Showing papers in "Inflammation Research in 1991"

Anti-inflammatory activity and inhibition of arachidonic acid metabolism by flavonoids.

Abstract: A group of flavonoids isolated from medicinal plants and which are selective inhibitors of lipoxygenase activityin vitro: sideritoflavone, cirsiliol, hypolaetin-8-O-β-d-glucoside, hypolaetin, oroxindin, quercetagetin-7-O-β-d-glucoside, gossypin, hibifolin and gossypetin, besides leucocyanidol, have been studied for their effects on acute responses induced by carrageenin in mice. The oral administration of flavonoids to mice inhibited dose-dependently the development of paw oedema at 1, 3 and 5 h after carrageenin injection. A similar administration of flavonoids induced a dose-dependent inhibition of leukocyte accumulation in inflammatory exudates following intraperitoneal injection of carrageenin into mice. Some of the flavonoids exhibited a potency against leukocyte infiltration similar to that seen for inhibition of carrageenin oedema at 3 h of induction. In agreement with data reported in rats, indomethacin was much more effective on inhibition of prostaglandin E2 (PGE2) formation than on leukocyte infiltration in mice. The selectivity of flavonoids towards lipoxygenase is not retainedin vivo since they behave as dual inhibitors of PGE2 and leukotriene B4 (LTB4) formation in peritoneal exudates. Our data support the inhibition of arachidonic acid metabolism as one of the mechanisms by which flavonoids exert their anti-inflammatory effects.

Leflunomide (HWA 486), a novel immunomodulating compound for the treatment of autoimmune disorders and reactions leading to transplantation rejection.

Abstract: Leflunomide has been shown to be very effective in preventing and curing several autoimmune animal diseases. Further, this agent is as effective as cyclosporin A in preventing the rejection of skin and kidney transplants in rats. Preliminary results from patients suffering from severe cases of rheumatoid arthritis demonstrated that clinical and immunological parameters could be improved with leflunomide therapy. Mode of action studies revealed that this substance antagonizes the proliferation inducing activity of several cytokines and is cytostatic for certain cell types. In this light, we could show that tyrosine phosphorylation of the RR-SRC peptide substrate and the autophosphorylation of the epidermal growth factor (EGF) receptor were, dose dependently, inhibited by leflunomide. EGF activates the intrinsic tyrosine kinase of its receptor, which stimulates the phosphorylation of a variety of peptides, the amino acid residue in all cases is tyrosine. These results indicate that much of leflunomide's activity could be due to the inhibition of tyrosine-kinase(s), which is an important general mechanism for the proliferation of various cell types. Thus, leflunomide, which is effective against autoimmune diseases and reactions leading to graft rejection, would seem to have a mode of action separating it from known immunosuppressive drugs.

Neurogenic and tissue-mediated components of formalin-induced edema: evidence for supraspinal regulation.

Abstract: Injection of formalin into a hind paw of rats produces localized inflammation and pain. The nociceptive effect of formalin, recorded as flinching/shaking of the injected paw, is biphasic. The present study shows that formalin-induced inflammation and edema (assessed by measurement of paw volume up to 24 h post-injection) is also biphasic, an early neurogenic component being followed by a later tissue-mediated response. Rapid initiation of edema is closely related to early phase nociception and is dependent on activity in primary afferent neurons and axon reflexes, but not on transmission of the noxious stimulus and the perception of pain itself. The major site responsible for down-regulating the inflammatory response, particularly in the later stages when tissue-mediated components are most heavily involved, appears to be located supraspinally. Down-regulation occurs principally by means of descending neuronal pathways but may also involve a secondary humoral component. The perhaps surprising dependence on neuronal mechanisms which this study demonstrates promotes spinal and peripheral sites as potential therapeutic targets in certain inflammatory conditions.

