The modulation of intra-articular inflammation, cartilage matrix and bone loss in mono-articular arthritis induced by heat-killed Mycobacterium tuberculosis.

Inflammation models for the assessment of anti-rheumatic drug activity utilize a variety of stimuli and sites. However, the determination of cartilage and bone degradation remains time consuming and problematic. A rapid rat model of Mycobacterium tuberculosis monoarticular arthritis with induces of inflammation, as well as patellar cartilage proteoglycan and bone degradation has been reported. This study characterizes this model with respect to the actions of anti-rheumatic drugs. Dexamethasone, cyclosporin and prednisolone inhibited all three parameters. Methotrexate inhibited joint inflammation alone, whilst azathioprine was without effect. Levamisole inhibited cartilage and bone degradation without affecting joint inflammation. NSAIDs were divided in their actions. Naproxen, piroxicam, diclofenac and tiaprofenic acid all inhibited joint inflammation and bone loss, but naproxen and piroxicam both significantly potentiated cartilage proteoglycan loss. This model appears to rely on cellular recruitment at this early stage, the anti-metabolites being ineffective. The modulation of inflammation can result in a protection against cartilage and bone damage in arthritis; however, certain NSAIDs are detrimental to cartilage integrity. The pharmacological manipulation of inflammatory arthritis can therefore dislocate inflammation from its effects on tissue destruction.

Mechanisms underlying reduced responsiveness of neonatal neutrophils to distinct chemoattractants.

Potential mechanisms underlying impaired chemotactic responsiveness of neonatal neutrophils were investigated. Two distinct chemoattractants were compared: bacterially derived N-formyl-methionyl-leucyl-phenylalanine (fMLP) and a unique chemotactic monoclonal antibody, designated DL1.2, which binds to a neutrophil antigen with an apparent molecular mass of 120 kDa. Chemotaxis of neutrophils toward fMLP, as well as DL1.2, was reduced in neonates when compared with adult cells. This did not appear to be a result of decreased fMLP receptor or DL1.2 antigen expression by neonatal neutrophils. fMLP, but not DL1.2, induced a rapid increase in intracellular calcium in adult and neonatal cells, which reached a maximum within 30 s. The calcium response of cells from neonates to fMLP was reduced when compared with adult cells, and an unresponsive subpopulation of neonatal neutrophils was identified. NF-kappaB nuclear binding activity induced by fMLP and DL1.2, as well as expression of the p65 NF-kappaB subunit and IkappaB-alpha, was also significantly reduced in neonatal cells, when compared with adult cells. In contrast, although fMLP, but not DL1.2, activated p42/44 and p38 mitogen-activated protein (MAP) kinases in neutrophils, no differences were observed between adults and neonates. Chemotaxis of adult and neonatal neutrophils toward fMLP and DL1.2 was also blocked to a similar extent by inhibitors of phosphatidylinositol 3-kinase, as well as an inhibitor of NF-kappaB. These findings indicate that reduced chemotactic responsiveness in neonatal neutrophils is a result of, at least in part, aberrations in chemoattractant-induced signaling. However, the biochemical pathways mediating this defect appear to be related to the specific chemoattractant.

Prooxidant and antioxidant functions of nitric oxide in liver toxicity.

