Anti-inflammatory and immuno-modulatory effects of novel retinoidlike 2,4,6,8-nonatetraenoic acids (NTA) in adjuvant-induced arthritis

Prophylactic treatment (p.o.) of rats with adjuvant-induced arthritis (AA) with two retinoid-like 2,4,6,8-nonatetraenoic acids (NTA), Ro 23-6457 and Ro 23-2895, significantly reduced hind paw swelling between days 10–23 and the level of plasma fibrinogen (MED 25 moles/kg). When given therapeutically (75 moles/kg between day 21 and 28) either NTA arrested the progression of the disease (MED, 25–75 moles/kg).Unseparated and adherent cell (AC) depleted spleen cells from rats with AA (day 12–15) responded poorly to the T cell mitogen, Con A (2.5 g/ml) and the B cell mitogen, LPS (10 g/ml). The responses were partially restored (30% of normal responses) in AC-depleted (but not unseparated) spleen cells from Ro 23-6457 treated rats (75 and 250 moles/kg/day). These data demonstrate an immunomodulatory effect of Ro 23-6457 in the adjuvant rat which may contribute to its anti-inflammatory activity in AA.     

Inhibition of a voltage-dependent Ca current by concanavalin A

Summary Incubation of the hypotrichous ciliateStylonychia mytilus in fluorescein-labeled concanavalin A (Con A, 0.1–0.5 g/ml) produced a strong fluorescence of its membranelles, but comparatively weak fluorescence of the other compound cilia and of the somatic membrane. Compared to untreated cells, the frequency of spontaneous backward movements was reduced in the presence of 0.5 g/ml ConA. In electrophysiological experiments Con A altered the excitability of the cell membrane. The two-peak action potential lost its second component which is associated with voltage-dependent Ca channels in the membranelles. The corresponding Ca current (Ca current I) was inhibited by low concentrations of Con A (0.2–0.5 g/ml). A second voltage-dependent Ca current (Ca current II) was not affected. Reducing the K outward current by intracellular Cs and/or extracellular tetraethylammonium, or changing the holding potential, did not restore the Con A-sensitive Ca current I. Con A also inhibited this current when Ca was replaced by Ba. The inhibitory effect of Con A on the voltagedependent Ca current I was prevented by 10–30 mM -methyl-d-mannoside, and the lectin wheat germ agglutinin (20 g/ml) did not affect the Ca currents, indicating that the Con A effect was mediated by binding to specific sugar residues on the excitable membrane. The succinylated dimeric derivative of Con A did not inhibit Ca current I up to concentrations of 5 g/ml. It is concluded that the two voltage-dependent Ca currents inStylonychia can be chemically isolated due to their different sensitivity to Con A, which appears to bind preferentially to sites near or at the Ca channel in the membranellar membrane.     

Lectins modulate prostaglandin E2 production by rat peritoneal macrophages

The effect of Aloctin A (Alo A), a lectin having anti-inflammatory activities, on prostaglandin (PG) E₂ production by activated rat peritoneal macrophages was compared with that of concanavalin A (Con A), wheat germ agglutinin (WGA), plsum sativum agglutinin (PSA) and soybean agglutinin (SBA). Alo A, WGA, Con A and PSA at 10 g per ml inhibited PG E₂ production. But SBA, even at a dose of 1 g per ml, stimulated PG E₂ production. The inhibition by Alo A treatment of the release of radioactivity from (³H)arachidonic acid-labeled macrophages and the stimulation of this release by SBA treatment were observed. The uptake of (⁵¹Cr)-labeled sheep red blood cells by the macrophage was inhibited by Alo A, Con A, and PSA, all at 10 g per ml and SBA at 1 g per ml, however, WGA at 10 g per ml stimulated the uptake of the sheep red blood cells. The mechanism of the anti-inflammatory properties of Alo A was discussed.To whom correspondence should be addressed.     

Modulation of human neutrophil and monocyte oxidative burst byLegionella pneumophila sonic extract

The effect of Legionella pneumophila sonic extract on human neutrophil and monocyte oxidative burst was studied by Superoxide anion release and luminol-enhanced chemiluminescence assays.Legionella pneumophila sonic extract by itself did not stimulate neutrophils and monocytes. The sonic extract at 8–2000g/ml primed neutrophils for enhanced Superoxide release and, at 8–62.5 g/ml, for enhanced chemiluminescence. Monocytes were only primed for enhanced chemiluminescence at very low extract concentrations (below 16g/ml). Monocyte Superoxide release was suppressed by extract concentrations higher than 2000g/ml and the chemiluminescence response of neutrophils and monocytes by concentrations higher than 250 and 125 g/ml, respectively. The priming activity was heat stable and present in fractions below 5 kDa. On the basis of these findings it is suggested that enhanced production of oxygen metabolites by neutrophils in contact with legionella components at low concentrations could contribute to the lung tissue damage seen in Legionnaires' disease, whereas the suppression of phagocyte oxidative burst by higher extract concentrations may be one of the mechanisms by which Legionella pneumophila survives intracellularly.     