The anaphylatoxins C3a and C5a are vasodilators in the canine coronary vasculature in vitro and in vivo

Abstract: The effect of complement fragments on coronary blood flowin vivo and the contraction of coronary arteriesin vitro was determined. In pentobarbital anesthetized dogs, intraarterial bolus injection of C3a and C5a, zymosan-activated serum and methylcholine in the coronary vascular bed caused transient and dose-dependent increases in coronary blood flow. Similar increases were obtained with 25 μg of C3a (104±13%,n=5) and 0.1 μg of methylcholine (102±4%,n=3). Smaller, increases in blood flow were elicited by 25 μg of C5a (41±18%,n=4) and 0.2 ml, of zymosan-activated serum (48±5%,n=4). None of these responses were associated, with significant changes in left ventricular contractile force measured with a strain gauge, arterial blood pressure, and heart rate. C3a dilated the coronary vascular bed in conscious dogs with an activity equal to or greater than that observed in anesthetized dogs. Isolated canine coronary arteries that were precontracted with serotonin relaxed in response to C3a, whether or not the endothelium was intact. Overall these data suggest that physiologically high doses of anaphylactic complement fragments vasodilate the canine coronary circulation.

Rat mast cells synthesize a nitric oxide like-factor which modulates the release of histamine.

Abstract: Rat serosal mast cells were evaluated for their capacity to generate a nitric oxide-like factor by two bioassays: inhibition of platelet aggregation and stimulation of mast cell guanylate cyclase. Incubation of mast cells with human washed platelets, both treated with indomethacin, inhibited thrombin-induced platelet aggregation which was potentiated by superoxide dismutase and reversed by oxyhaemoglobin. When mast cells alone were stirred at 1000 rpm, a time dependent increase in the levels of their cGMP but not cAMP was observed. Preincubation of mast cells with NG-monomethyl-l-arginine significantly enhanced E. coli lipopolysaccharide-evoked histamine release. Our results show that mast cell histamine release can be modulated by an intrinsically generated nitric oxide-like factor.

Mechanisms of oxidant-induced changes in erythrocytes.

Abstract: There is an increasing body of experimental studies demonstrating the toxic effects of oxygen-derived free radicals. Evidence supports an important role for free radicals in ischemic injuries, inflammation, and chemical-induced tissue injury. Free radicals are involved in normal biochemical processes like oxidative reduction and cellular metabolism; however, they also mediate disease processes. The participation of oxygen free radicals in lysis of red cells is important in some situations of intravascular hemolysis. This article will review neutrophil-derived oxygen free radicals, emphasizing: (1) their effects on the erythrocyte and (2) how these effects may be attenuated.

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophils in vitro. Evidence of formation of antiinflammatory compounds.

Abstract: Enzymatic transformation of then-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B4 (LTB4), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of othern-6 fatty acids on the formation of LTB4 by human neutrophils and to determine if thesen-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 μM) and different concentrations (0–100 μM) of then-6 fatty acids linoleic acid (LA) and dihomo-gammalinolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB4 was dose dependently inhibited by both LA (IC50=45 μM) and DGLA (IC50=40μM). This inhibition of LTB4 formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB4 formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB4 formation (IC50=7.5 μM and IC50=0.2 μM). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB4 formation (IC50=0.75 μM). The results show that the addition of LA and DGLA to neutrophils results in an inhibition of LTB4 formation and simultaneously to the formation of 13-HODE and 15-HETrE, that also inhibits LTB4 formation. Therefore, dietary supplementation or topical application of LA and DGLA or preferentially their respective 15-LO products, may have a therapeutic effect in inflammatroy diseases in which LTs are suspected to play a pathogenic role.

Induction of vascular smooth muscle bradykinin B1 receptors in vivo during antigen arthritis.

Abstract: Antigen arthritis in rabbits was associated with induction of bradykinin B1 receptors in isolated aorta smooth muscle 24 h following intra-articular injection of antigen in sensitized animals. Control tissues developed responsiveness to desArg9-bradykinin or bradykinin during 3 h incubation, but failed to respond to either kinin at the beginning of experiments. Aorta from rabbits 24 h after induction of arthritis not only developed responsiveness to kinins more rapidly than controls, but also responded at the outset of experiments. Antigen arthritis was characterized by acute phase protein synthesis and joint swelling. This is the first demonstration of induction of smooth muscle responsiveness to desArg9-bradykinin during an immune complex disease.