In response to tissue damage and inflammation induced by a variety of xenobiotics including acetaminophen, carbon tetrachloride, ethanol, galactosamine, and endotoxin, as well as disease states such as viral hepatitis, and postischemic and regenerative injury, the liver produces large quantities of nitric oxide. Indeed, nearly all cell types in the liver including hepatocytes, Kupffer cells, stellate cells, and endothelial cells have the capacity to generate nitric oxide. Thus, these cells, as well as infiltrating leukocytes, may indirectly augment tissue injury. In many models of liver damage, nitric oxide and its oxidation products such as peroxynitrite contribute to the injury process by directly damaging the tissue or by initiating additional immunologic reactions that result in damage. In some models, nitric oxide donors or peroxynitrite can mimic the cytotoxic actions of liver toxins. Moreover, agents that prevent the generation of nitric oxide or antioxidants that bind reactive nitrogen intermediates, or knockout mice with reduced capacity to produce nitric oxide, are protected from xenobiotic-induced tissue injury. In contrast, there have been reports that blocking nitric oxide production enhances xenobiotic-induced tissue injury. This has led to the concept that nitric oxide either inactivates proteins critical for xenobiotic-induced tissue injury or acts as an antioxidant, reducing cellular levels of cytotoxic reactive oxygen intermediates. Whether or not nitric oxide or secondary oxidants generated from nitric oxide act as mediators of tissue injury or protect against toxicity is likely to depend on the precise targets of these reactive nitrogen intermediates, as well as levels of superoxide anion present and the extent to which tissue injury is mediated by reactive oxygen intermediates. In addition, as toxicity is a complex process involving a variety of cell types and many soluble mediators, the contribution of each of these factors must be taken into account when considering the role of nitric oxide as a determinant of tissue injury.

Minimal amidine structure for inhibition of nitric oxide biosynthesis.

Pharmacological modulation of nitric oxide synthase activity has been achieved using structural analogs of arginine. In the present studies, we demonstrated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cultures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a concentration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expressing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidine had no effect on gamma-interferon-induced expression of nitric oxide synthase protein, its enzymatic activity was blocked. Kinetic analysis revealed that formamidine acts as a simple competitive inhibitor with respect to arginine (K(i) formamidine approximately 800 microM). Using a polarographic microsensor to measure real-time flux of nitric oxide release from RAW 264.7 macrophages, formamidine was found to require 30-90 min to inhibit enzyme activity, suggesting that cellular uptake of the drug may limit its biological activity. Our data indicate that formamidine is an effective inhibitor of nitric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased production of nitric oxide.

Phasic production of urinary pyridinium crosslinks in mice: the effect of ovariectomy.

Considerable sample to sample variability in deoxypyridinoline crosslink/creatinine (Dpd/CREAT) ratio values was confirmed when twice-weekly sampling for 77 days was performed in C57 mice. Analysis of samples from individual mice indicated that, in the majority of mice in a given group (54-67%), phasic changes occurred with periodic peaks as much as 4-5 times basal values. Alignment of peaks in the individual time courses of mice revealed a clear cyclic crosslink production (periodicity 12-14 days) for the population, although not all mice gave a peak in every case. Ovariectomy (OVX) (compared to sham-operated mice) increased mean values of crosslink production by either C57 or C57 x 129 F1 mice from about 10 days after operation with highest values between 21 and 35 days, and then a decrease in the difference between sham and OVX, particularly in C57 mice. Analysis of both time courses for individual mice and distribution curves of the data from groups of mice indicated that OVX consistently increased basal crosslink values (6-9 ratio units) with phasic peak values superimposed. The peak alignment analysis reinforced this observation. The influence of the background variability was avoided by pooling data over one cycle time and the use of nonparametric statistics, and the effect of OVX was analyzed in several strains of mice. Crosslink levels and phasic production declined with age but were also influenced by manipulation of the mice, suggesting that experimental conditions should be rigorously controlled when this urinary crosslink measure is used as a parameter in mouse models of OVX-induced osteoporosis.

Mechanisms involved in prostaglandin-induced increase in bone resorption in neonatal mouse calvaria.