Effect of auranofin,a new antiarthritic agent,on immune complex-induced release of lysosomal enzymes from human leukocytes

Auranofin, an oral chrysotherapeutic agent effective in the treatment of rheumatoid arthritis (RA), was found to be a potent, noncytotoxic inhibitor of IgG-RF immune complex-induced lysosomal enzyme release (LER) from human leukocytes. At a concentration of 1 g Au/ml (5M), auranofin produced a marked reduction in-glucuronidase (100%), acid phosphatase (88%), and lysozyme (72%) release. In contrast, gold sodium thiosulfate (GST, an injectable gold compound) had no inhibitory activity on LER at equivalent gold concentrations (i.e., 1g Au/ml) and only modest activity (< 36% inhibition) at concentrations as high as 40g Au/ml. The 50% inhibitory dose (ID₅₀) of auranofin on LER was calculated to be 3–4M (0.6–0.8g Au/ml). Blood gold levels in auranofin-treated RA patients were found to be within the range required for in vitro inhibition of LER, and correlated with decreases in IgG, RF titers, and IgG-RF immune-complex formation in vitro. These results suggest that the therapeutic action of auranofin may be caused, at least in part, by inhibition of LER and/or decreases in immune-complex formation.SK&F D-39162 (2,3,4,6-tetra-O-acetyl-1-thio--D-glucopyranosato-S) (triethylphosphine) Gold.     

Tension development and calcium sensitivity in skinned muscle fibres of the frog

Calcium activated isometric tension development was measured in single skinned muscle fibres of the ileofibularis muscle of the frog. The experiments were carried out at 5°C, pH=6.9, 1 mM free Mg²⁺ and an ionic strength of 160 mM. A Hill curve was fitted to the isometrically developed tension at different Ca²⁺ concentrations by means of a non-linear least mean square approximation. At a sarcomere length of 2.15 m, the Ca²⁺ concentration for half maximum tension (K) was 1.6 M. This Ca²⁺ concentration decreased with increasing sarcomere length; at 2.7 m, K was 1.1 M and at 3.1 m, K was 0.9 M. Therefore, Ca sensitivity is increased at larger sarcomere lengths. Consequently, the optimal sarcomere length for tension development shifted to larger values when the Ca²⁺ concentration was lowered. Osmotic compression of the fibre at 2.15 m by means of 5% Dextran also caused an increase in Ca sensitivity (K was 1.0 M). At 2.7 m, addition of 5% Dextran hardly affected the Ca sensitivity. The possible role of the interfilament spacing in the explanation of these results discussed.     

High-resolution chromosome R-banding in lymphoblastoid cell lines by the combined use of cell synchronization and ethidium bromide treatment

Summary A reliable method for obtaining high-resolution R-banded chromosomes from lymphoblastoid cell lines is described. The cell cultures are subjected to S-phase synchronization in the presence of excess thymidine (300 g/ml) for 17 to 19 hr, followed by BrdU treatment (30 g/ml) for 6.5 hr. Prior to harvest, they are exposed to ethidium bromide (7.5 g/ml) for 1.5 hr and Colcemid (0.02 g/ml) for 30 min. Using this method, high-resolution R-banded chromosomes at the 550–850 band level were obtained with frequencies at high as 70% of all mitotic cells.     

Analysis of mutations introduced into the chromosomal immunoglobulin μ gene

We have introduced a pSV2neo-derived vector that contains a 2-base-pair (bp) deletion in its immunoglobulin gene constant region into hybridoma cells bearing a single copy of the wild-type chromosomal immunoglobulin gene. Homologous recombination between the transferred mutant C region and the wild-type chromosomal C region is expected to introduce the 2-bp deletion into the chromosomal gene, generating recombinant cells synthesizing noncytolytic IgM. Analysis of the DNA in independent noncytolytic transformants indicates that in one case the gene has the structure expected for correct homologous recombination. Unexpectedly, the remaining transformants, bear chromosomal gene deletions.     

