate population spike; however, it did reverse the effects of the selective kappa-1 agonist U69,593 when co-perfused over the slices. In the CA3, GR89,696 inhibited the NMDA receptor-mediated synaptic current. The inhibition was antagonized by naloxone. The EC50 for GR89,696 on the NMDA current was 41.7 nM (95% CL, 7.0-248 nM). These findings indicate that GR89,696 is an agonist for kappa-2 opioid receptors and an antagonist at kappa-1 receptors in the guinea pig hippocampus.     

Circulating levels of interleukin 1 beta and of interleukin 1 receptor antagonist in systemic juvenile chronic arthritis

OBJECTIVE: To measure circulating interleukin 1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1Ra) levels in patients with systemic juvenile chronic arthritis (JCA) and to evaluate their correlation with disease activity. METHODS: IL-1 beta and IL-1Ra levels were measured by ELISA in 45 patients with JCA (20 systemic, 10 polyarticular and 15 pauciarticular) and in 15 healthy controls. RESULTS: Plasma IL-1 beta levels were undetectable in the majority of patients with systemic JCA, and detectable levels were not associated with different treatments or with parameters of disease severity. Serum IL-1Ra levels were markedly increased in patients with systemic JCA and significantly correlated with the persistence of systemic features, the extent and severity of joint involvement, and with C-reactive protein concentrations. Serum IL-1Ra levels were also significantly correlated with IL-6 levels. CONCLUSION: These results argue against a relevant role of IL-1 in systemic JCA. The increase in IL-1Ra levels does not appear to reflect an increase in IL-1 production, but may rather be induced by IL-6.     

Pharmacology and toxicology of ATOA, an AMPA receptor antagonist and a partial agonist at GluR5 receptors

(RS)-2-Amino-3-[3-(carboxymethoxy)-5-tert-butyl-4-isoxazolyl]propi onic acid (ATOA) has previously been described as an antagonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors with an IC50 value of 150 microM towards AMPA-induced depolarisation in the rat cortical wedge preparation. ATOA has now been shown also to be a partial agonist at recombinant GluR5 receptors, expressed in Xenopus oocytes, with an EC50 value of 170 microM and a relative efficacy of 0.17 +/- 0.04 compared with responses produced by kainic acid (1.0). Using cultured cerebral cortical neurones as a test system and leakage of lactate dehydrogenase (LDH) as an indicator of cell damage, ATOA was shown to be cytotoxic (ED50 > 300 microM), though much less toxic than the structurally related dual AMPA and GluR5 agonist, (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid (ATPA) (ED50 = 14 +/- 2 microM). The toxic effect of ATPA was sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX) but was not significantly reduced by the selective AMPA receptor antagonist, (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). The toxicity of ATOA (1 mM) could not be significantly attenuated by co-administration of AMOA (300 microM) or DNQX (25 microM). A structure-activity analysis indicates that the tert-butyl group of ATPA and ATOA facilitates the interaction of these compounds with GluR5 receptors.     

Monomeric isomers of human interleukin 5 show that 1:1 receptor recruitment is sufficient for function

The normally dimeric human interleukin 5 (IL-5) was re-engineered into two monomeric isomer forms to investigate mechanistic features of receptor recognition. One form, denoted GM1-IL-5, is a CD-loop expanded form, in which an 8-residue linker designed for flexibility was inserted between residues 85 and 86. The second, denoted DABC-IL-5, is a circularly permuted form of human IL-5 in which a chain discontinuity was introduced in the CD loop and the two consequent chain fragments were joined at the normal N and C termini by a di-glycyl linker. Both IL-5 isomers folded into stable monomers in solution as shown by sedimentation equilibrium and CD and formed an intrachain disulfide bond predicted from the structure of wild type IL-5. From titration microcalorimetry and optical biosensor analyses, both monomers were shown to interact with the IL-5 receptor alpha chain with 1:1 stoichiometry and affinities 30- to 40-fold weaker than for the dimeric wild type protein. And both monomers stimulated cell proliferation of human IL-5 receptor positive cells with a concentration dependence close to that of wild type. The data show that both monomeric and dimeric forms of IL-5 function through similar 1:1 receptor alpha chain recruitment processes and that it is the helical packing of the monomeric four-helix bundle unit in IL-5, rather than the helical connectivity itself, that appears to play the major role in presenting structural epitopes to trigger functional receptor activation.     