Prostaglandins (PG) E1, E2 and F2alpha induce bone resorption in isolated neonatal parietal bone cultures, and an associated increase in interleukin-6 (IL-6) production. Indomethacin had little effect on the response to PGE2, or the relatively non-selective EP receptor agonists 11-deoxy PGE1 and misoprostol, but blocked the effects of PGF2alpha and the F receptor agonist fluprostenol, indicating an indirect action via release of other prostaglandins. It is more likely that there is positive autoregulation of prostaglandins production in this preparation mediated via stimulation of F receptors. The effects of selective EP receptor agonists sulprostone (EP1,3) and 17-phenyl trinor PGE2(EP1), indicated the involvement of EP2 and/or EP4 receptors, which signal via cAMP. The relatively weak increase in IL-6 production by misoprostol (with respect to resorption) suggests that these responses are controlled by different combination of EP2 and EP4 receptors. The PKA activator, forskolin, induced small increases in bone resorption at lower concentrations (50-500 ng/ml) but a reversal of this effect, and inhibition of resorption induced by other stimuli (PTH, PGE2), at higher concentrations (0.5-5 microg/ml). IL-6 production was markedly increased only at the higher concentrations. The inhibitory effect of forskolin may be a calcitonin-mimetic effect. PMA induced both resorption and IL-6 production which were both blocked by indomethacin, indicating a role for PKC in the control of prostaglandin production.

Factors influencing the effect of the soluble IL-6 receptor on IL-6 responses in HepG2 hepatocytes.

The soluble IL-6 receptor (sIL-6R) can increase IL-6-induced signalling by forming a complex with IL-6 and membrane-bound gp130 (the receptor beta chain which transduces signals). The conditions affecting this response to sIL-6R were studied using fibrinogen release from HepG2 hepatocytes. Exogenous sIL-6R had no effect alone or in the presence of a submaximal concentration of IL-6, but increased responses to supramaximal IL-6 concentrations in a concentration-related manner. Dexamethasone increased the expression of the membrane IL-6R and endogenous sIL6R release, and increased responses to supramaximal but not submaximal IL-6 concentrations. The amount of endogenous sIL-6R released is relatively small and is unlikely to influence the effects of the exogenous sIL-6R. The observed concentration-related decrease in sIL-6R production in the presence of IL-6 may indicate internalization of ligand/receptor complexes. This would significantly decrease the amount of IL-6R (soluble or membrane) available for signalling and limit continued functional response later in the cultures. These data indicate that the major factor influencing responses to exogenous sIL-6R is an excess of IL-6 which is necessary to form complexes with the sIL-6R, which can then interact with gp130 to increase signalling.

The use of TNF family ligands and receptors and agents which modify their interaction as therapeutic agents.

The earlier known TNF family cytokines have fairly wide physiological actions, mainly in inflammation and immune responses. It is now considered feasible to develop these large proteins themselves as therapeutic agents, but in addition, modular organisation of structures of biological proteins, and the identification of localised ligand-receptor interaction sites, allow rational design of smaller, preferably nonpeptide molecules which interfere with these protein:protein interactions. Neutralising anti-TNF antibodies and soluble TNF receptor preparations were shown to have striking anti-inflammatory activities in clinical studies, particularly in rheumatoid arthritis. As the TNF beta:TNFR1 co-crystal structure was the first in the family to be solved, rational drug design based on the ligand:receptor interaction sites is more advanced. Ligand mutations and a peptide sequence from TNF-alpha have given useful information regarding ligand-receptor interactions. Small peptide sequences from TNFR I which interact with the ligand have shown some activity in blocking the biological actions of TNF. The physiological activities of several recently-discovered ligands are more limited, giving possibilities for selective treatment of several diseases. For example, TRAIL can induce apoptosis in a wide range of tumour cells with little effects on normal tissues, both in vitro and in vivo. The co-crystal structure of TRAIL with one of its signalling receptors TRAILR 2 has been solved, opening the way to rational small molecule drug design. TRANCE (RANKligand) has modulatory effects on the dendritic cell:T cell interaction in immune responses. However, it plays a more major controlling role in the development of osteoclasts and their bone resorbing activity. In this way, it is a very interesting drug development target for the treatment of bone disorders such as osteoporosis. A recombinant secreted inhibitory receptor, osteoprotegerin (OPG), is in Phase 1 clinical trial for the treatment of hyper-resorptive bone diseases. However, OPG also blocks TRAIL and may not be sufficiently specific in long term therapy, but it is hoped that inhibitors of the interaction of TRANCE and its specific signalling receptor, RANK, can be rationally designed.