Effects of anti-inflammatory drugs on cartilage recovery from catabolin-induced degradation

Effects of aspirin (200 g/ml), hydrocortisone (10 g/ml), sodium aurothiomalate (100 g/ml), and indomethacin (10 g/ml) on recovery of cartilage from interleukin 1 or catabolin-induced degradation were examined in this initialin vitro study. The experimental protocol involved a degradative phase of eight days during which cartilage plugs were incubated in the presence or absence of spent human rheumatoid synovial culture media. A recovery period of six days followed during which the effects of the aforementioned drugs on treated cartilage were analyzed. Incorporation of [³⁵S]sulfate and [³H]proline precursors, and total contents of hydroxyproline and glycosaminoglycan in cartilage were determined two, four, and six days after insult. Aspirin treatment caused a rise in total proteoglycan content over degraded controls (p<0.002), however, this increase was not associated with increased [³⁵S]sulfate incorporation into glycosaminoglycans. Hydrocortisone resulted in a delayed rise in proteoglycan content concommitant with increased [³⁵S]sulfate uptake, whereas sodium aurothiomalate treatment was without effect on proteoglycans. Indomethacin treatment was associated with an increased release of newly synthesized macromolecules by cartilage into the media (p<0.01). These results suggest that common anti-inflammatory drugs may exhibit distinctly different effects on thein vitro synthesis and retention of proteoglycans by cartilage explants previously exposed to a degradative phase. Further work is necessary to assess the influence of drug concentration in this experimental system.     

Comparison of histamine release from peritoneal mast cells derived from diabetic and control rats

Clinical and experimental diabetes are associated with an increased number of mast cells and elevated tissue histamine concentrations. This study compared histamine release from peritoneal mast cells derived from diabetic and control rats. Experimental diabetes was induced by a single i.v. injection of streptozotocin (50 mg/kg body weight). Measurement of plasma glucose levels confimed the diabetic state. Peritoneal mast cells were stimulated for 10 min with the lectin concanavalin A (0.5–100 g/ml) in the presence or absence of phosphatidylserine, clinical dextran (0.6–1200 g/ml) in the presence of phosphatidylserine, the calcium ionophore A23187 (0.1–1 M) or the basic releasing agent compound 48/80 (0.1–10 g/ml). Histamine release induced by these agents was similar in both populations. Further studies will compare the differences in histamine release from mast cells isolated from different tissues, e.g. heart and lung. In addition, physiological stimuli which are altered in the diabetic state (e.g. hyperosmolalar solutions and free radical generating systems) are under investigation.     

Effects of purinergic stimulation on the Ca current in single frog cardiac cells

Ca current (I _Ca) was measured by whole-cell voltage clamp in single cells isolated from frog ventricle, in which the Na current was inhibited by tetrodotoxin (0.3 M) and K currents were blocked by substituting K with 120 mM intracellular and 20 mM extracellular Cs. The influence of stimulation by ATP (0.1–100 M) was assessed in the presence of propranolol (1 M) or pindolol (0.1 M), prazozin (0.1 M) and atropine (10 M). ATP, in the micromolar range, had two types of effect. Like other P₁-purinoagonists, it antagonized the increase in I _Ca elicited by -adrenostimulation. When added alone, 1 M ATP could increase I _Ca up to twofold. An increase in I _Ca was also observed even after it had been maximally enhanced by intracellularly applied cAMP (50 M). Voltage dependence and kinetics of I _Ca were not affected. These effects were considered to be related to P₂-purinoceptor activation. At higher ATP concentrations the increase in I _Ca was less; at 100 M, ATP reduced I _Ca. The ATP-induced increase in I _Ca was prevented by internal perfusion of the cells with GDP [-S] or neomycin, respectively, to block signal transduction to phospholipase C or its phosphodiesterase activity on the polyphosphoinositides. We conclude that P₂purinoceptor stimulation increases the Ca current in frog ventricular cells by a pathway that might involve phosphoinositide turnover.     

In vitro activity of the aryl-fluoroquinolones A-56619 and A-56620 and evaluation of disk susceptibility tests

The activity of two new quinolones, A-56619 and A-56620, was compared in vitro to that of norfloxacin and ciprofloxacin against 6,699 bacterial isolates in four separate clinical laboratories. The overall percentage of strains susceptible to designated concentrations were as follows: 99.1% for norfloxacin (MIC4.0 g/ml), 96.1% for ciprofloxacin (MIC1.0 g/ml), 96.8% for A-56620 (MIC 2.0 g/ml) and 96.1% for A-56619 (MIC 4.0 g/ml). For disk diffusion susceptibility tests 10 g A-56619 disks are tentatively recommended with interpretive standards of 18mm for susceptibility and 13mm for resistance; 5 g A-56620 disks may be used with tentative standards of 19mm for susceptibility and 14mm for resistance.     