Kinetic study of the hepatobiliary transport of a new prostaglandin receptor agonist

To investigate whether acetylcholine is released in a similar fashion in different regions of the cortex, in vivo microdialysis was used to measure acetylcholine efflux simultaneously in the medial prefrontal and the frontoparietal cortex, under both basal conditions and following tactile stimulation. Additionally, the effects of including two different concentrations (0.05 microM and 0.5 microM) of a cholinesterase inhibitor (neostigmine) in the perfusion fluid were assessed. Basal levels of acetylcholine (i.e. during non-stimulated sessions) were similar in medial prefrontal and frontoparietal areas. Tactile stimulation reliably increased acetylcholine efflux in a similar fashion (up to 140% increase above baseline) in both cortical areas studied. Predictably, the higher concentration of neostigmine (0.5 microM) increased basal acetylcholine efflux by about 150% from levels observed with the lower neostigmine concentration (0.05 microM), but the concentration of local neostigmine had no effect on either the magnitude or the duration of the increased acetylcholine efflux following tactile stimulation. These results suggest that the pattern of acetylcholine release may be comparable in different areas of the cortex, supporting the idea that cholinergic projections from the basal forebrain to the cortex represent a globally regulated system. Furthermore, while the inclusion of neostigmine in perfusion fluid must be taken into account when interpreting acetylcholine efflux data, it appears that concentrations of up to 0.5 microM do not interfere fundamentally with the lability of cortical acetylcholine efflux in response to behavioural stimulation.     

Binding of an adenosine A1 receptor agonist and adenosine A1 receptor antagonist to sheep pineal membranes

The pineal organ of vertebrates produces melatonin and adenosine. In lower vertebrates, adenosine modulates melatonin production. We report herein that 2-chloro-cyclopentyl-[3H]-adenosine ([3H]CCPA: adenosine A1 receptor agonist) and [3H]-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX: adenosine A1 receptor antagonist), bind specifically to sheep pineal membranes. Binding of [3H]CCPA reached equilibrium at 90 min and dissociation revealed the presence of two components. Saturation analysis suggested the presence of a single population of binding sites (Kd = 1.67 +/- 0.06 nM, Bmax = 2386 fmol/mg protein). Binding was sensitive to GTP and GTPgammaS. Binding of [3H]DPCPX reached equilibrium at 60 min and dissociation was monophasic. Saturation analysis revealed a single population of binding sites (Kd = 5.8 +/- 1.12 nM, Bmax = 1116 fmol/mg protein). The specificity of the [3H]-analogues used and the rank order potency of the competitors tested in the competition experiments suggested the presence of A1 receptors. Future investigations are necessary to elucidate the significance of the differences observed between the binding properties of the adenosine A1 receptor agonist and adenosine A1 receptor antagonist.     

Dopamine D1B receptor chimeras reveal modulation of partial agonist activity by carboxyl-terminal tail sequences

NNC 01-0012, a second-generation benzazepine compound, pharmacologically differentiates multiple vertebrate D1 receptor subtypes (D1A, D1B, D1C, and D1D) and displays high selectivity and affinity for dopamine D1C receptors. Functionally, whereas NNC 01-0012 acts as a full or poor antagonist at D1C and D1A receptor-mediated cyclic AMP production, respectively, it exhibits partial agonist activity at the D1B receptor. To define some of the structural motifs that regulate the pharmacological and functional differentiation of vertebrate dopamine D1 receptors by NNC 01-0012, a series of receptor chimeras were constructed in which the divergent carboxyl-terminal (CT) receptor tails were replaced with the corresponding sequences of D1A, D1B, or D1C receptors. Substitution of the vertebrate D1B carboxyl-terminal-tail at position Tyr345 with carboxyl-terminal-tail sequences of the D1A receptor abolished the partial agonist activity of NNC 01-0012 without affecting dopamine-stimulated cyclic AMP accumulation. At vertebrate D1B/D1CcT-tail receptor mutants, however, the intrinsic activity of the partial agonist NNC 01-0012 (10 microM) was markedly enhanced (approximately 60% relative to 10 microM dopamine) with no concomitant alteration in the molecule's ligand binding affinity or constitutive activity of the chimeric receptor. Similar results were obtained with other benzazepines such as SKF-38393 and SCH-23390, which act as partial agonists at vertebrate D1B receptors. Substitution of D1A and D1C receptor carboxyl-terminal tails with sequences encoded by the D1B receptor carboxyl-terminal tail did not, however, produce receptors with functional characteristics significantly different from wild type. Taken together, these data clearly suggest that in addition to well-characterized domains and amino acid residues in the third cytoplasmic loop, partial agonist activity at the D1B receptor is modulated by sequence-specific motifs within the carboxyl-terminal tail, a region that may underlie the possible structural basis for functionally divergent roles of multiple dopamine D1-like receptors.     