Dual action of the benzodiazepine receptor inverse agonist RU34347 on responses to exogenously applied GABA in the rat cerebellar slice.

The benzodiazepine receptor inverse agonist has been shown to produce agonist-like effects at low concentrations. RU34347 has both inverse agonist (attenuation of GABA-responses) and agonist-like (reduction of spontaneous Purkinje cell firing rate) in the cerebellar slice preparation. The benzodiazepine antagonist flumazenil prevented the inverse agonist actions, but only partially reduced the agonist-like effects. Further, brief application of RU34347 to slices mimicked the response to GABA, and pharmacological investigation determined that this action was mediated through increased GABA through action at a site proximal to the parallel fiber-basket cell synapse, at an as yet undetermined receptor.

Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: alterations in polyamine biosynthesis and the expression of p21/CIP1/WAF1.

We examined the effects of epidermal growth factor (EGF) on MDA-MB-468 cells to understand its mechanism of action in an EGF receptor-rich breast cancer cell line. EGF inhibited the growth of MDA-MB-468 cells with an IC50 of 1.5 +/- 0.5 nM, as determined by measurements of DNA content of cells in culture over a period of 4 to 6 days. This growth inhibition included apoptosis 24 h after EGF addition, as detected by an enzyme-linked immunosorbent assay (ELISA) and Hoechst 33342 staining. In EGF-treated cells, peak activities of two key enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC), were reduced by 57% and 83%, respectively. EGF treatment also caused a 30 to 50% decrease in cellular putrescine at all time points tested (12 to 48 h). EGF-induced inhibition of DNA synthesis was also partially reversed by the addition of putrescine or spermidine, but not by spermine. Western blot analysis of cell cycle regulatory proteins showed that EGF-mediated growth inhibition was associated with the induction of p21, an inhibitor of cyclin-dependent kinases. However, EGF had no significant effect on the expression of cyclin D1 or cyclin E. Furthermore, putrescine reversal of EGF effects was associated with the down-regulation of EGF-induced p21. These results suggest that the mechanism of growth inhibition by EGF in MDA-MB-468 cells include a down-regulation of polyamine biosynthesis and the induction of p21. Identification of growth regulatory pathways in breast cancer cells might be useful in the development of novel targets for therapeutic intervention.

Dual mechanisms of action of interferon-gamma in potentiating responses to LPS in mice: IL1, TNFalpha and IL6 production in serum and hypothermia.

IFNgamma potentiates the production of serum cytokines and mortality induced by LPS, but these responses do not change in parallel, and the underlying mechanisms are not clear. Pretreatment of mice with 15 microg rrIFNgamma intraperitoneally (IP) resulted in potentiation of LPS-induced serum cytokine production and hypothermia, but these changes depended on the pretreatment time and did not occur in parallel. TNFalpha and IL1beta levels showed peak potentiation after 8-h-IFNgamma pretreatment which may result from a process of sensitization of mechanisms involved in LPS responses. IL6 levels were most markedly potentiated after 3- and 6-h-IFNgamma-pretreatment and hypothermia was markedly potentiated after 0-8 h pretreatments. These effects may result from an additional synergistic action of IFNgamma with other mediators when it is present at significant levels earlier after its injection, given that IFNgamma had little (hypothermia) or no effect (cytokines) alone. The degree of potentiation induced by 18-h-IFNgamma pretreatment was related to the dose of LPS, the maximum response having been increased. Two injections of IFNgamma at 42 and 18 h prior to LPS induced greater increases in TNFalpha and IL1beta production than 18-h pretreatment alone, but not in IL6 production or hypothermia. There may be a maximum level of IL6 production which was surpassed under these conditions. These findings suggest that a balance of sensitizing and synergistic actions of IFNgamma with other mediators such as IL1 and TNFalpha, are the major mechanisms underlying its potentiation of LPS responses in mice.