Toxicological properties of carrageenan

Abstract: Food-grade carrageenan is a safe natural product prepared from seaweed. Its addition to food imparts many desirable characteristics which have allowed it to be used continuously for centuries. The long safe history of this natural food additive is confirmed by negative results in subchronic and chronic feeding studies in many animal species, mutagenicity studies and reproductive toxicity studies.

Evaluation of an interleukin-1 receptor antagonist in the rat acetic acid-induced colitis model.

Abstract: The anti-inflammatory activity of the IL-1 receptor antagonist, IL-1ra, was evaluated in the acetic acid (HOAc)-induced model of colitis in rats. Animals treated with 10 mg/kg IL-1ra or vehicle were evaluated for general health, acute phase response, and colonic in flammation 24 hours after the initiation of inflammation. A significant decrease in the accumulation of neutrophils in the colonic mucosa as measured by myeloperoxidase activity was seen in animals with HOAc induced colitis that were treated intraperitoneally with IL-1ra when compared to animals with colitis that had been treated with vehicle. IL-1ra also reduced colonic necrosis measured grossly, although there was no effect on the histology IL-1ra had a modest effect on the HOAc-induced acute phase response, as indicated by changes in the serum iron, albumin and transferrin, but the results were not statistically significant. The number of circulating erythrocytes and neutrophils was significantly increased in animals with HOAc-induced colitis and treated with IL-1ra, suggesting that IL-1ra under these experimental conditions inhibited the migration of neutrophils to the injured colon and also the overall intestinal necrosis in the colon as assessed by gross pathology. IL-1ra may be useful as an intestinal anti-inflammatory agent.

The effect of nitric oxide generators on ischemia reperfusion injury and histamine release in isolated perfused guinea-pig heart

Abstract: Experiments were carried out to provide evidence of the effect of L-arginine (L-Arg), its analogue NG-monomethyl-L-arginine (MeArg) and of some nitrovasodilators (sodium nitroprusside, NaNP; 3-morpholino-sydnonimine, SIN-1) which spontaneously release nitric oxide (NO) on ischemia-reperfusion injury, histamine release and mast cell degranulation, occurring after multiple ligature and release of the left anterior descending (LAD) coronary artery in isolated perfused guinea-pig hearts. The reopening of the LAD coronary artery leads to a release of histamine related to a decrease in microdensitometry of cardiac mast cells and to calcium overload. The perfusion of the heart with NO-donors significantly reduces either the release of histamine, the loss of mast cell metachromasia and the overload of calcium. These effects were potentiated by SOD. The results suggest that the endogenous formation of NO and molecules able to generate NO have a role in the prevention of post-ischemic tissue injury.

Stimulation of CRP secretion in HepG2 cells: cooperative effect of dexamethasone and interleukin 6.

Abstract: This report described the capability of the human. human acute phase reactant, C-reactive protein (CRP). Its secretion is stimulated by interleukin 6 (IL-6) in a dose-dependent fashion and can further be positively modulated by dexamethasone. The way in which this glucocorticoid influences the CRP response depends on its time of application. Incubation of HepG2 cells simultaneously with IL-6 and dexamethasone increases the magnitude of CRP release significantly above that seen with IL-6 alone. After preincubation with dexamethasone, the kinetics of CRP release, induced by IL-6, are increased and approach that observed in the case of alpha 1-acid glycoprotein (alpha 1-AGP) without dexamethasone pretreatment. Conditions for optimal secretion of CRP were determined.

Crystal-neutrophil interactions lead to interleukin-1 synthesis.