Combined treatment with a 5HT1A receptor agonist and a muscarinic acetylcholine receptor antagonist disrupts water maze navigation behavior

In the present study, the effects of PACAP27, PACAP38 and VIP in a concentration range from 10(-13) to 10(-6) M were studied in vitro on the spontaneous and directed mobility of lymphocytes from rat spleen and thymus. The results show that VIP and both PACAPs inhibit significantly and in a similar way the mobility of lymphocytes from thymus and spleen, and the maximal effects were observed at 10(-9) M and 10(-8) M. The three neuropeptides significantly increased cAMP concentrations. Moreover, incubation with increasing PMA concentrations showed a progressive enhancement of chemotaxis of lymphocytes, which was partially prevented by VIP, and both PACAPs. Incubation with forskolin caused decrease in the chemotaxis of thymocytes and splenocytes, and the presence of VIP or PACAP peptides was not synergistic in the inhibitory effect on lymphocyte chemotaxis, suggesting that the three neuropeptides and forskolin mediate their actions by the same intracellular pathway. This study showed the ability of the VIP receptor antagonist (N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2 to partially reverse the inhibitory effect of both PACAPs and VIP on chemotaxis, suggesting that PACAP receptors are identical or very similar to VIP receptors in both thymocytes and splenocytes. These data suggest that PACAP27 and PACAP38 can be included as two novel immunoregulatory peptides that can modulate cell mobility on central and peripheral lymphoid organs.     

Porcine hepatic response to sepsis and its amplification by an adrenergic receptor alpha1 agonist and a beta2 antagonist

CDK9 has been recently shown to have increased kinase activity in differentiated cells in culture and a differentiated tissue-specific expression in the developing mouse. In order to identify factors that contribute to CDK9's differentiation-specific function, we screened a mouse embryonic library in the yeast two-hybrid system and found a tumor necrosis factor signal transducer, TRAF2, to be an interacting protein. CDK9 interacts with a conserved domain in the TRAF-C region of TRAF2, a motif that is known to bind other kinases involved in TRAF-mediated signaling. Endogenous interaction between the two proteins appears to be specific to differentiated tissue. TRAF2-mediated signaling may incorporate additional kinases to signal cell survival in myotubes, a cell type that is severely affected in TRAF2 knockout mice.     

The agonist selectivity of a mouse B1 bradykinin receptor differs from human and rabbit B1 receptors

A genomic clone encoding the mouse B1 receptor was isolated by homology to the human B1 receptor cDNA. The deduced amino acid sequence of the mouse B1 receptor is 72% identical to the human B1 receptor and 73% identical to the rabbit B1 receptor. Ligand binding studies of the mouse B1 receptor expressed in COS cells indicate that it has the pharmacological properties associated with the B1 receptor subtype. However the pharmacology of the mouse receptor is unique in that it possesses a 2-3-fold selectivity for the 'classical' B1 agonist des-Arg9BK over the agonist des-Arg10 kallidin. In contrast, the human and rabbit B1 receptors exhibit an approx. 2000- and 150-fold selectivity, respectively, for des-Arg10kallidin over des-Arg9BK. Thus relative to the human and rabbit B1 receptors the mouse B1 receptor has the opposite selectivity for kinin agonists. The DNA sequence of the region encoding bradykinin was determined for two different mouse kininogen cDNA clones, both encode the sequence Arg-BK. Antipeptide antibodies directed against a C-terminal peptide of the human B1 receptor were produced. Initial characterization of this antibody indicates that it detects specific bands by Western blot analyses that are present in membranes prepared from COS cells transfected with the human B1 receptor cDNA but not from mock transfected COS cells.     

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.     

Induction of mitogen-inducible nuclear orphan receptor by interleukin 1 in human synovial and gingival fibroblasts

High levels of interleukin 1 (IL-1) found in inflammatory diseases such as rheumatoid arthritis and periodontitis act on the local fibroblasts, resulting in an altered phenotype characterized by hyperplasia and the production of inflammatory mediators and destructive enzymes. The goal of this study was to identify genes induced as an early response to IL-1 in synovial and gingival fibroblasts which might play a regulatory role in the cascade of events leading to their activation. Using the technique of mRNA differential display, we have identified the mitogen-inducible nuclear orphan receptor (MINOR) as a gene up-regulated by IL-1 in human synovial and gingival fibroblasts. The rapid induction of both mRNA and DNA binding activity suggests that MINOR may play an important early role in regulating the response of fibroblasts to inflammation.     

Sulpiride anxiogenic-like effect inhibition by a D1 dopamine receptor antagonist

The increased latency to explore a white compartment from a black one into which a mouse has been introduced, appears to be correlated with its anxiety level. In this test, the D2 specific dopamine receptor antagonist (+/-) sulpiride dose dependently (5-40) mg kg-1 i.p.) increased this latency. This effect was suppressed by the D1 specific dopamine receptor antagonist SCH 23390 (25 micrograms kg-1 s.c.) which intrinsically displayed an apparent anxiolytic effect.     

A His19 to Ala mutant of melanoma growth-stimulating activity is a partial antagonist of the CXCR2 receptor

Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.