Inhibition of macrophages with gadolinium chloride alters intercellular adhesion molecule-1 expression in the liver during acute endotoxemia in rats.

Cell adhesion molecules are important for localized accumulation of phagocytes at sites of tissue damage. In the present studies, we analyzed the effects of blocking hepatic macrophages on expression of beta2 integrins and intercellular adhesion molecule-1 (ICAM-1) adhesion molecules on liver cells during acute endotoxemia. Flow cytometric analysis revealed distinct subpopulations of macrophages from control animals that varied on the basis of their size and density. In contrast, hepatocytes and endothelial cells were relatively homogeneous. Treatment of rats with endotoxin (5 mg/kg, intravenously) resulted in a time-dependent increase in the percentage of small, dense macrophages and a progressive loss of larger, less-dense cells. In contrast, no major effects were observed on the physical properties of hepatocytes or endothelial cells. ICAM-1 was found to be constitutively expressed on endothelial cells and hepatocytes, as well as on macrophages. Induction of acute endotoxemia resulted in a time-dependent increase in ICAM-1 expression on hepatocytes, which was observed within 3 hours and reached a maximum after 24 hours. An increase in ICAM-1 expression was also observed on endothelial cells and on macrophages at 3 hours, followed by a decrease at 24 to 48 hours. Macrophages and endothelial cells also constitutively expressed beta2 integrins. Induction of acute endotoxemia had no effect on beta2 integrin expression by these cells. Pretreatment of rats with gadolinium chloride (GdCl3), a macrophage inhibitor known to block endotoxin-induced liver injury, abrogated the effects of endotoxin on ICAM-1 expression by hepatocytes and macrophages. In contrast, ICAM-1 expression on endothelial cells increased. Interestingly, treatment of rats with GdCl3 alone resulted in a marked increase in expression of ICAM-1 on endothelial cells and hepatocytes, and of beta2 integrins on macrophages and endothelial cells. Taken together, these data suggest that ICAM-1 is involved in mediating macrophage adherence and accumulation in the liver during endotoxemia. Furthermore, macrophages appear to regulate expression of this cell adhesion molecule on parenchymal cells.

Inhaled nitric oxide primes lung macrophages to produce reactive oxygen and nitrogen intermediates.

Inhaled nitric oxide is a selective pulmonary vasodilator used for the treatment of pulmonary hypertension. The potential adverse effects of inhaled nitric oxide are unknown and represent the focus of the present studies. Whereas inhalation of nitric oxide (10 to 100 ppm, 5 h) by Balb/c mice had no effect on the number or type of cells recovered from the lung, a dose-related increase in bronchoalveolar lavage protein was observed, suggesting that nitric oxide induces alveolar epithelial injury. To determine if this was associated with altered alveolar macrophage activity, we quantified production of reactive oxygen and nitrogen intermediates by these cells. Interferon-gamma, alone or in combination with lipopolysaccharide (LPS), induced expression of inducible nitric oxide synthase (iNOS) protein and nitric oxide production by alveolar macrophages. Cells from mice exposed to 20 to 100 ppm nitric oxide produced significantly more nitric oxide and expressed greater quantities of iNOS than cells from control animals. Superoxide anion production and peroxynitrite generation by alveolar macrophages were also increased after exposure of mice to nitric oxide. This was correlated with increased antinitrotyrosine antibody binding to macrophages in histologic sections. Taken together, these data demonstrate that inhaled nitric oxide primes lung macrophages to release reactive oxygen and nitrogen intermediates. Increased production of these mediators by macrophages following inhalation of nitric oxide may contribute to tissue injury.

Selective enhancement of LPS-induced serum TNF-alpha production by carrageenan pretreatment in mice.