Abstract: Normal human blood neutrophils were studied for their capacity to synthesize and release interleukin-1 (IL-1) species after phagocytosis of triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate crystals (CPPD). MSU crystals were more potent inducers of IL-1 generation than CPPD or unopsonized zymosan. Microcrystal-stimulated neutrophils characteristically secreted most of the newly synthesized IL-1. Colchicine partly inhibited the secretion of IL-1 by neutrophils during phagocytosis of solid particles. However, colchicine selectively inhibited IL-1 synthesis induced by microcrystals. These results suggest that neutrophil-derived IL-1 may contribute to the pathogenesis of crystal-induced arthritis.

The pharmacology of ebselen.

Abstract: Ebselen has been demonstrated to be an effective anti-inflammatory agent in a variety of experimental modelsin vivo which differ from classical tests in that the aetiological roles of hydroperoxides and/or lipoxygenase products appear to be greater. Indeed, ebselen exhibits only weak anti-inflammatory activity in the traditional prostaglandin-dominated models, such as carrageenan paw oedema, adjuvant arthritis and yeast paw hyperalgesia [50]. The major targets of this anti-inflammatory activity appear to be plasma exudation and infiltration, possibly as a result of the inhibition of the hydroperoxide and/or leukotriene effects on leukocyte-endothelium interactions. Both reactive oxygen species [24] and LTB4 [51, 52] enhance granulocyte adhesiveness to endothelium and ebselen inhibits the generation of reactive oxygen species, catalyses the breakdown of hydroperoxides, inactivates LTB4 by isomerization and inhibits 5-lipoxygenase [9–11, 13–16, 27–29]. Consequently, any or all of these mechanisms of action, together with inhibition of hypoxic-reperfusion injury [53], could contribute to the anti-inflammatory activity of ebselen.

The effect of in vitro and in vivo dexamethasone on human neutrophil function.

Abstract: There is a significant fall in PMN chemotaxis to the peptide FMLP in response to increasing concentrations of dexamethasonein vitro. The response fell in a dose related manner from a control value of 53.7 SE±9.6 cells per high power field (cpf) to 47.3 SE±8.1 at 10−6M (p<0.05) and 24.7±8.9 at 10−3M (p<0.025). A similar response was observed for the chemoattractants zymosan activated serum and the sol phase of purulent sputum. The effect was independent of protein synthesis or the period of incubation. Twelve milligrams of dexamethasone taken daily by 6 healthy volunteers resulted in a significant (p<0.025) reduction in the chemotactic response of PMN to 10−8M FMLP (from 29.5±1.55 to 13.7±1.8 cpf) which was apparent within 2 hours of taking the first dose. This effect was sustained for the three days on which dexamethasone was taken but returned to normal 7 days after the last dose had been administered.

Analgesic activity of a novel bivalent opioid peptide compared to morphine via different routes of administration.

Abstract: A novel bivalent opioid tetrapeptide, biphalin (Tyr-D-Ala-Gly-Phe-NH)2, was synthesized based on structure-activity relationships. The analgesic activity of biphalin was assessed in comparison to morphine in rats. Drugs were administered subcutaneously (s.c.), intravenously (i.v.) and intrathecally (i.t.). Tail flick and tail pinch were used as tests for analgesia. Biphalin s.c. showed negligible analgesic activity, but when given i.v. produced significant analgesia, although less potent than morphine via this route. In contrast, intrathecal biphalin was more potent than morphine. These results indicate that biphalin has intrinsic activity that is compromised by enzymatic degradation or redistribution in the periphery, properties that may render it useful in exploring analgesic actions of locally applied opioids in the periphery without the likelihood of unwanted central effects.

Protective effect of vitamin D3 analogues on endotoxin shock in mice.