1. Bacterial lipopolysaccharide (LPS)-induced increases in serum cytokines (TNF-alpha, IL-1 and IL-6) and hypothermia were studied in mice sensitized by carrageenan pretreatment and compared with mice sensitized with heat-killed P. acnes or IFN-gamma, all given IP at appropriate intervals (24 hr, 7 days and 12-18 hr, respectively) before LPS. 2. In mice with localized peritoneal inflammation induced by carrageenan, peak TNF-alpha levels (1.5-2 h after LPS) were markedly enhanced after both doses of LPS tested (50 and 200 microg/mouse IP). However, IL-1beta levels were not changed and IL-6 levels were decreased only after the higher dose of LPS. Hypothermia showed weak and inconsistent changes in carrageenan-sensitized mice. 3. IL-1beta levels in spleen lysates were higher but paralleled those in the serum, being increased in IFN-gamma-sensitized but not in carrageenan-sensitized mice. The levels of both TNF-alpha and IL-1beta were high in the peritoneum of carrageenan-sensitized mice, suggesting that the increased serum TNF-alpha did not emanate from the peritoneum. 4. In mice sensitized with the other two agents, as expected, the levels of all three cytokines increased, but peak levels were attained at the same times post-LPS (TNF-alpha: 1-1.5 hr; IL-1: 3-4 hr; IL-6: 3-4 hr). In addition, hypothermia was increased with both of these methods of sensitization. 5. The lack of consistent correlation of the levels of cytokines studied, particularly TNF-alpha, with the degree of hypothermia, raises questions as to their causative role in its induction in these models. 6. The mechanisms underlying these models of sensitization are clearly different, and further understanding of these mechanisms would aid in the interpretation of the effects of drugs in the models.

Role of nitric oxide in acetaminophen-induced hepatotoxicity in the rat.

Acetaminophen is a mild analgesic and antipyretic agent known to cause centrilobular hepatic necrosis at toxic doses. Although this may be due to a direct interaction of reactive acetaminophen metabolites with hepatocyte proteins, recent studies have suggested that cytotoxic mediators produced by parenchymal and nonparenchymal cells also contribute to the pathophysiological process. Nitric oxide is a highly reactive oxidant produced in the liver in response to inflammatory mediators. In the present studies we evaluated the role of nitric oxide in the pathophysiology of acetaminophen-induced liver injury. Treatment of male Long Evans Hooded rats with acetaminophen (1 g/kg) resulted in damage to centrilobular regions of the liver and increases in serum transaminase levels, which were evident within 6 hours of treatment of the animals and reached a maximum at 24 hours. This was correlated with expression of inducible nitric oxide synthase (iNOS) protein in these regions. Hepatocytes isolated from both control and acetaminophen-treated rats were found to readily synthesize nitric oxide in response to inflammatory stimuli. Cells isolated from acetaminophen-treated rats produced more nitric oxide than cells from control animals. Production of nitric oxide by cells from both control and acetaminophen-treated rats was blocked by aminoguanidine, a relatively specific inhibitor of iNOS. Arginine uptake and metabolism studies revealed that the inhibitory effects of aminoguanidine were due predominantly to inhibition of iNOS enzyme activity. Pretreatment of rats with aminoguanidine was found to prevent acetaminophen-induced hepatic necrosis and increases in serum transaminase levels. This was associated with reduced nitric oxide production by hepatocytes. Inhibition of toxicity was not due to alterations in acetaminophen metabolism since aminoguanidine had no effect on hepatocyte cytochrome P4502E1 protein expression or N-acetyl-p-benzoquinone-imine formation. Taken together, these data demonstrate that nitric oxide is an important mediator of acetaminophen-induced hepatotoxicity.

Increases in osteocalcin after ovariectomy are amplified by LPS injection: strain differences in bone remodelling.

1. LPS (Escherichia coli serotype 0111:B4, 300 micrograms/mouse IP) increases serum osteocalcin in normal female C57B16 mice from 2 to 6 hr after its injection, with peak levels at 2-4 hr after LPS. 2. Both basal and LPS-stimulated serum osteocalcin were markedly inhibited by dexamethasone (10 mg/kg IP). 3. When observed 3 hr after LPS injection, serum osteocalcin was increased by ovariectomy (OVX) (with respect to sham-operated mice) and this increase was amplified in LPS-treated mice. This increase in osteocalcin was maximal 14 days after OVX, whereas urinary deoxypyridinoline cross-link levels were increased at all observation times (11-28 days). 4. All these changes were also observed in Balb/c mice but their magnitudes were consistently lower than those in C57B16 mice. 5. We propose that, (1) osteocalcin is a useful marker of bone remodelling in mice and the precision of measurement of changes in its levels after OVX is increased by LPS treatment and (2) C57B16 mice give greater magnitude and more consistent changes in both serum osteocalcin and urinary deoxypyridinoline cross-links after OVX, and may be a better strain for development of an in vivo model of post-menopausal osteoporosis.

Comparison of responses of spontaneously active cells in the cerebellar Purkinje layer to parallel fibre stimulation in slice preparations and urethane-anaesthetised rats: effects of benzodiazepine receptor ligands.

1. GABA-mediated inhibitory responses were induced in spontaneously active Purkinje cells by parallel fibre stimulation in cerebellar slices or in urethane-anaesthetised rats. Effects of agonist and inverse agonist benzodiazepine (BDZ) receptor ligands were compared in the preparations. 2. Purkinje cells fired simple spikes at higher rates in slice preparations while complex spikes were seldom (in vivo) or never observed (slice). Cells fired more regularly in vivo resulting in the occurrence of rhythmic postinhibitory responses in the PSTH analysis in some preparations. 3. Single pulse stimulation of parallel fibres at just suprathreshold intensity induced inhibition of Purkinje cell activity in both preparations. At lower firing rates there was a marked increase in the duration of this response, which was more evident in vivo where more slowly firing cells were encountered. 4. BDZ receptor ligands modified inhibitory responses in slice preparations with only weak effects on the firing rates of the cells. These compounds predominately induced changes in firing rate in the anaesthetised rat with little evidence of direct modification of GABA-mediated synaptic transmission. 5. In a few experiments, following injection of the partial inverse agonists beta-CCE and beta-CCM, block of the inhibitory response was observed independent of changes in firing rate. Bidirectional efficacy of BDZ receptor ligand (agonists decrease firing and increase inhibitory response, inverse agonists increase firing and decrease inhibitory response) was demonstrated for modulation of inhibitory responses in slices and for changes in firing rate in vivo. The increased firing rate response in vivo was biphasic the magnitude of the later phase being correlated with efficacy of inverse agonists. 6. It is concluded that cerebellar slice preparations are more appropriate for studying direct effects of BDZ receptor ligands on GABA-mediated synaptic inhibition than in vivo preparations.

Effects of SK&F 86002 on cytokine-stimulated IL6 production in cultured neonatal mouse calvaria and SaOS2 osteoblastic cells: the role of prostaglandins and other mechanisms of action.

The cytokine-suppressant anti-inflammatory drug (CSAID) SK&F 86002 inhibits bone resorption in vitro and this effect cannot be totally explained by its inhibition of IL 1 beta and TNF alpha release. IL6 is another cytokine important in the mechanisms of bone resorption and could be a target for the actions of SK&F 86002. IL6 release and resorption (45Ca release) were induced by IL 1 beta in neonatal mouse calvaria bones in culture. Both indomethacin (5 x 10(-8)-5 x 10(-6) M) and SK&F 86002 (5 x 10(-7)-10(-5) M) markedly inhibited the IL6 release and totally inhibited resorption at all concentrations tested. This may result from inhibition of prostaglandin production by both compounds. In human osteoblastic cells (SaOS2) both basal and TNF alpha-stimulated IL6 production were inhibited in a concentration-related manner by SK&F 86002 but not by indomethacin. The effect of SK&F 86002 was greatest in 6 h cultures where relatively low levels of IL6 are produced and progressively less in 24 and 48 h cultures which produce higher levels of IL6. This is unlikely to be an indirect effect via inhibition of IL 1 beta production as no IL 1 beta (< 0.3 pg/ml) was detected in non-stimulated or stimulated culture supernatants. Therefore, SK&F 86002 may inhibit IL6 production in osteoblastic cells via a more direct mechanism, possibly involving inhibition of the p38 MAP kinase, the mechanism proposed for its inhibition of IL 1 beta and TNF alpha release.