Abstract: The effect of vitamin D3 analogues on endotoxin shock in mice was investigated. Male ICR mice were orally administered vitamin D3 analogues or vehicle, accompanied by an intraperitoneal injection of endotoxin (E. Coli lipopolysaccharide, LPS, 20 mg/kg). Endotoxin caused a decrease in survival rate in a time-dependent manner. Increases in plasma immunoreactive (i) eicosanoid and hepatic malondialdehyde (MDA) levels were also observed. Administration of 1α-hydroxyvitamin D3 (1α-OH-D3) improved the survival rate 24 to 48 h after endotoxin treatment. The effects were markedly observed at a dose of 20 ng/kg. In addition, 1α-OH-D3 restored the plasma iTXB2 and hepatic MDA levels 8 h after endotoxin injection. However, it did not affect plasma iPGE2, i6-keto-PGF1α and blood iLTB4 levels. At a dose of 20 ng/kg, both 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and 1,24(R)-dihydroxyvitamin D3 (1,24(R)-(OH)2D3) restored the survival rate, the plasma iTXB2 and hepatic MDA levels. These results suggest that vitamin D3 analogues may inhibit endotoxemia through regulation of the formation of TXA2 and free radicals.

Histidine decarboxylase inhibition induced by alpha-fluoromethylhistidine provokes learning-related hypokinetic activity.

Abstract: The influence of brain histamine (HA) on learning and memory is not well understood, although some reports indicate that HA improves memory consolidation. We have studied the effects of α-fluoromethylhistidine (FMH) (100 mg/Kg, i.p.), which reduced by 60–80% the concentration of hypothalamic HA, on rat locomotor activity (LA) and learning in several experimental conditions in a computerized system. FMH reduced LA in an open field paradigm (OFP) 3h after injection and tended to inhibit motor habituation after a 3-day trial. In a maze paradigm (MP), where the animals had to learn to avoid a foot-shock (1 mA), FMH reduced LA, rearing (2F), and jumping activity (JA). The peripheral administration of HA to control-trained rats reduced a learning index (N/Sts ratio) by 50%. According to these results, FMH diminished LA, 2F, and JA with no effect on habituation in MP. These observations might indicate that a moderate reduction in the levels of brain HA might enhance attention in solving visuo-spatial tasks under stressful stimuli.

Histamine influences the expression of the interleukin-6 receptor on human lymphoid, monocytoid and hepatoma cell lines

Abstract: Based on our earlier data on the enhancing effect of histamine on the action of interleukin-6 (IL-6), we have studied the molecular mechanisms of these interactions. The effect of histamine was investigated on the binding of125I-IL-6 by B lymphoma cell line CESS, monocytoid cell line U937 and hepatoma cell line HepG2. Histamine increases the IL-6 binding by CESS cells and inhibits that by U937 and HepG2 cells. Using H1 receptor (cetirizin and loderix) and H2 receptor (cimetidine and ranitidine) specific antagonists, an H1-dependent stimulation of IL-6 binding by CESS cells was found. In contrast, down-regulation of IL-6 binding by histamine was clearly mediated through H2 receptors.

Properties of tuberomammillary histamine neurones and their response to galanin.

Abstract: Histaminergic neurones in the tuberomammillary nucleus possess electrophysiological properties which distinguish them from other neurones in their neighborhood Their resting potential is −50 mV and they are spontaneously active at about 2 Hz in a slice preparation They display a transient outward rectification and an anomalous inward rectification Bath application of galanin (01 μM) reduced their firing rate significantly and hyperpolarized them slightly

Biological effects of histamine: an overview.

Abstract: It is now some eighty years since the pioneering studies of Dale, Barger, Laidlaw and others defined the immediate pharmacological actions of histamine. In that period, it has become apparent that endogenous histamine is implicated in a diversity of physiological processes as well as in immediate hypersensitivity reactions and injury. Thus, the amine is intimately involved in the control of gastric acid secretion, acts as a neurotransmitter or modulator in the brain, and influences cell growth and immune function. The definition of distinct receptor subtypes for the autacoid has found therapeutic application in the treatment of allergy and peptic ulcer disease and holds promise for the management of problems of sleep and wakefulness. Indeed, the discovery of H2-receptors, and more recently H3-receptors, has led to a considerable resurgence in interest in histamine in biology and clinical medicine. It is hoped that this interest will continue and prosper.

A novel source of mast cells: the human placenta.