Hypothermia as an indicator of the acute effects of lipopolysaccharides: comparison with serum levels of IL1 beta, IL6 and TNF alpha.

1. Hypothermia was investigated as a parameter indicating the severity of the acute effects of lipopolysaccharides (LPS) in BALB/c mice, and was compared with the induction of serum levels of IL1 beta, TNF alpha and IL6. 2. Hypothermia induced by low doses of LPS (10-50 micrograms/mouse IP LPS E. coli 0111:B4) peaked at 2 hr after LPS and then either plateaued (50 micrograms) or declined. LPS, 100 and 300 mu, induced greater degrees of hypothermia that plateaued or continued to increase with time for 8 hr. Higher doses of LPS induced similar levels of hypothermia until 4 hr but then continued to increase markedly until 8 hr. 3. TNF alpha levels peaked early (1-2 hr) and declined rapidly, IL6 levels peaked at 3 hr and then declined slowly, and IL1 beta levels peaked at 4 hr, declined at lower doses of LPS, plateaued at higher doses and continued to slowly increase at highest doses. 4. The peak levels of the cytokines (IL1 beta up to 4 hr) and hypothermia (4 hr) increased in relation to the dose of LPS and maximum responses were apparently achieved in all cases at 300-1000 micrograms LPS. 5. A similar parallel between hypothermia and induction of cytokines was observed in C57BL6 and OF1 mice, which were good and poor responders to LPS, respectively, and with the more potent Shigella dysenteria LPS in BALB/c mice. 6. In conclusion, hypothermia is a useful parameter for indicating the strength of the acute effects of LPS. Further studies are necessary to determine whether or not the cytokines studied here play a causative role in hypothermia.

Inhibition of ozone-induced nitric oxide synthase expression in the lung by endotoxin.

Inhalation of the pulmonary irritant ozone is associated with an accumulation of macrophages in the lung. These cells, along with type II epithelial cells, are activated to release increased quantities of hydrogen peroxide and nitric oxide, two reactive mediators that have been implicated in tissue injury. In the present studies we determined whether pretreatment of rats with bacterially derived endotoxin, which modulates oxidant levels in tissues, could abrogate the effects of ozone on lung injury and nitric oxide production. Acute exposure of rats to ozone (2 parts per million, 3 h) resulted in nitric oxide production in the lung as measured by electron paramagnetic resonance spin trapping. This was correlated with expression of inducible nitric oxide synthase (iNOS) mRNA in the lung as determined by in situ hybridization. Particularly high levels of iNOS were evident in alveolar macrophages and type II cells. Alveolar macrophages isolated from ozone-treated rats also expressed increased iNOS mRNA and protein as measured by Northern and Western blotting, respectively, and produced more nitric oxide compared with cells from air-exposed animals. Treatment of rats with endotoxin (5 mg/kg, intravenously), 30 min prior to ozone, was found to abrogate ozone-induced increases in iNOS mRNA and protein expression, as well as nitric oxide production by alveolar macrophages. This was associated with a reduction in ozone-induced tissue injury as determined by levels of lung lavage fluid protein. Ozone inhalation also resulted in a reduction in intracellular glutathione in alveolar macrophages, an effect that was blocked by endotoxin administration. Taken together, these data provide evidence that the protective effects of endotoxin against ozone-induced injury are mediated, at least in part, by alterations in levels of lung oxidants and antioxidants.