Abstract: The presence of moderate amounts of histamine in the human placenta was confirmed (0.72 +/- 0.10 microgram/g wet weight), and the hitherto unknown storage site of this biogenic amine was elucidated. Mast cells were identified by their characteristic morphology, staining reactions and secretory activity measured in terms of histamine release. Human placental tissue contains 7.6 x 10(5) mast cells/g wet weight, identified by staining with toluidine blue or alcian blue, and these cells were positive for chloro-acetate-esterase. Light microscope studies of placental tissue stained with HRP-conjugated anti-human IgE demonstrated cells with a typical 'halo' effect indicating cell-bound IgE, and electron microscopy revealed cells containing membrane-bound electron dense granules. A single mast cell was calculated to contain approximately 1 pg of histamine. Enzymatic digestion of placental tissue with collagenase (1.5 mg/ml) yielded viable cell suspension. containing mast cells in a purity of 0.6% which exhibited a low spontaneous output of histamine (12%). Placental mast cells released histamine in a concentration dependent manner upon challenge with anti-human IgE and the calcium ionophore A23187. Also, unlike other human mast cells so far studied. with the exception of skin, those dispersed from human placenta were responsive to the polybasic secretagogue compound 48/80. These findings represent a novel source of human mast cells and, since placentas are readily available in quantity, such tissue is proposed as an ideal source of mast cells for biochemical and pharmacological use.

Plasma histamine and clinical tolerance to infused histamine in normal, atopic and urticarial subjects.

Abstract: Five subjects with a recent history of urticaria (U), five atopic (A) subjects and a non-atopic (NA) control group were given intravenous infusions of histamine starting at 0.05 μg/kg/min, increasing by 0.05 μg/kg/min every 30 minutes to a maximum of 0.35 μg/kg/min. Plasma histamine levels were monitored every 15 minutes. The infusion was stopped when an objective clinical endpoint was reached, involving either evidence of peripheral vasodilatation (rise of skin temperature by at least 1 °C) or a 20% fall of peak expiratory flow rate.

Sucralfate induces proliferation of dermal fibroblasts and keratinocytes in culture and granulation tissue formation in full-thickness skin wounds.

Abstract: Sucralfate is used to induce healing of gastrointestinal tract ulcers. We evaluated its potential utility in the healing of skin wounds. Initial experiments examined the effects of the sucralfate on proliferation of cultured dermal fibroblasts and keratinocytes. Sucralfate induced proliferation in quiescent cultures of both cell types. Additionally, sucralfate enhanced prostaglandin E2 synthesis in basal keratinocytes and in interleukin-1-stimulated keratinocytes and dermal fibroblasts. Basal interleukin-1 and 6 release were not affected by sucralfate, but the agent enhanced interleukin-1-stimulated interleukin-6 release from fibroblasts. When applied daily to full-thickness wounds in rats, sucralfate increased the thickness of granulation tissue when assessed at day 12.

Effect of Zileuton (A-64077) on the 5-lipoxygenase activity of human whole blood ex vivo.

Abstract: The potency and reversibility of a new orally active 5-lipoxygenase (5-LO) inhibitor were evaluated in human volunteers. Zileuton (A-64077) 600 mg q.i.d. was administered to volunteers for 14 days in a phase I study, and blood samples were withdrawn, stimulated with ionophore A23187 and LTB4 levels were determined using both reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA). The drug significantly inhibited (above 70%) LTB4 biosynthesis in whole blood stimulated with A-23187 throughout the 14 days. The activity of 5-LO was also measured one week after stopping the medication and was returned to control levels. Measurement of LTB4 levels using either RP-HPLC or RIA gave similar percentage of inhibition although RIA appeared to underestimate by half the absolute amounts of LTB4 in the blood samples. These results show that Zileuton is a highly active and reversible 5-LO inhibitor in human.

Anti-tumoral and anti-inflammatory effects of biological stains.

Abstract: The biological stains, methylene blue and its metabolite azure B, were evaluated as anti-tumor and anti-inflammatory agents. Azur B, administered in drinking water to tumor-bearing mice, inhibited the growth of transplanted tumors and the growth of primary tumors induced by methylcholanthrene. Inhibition of growth of primary tumors was observed only in female mice. Azure B also reduced the wet weight of carrageenin-induced granulomas in rats. Azure B, given intravenously to BCG-sensitized mice 15 minutes prior to challenge with lipopolysaccharide, decreased TNF production (to 10% of control values) and prevented death from endotoxic shock. Methylene blue decreased TNF production (to 50% of control values) but did not protect the animals from endotoxic shock. Our results suggest that some of the effects previously ascribed to methylene blue are probably mediated via its metabolite, i.e. azure B. Low toxicity and easy administration of the dyes explain their use in clinical settings.

Modulation of cellular processes by H7, a non-selective inhibitor of protein kinases.

Abstract: H7 has been described as a potent inhibitor of protein kinase C (PKC) and has been widely used to investigate the regulatory role of this enzyme in intact cell systems. In this comparative study between H7 and the microbial alkaloid, staurosporine, we found that the former inhibited rat brain PKC and cAMP dependent protein kinase with IC50 values of 18 and 16 μM respectively whereas the latter was a much more potent inhibitor of both kinases with IC50 values of 9.5 nM and 42 nM respectively. H7, at concentrations up to 100 μM, failed to block cellular events induced by phorbol esters, agents which specifically stimulate PKC, yet was a potent inhibitor of IL-2 induced T cell proliferation with an IC50 value of 19 μM. In contrast, staurosporine was a potent inhibitor of both phorbol ester induced p47 phosphorylation in platelet (I50 value=540 nM) and also CD3 and CD4 down-regulation in T cells (I50 values 200 nM and 50 nM respectively). Staurosporine was also a potent inhibitor of IL-2 induced T cell proliferation I50 value=9 nM). These results provide a strong argument against the use of H7 to probe for PKC involvement in cellular processes.

Cytokine production in whole blood ex vivo.

Abstract: Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) have been reported to contribute to the pathogenesis of many inflammatory diseases, e.g., rheumatoid arthritis. As monocytes are believed to be the primary source of these cytokines in peripheral blood, the present study was conducted to establish ranges and patterns of IL-1β and TNF-α secretion. Using heparinized unseparated whole blood obtained from normal human volunteers, peripheral blood monocytes were stimulated withSal. minnesota LPS or BSA/anti-BSA immune complex-coated beads (BSA-beads). ELISAs for IL-1β and TNF-α were employed to quantitate cytokine levels in blood plasma without performing arduous and time-consuming extraction procedures. Over the course of a 6 hr incubation, LPS elicited a dose-dependent increase in TNF-α and IL-1β production. Preincubation of whole blood with interferon-γ prior to the addition of a suboptimal dose of LPS or BSA-beads resulted in a synergistic potentiation of IL-1β/TNF-α production. Dexamethasone, utilized in the treatment of rheumatoid arthritis, proved to be a potent inhibitor of cytokine biosynthesis in whole bloodex vivo. The measurement of cytokine biosynthesis in a relevant physiologic environment not only avoids non-specific monocyte activation, but also may increase our ability to predict clinical outcomes in rheumatoid arthritis and/or other inflammatory diseases.

Leukotriene B4 up-regulates IL-6 rather than IL-1 synthesis in human monocytes

Abstract: Leukotriene B4 (LTB4) preferentially induced IL-6 mRNA accumulation and IL-6 protein release as assessed by ELISA and the B9 cell bioassay. In contrast, minimal IL-1 mRNA or protein was induced by LTB4 either in the absence or presence of muramyl dipeptide. Supernatants of LTB4-treated monocytes consistently showed enhanced thymocyte costimulatory activity and this was abrogated by 75–80% by anti-IL-1 antibody. Baseline production of IL-1 appeared however to be sufficient for a synergistic stimulation of thymocytes in the presence of IL-6. Our results now help clarify that LTB4 stimulated preferentially IL-6 production and that the observed LTB4-induced augmentation in thymocyte responses to monocyte supernatants is due to augmented IL-6 contents in the presence of baseline minimal IL-1 production.