Neural nicotinic acetylcholine responses in solitary mammalian retinal ganglion cells

Using the patch-clamp technique,whole-cell recordings from solitary rat retinal ganglion cells in culture have established the nicotinic nature of the acetylcholine responses in these central neurons. Currents produced by acetylcholine (5–20 mol/l) or nicotine (5–20 mol/l) reversed in polarity near –5 mV and were unaffected by atropine (10 mol/l). Agonist-induced currents were blocked by low doses(2–10 mol/l) of the classical ganglionic antagonists hexamethonium and mecamylamine, as well as by d-tubocurarine and dihydro--erythroidine (the latter two do not discriminate clearly between ganglionic and neuromuscular junction receptors). Treatment with the potent neuromuscular blocking agent -bungarotoxin (10 mol/l) did not affect the cholinergic responses of these cells, while toxin F (0.2 mol/l), a neural nicotinic receptor antagonist, readily abolished acetylcholine-induced currents. Thus, the experiments performed to date show that the nicotinic responses of retinal ganglion cells in the central nervous system share the pharmacology of autonomic ganglion cells in the peripheral nervous system. The ionic current carried by the nicotinic channels was selective for cations, similar to that described for nicotinic channels in other tissues. In addition, single-channel currents elicited by acetylcholine were observed in whole-cell recordings with seals > 5 G as well as in occasional outside-out patches of membrane. These acetylcholine-activated events, which had a unitary conductance of 48 pS and a reversal potential of 0 mV, represent the ion channels that mediate the neural nicotinic responses observed in these experiments on retinal ganglion cells.     

Mycoplasma fermentans—Induced Inflammatory Response of Astrocytes: Selective Modulation by Aminoguanidine, Thalidomide, Pentoxifylline and IL-10

Exposure of primary rat glial cells, mostly astrocytes, to heat-inactivated Mycoplasma fermentans triggers the production of tumor necrosis factor (TNF) nitric oxide (NO) and prostaglandin E₂ (PGE₂). To attenuate the production of these proinflammatory mediators, four agents: aminoguanidine, pentoxifylline, thalidomide and IL-10 were added to astrocyte cultures. Aminoguanidine (1 and 3 mM), an inhibitor of inducible nitric oxide synthase (iNOS), suppressed the production of the three mediators. TNF was the most sensitive to thalidomide, showing dose-response inhibition at concentrations of 20 g/ml, 50 g/ml and 250 g/ml. PGE₂ was affected only by concentrations of 50 g/ml and 250 g/ml, whereas NO responded solely to the highest amount of this inhibitor. The cytokine IL-10, at 10 U and 50 U, inhibited only TNF production. Our results imply that selective suppression of proinflammatory mediators by various agents may prove feasible for amelioration of central nervous system inflammatory diseases.     

Regulation of interleukin-1β and tumor necrosis factor secretion by the human monocytic leukemia cell line,THP-1

Activated macrophages synthesize and release the potent polypeptides, interleukin-1 (IL-1) and tumor necrosis factor (TNF). In an effort to identify the cellular signals which control cytokine production by activated macrophages, we have developed anin vitro model employing the human THP-1 cell line. In the present study, THP-1 cells primed by 1.6 M phorbol 12-myristate-13-acetate (TPA) for 4 hr demonstrated a dose-and time-dependent release of IL-1 and TNF upon activation by 20 g/ml LPS. BSA/anti-BSA-coated latex beads were also a potent stimulus for IL-1 secretion. Moreover, the combination of a suboptimal concentration of LPS (200 ng/ml) plus interferon- (0.03–333 U/ml) greatly enhanced IL-1 production. Resting THP-1 monocytes not primed by TPA did not secrete IL-1 or TNF. These distinct patterns of cytokine production may be related to the developmental stages of macrophage activation.     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibers

The histamine-receptor-subtype-mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibers were investigated using H₁-and H₂-selective agonists and antagonists.In electrically stimulated Purkinje fibers, histamine (3 mol/l) increased the action potential plateau height, decreased the action potential duration measured at a repolarization level of –60 mV and enhanced the pacemaker activity. These effects were abolished by the H₂-selective antagonist cimetidine (30 mol/l), but were not impaired by the H₁-selective antagonist dimetindene (0.3 mol/l).In spontaneously active Purkinje fibers, histamine (10 mol/l) increased the spontaneous rate by 24%, the slope of diastolic depolarization by 45% and shortened the duration of the diastole by 32% of the respective control measurements. These effects were blocked by 30 mol/l cimetidine, but remained unchanged in the presence of 0.3 mol/l dimetindene.Concentration-response curves of histamine were shifted to the right by approximately 2 logarithmic units in the presence of 30 mol/l cimetidine, but were not influenced in the presence of 0.3 mol/l dimetindene. The H₂-selective agonist impromidine (0.001–0.3 mol/l) had similar actions as histamine on spontaneously active Purkinje fibers, while the H₁-selective agonist 2-(2-pyridyl-)ethylamine was ineffective. It is concluded that the pronounced stimulatory action of histamine on spontaneous activity in sheep cardiac Purkinje fibers is exclusively mediated by H₂ receptors.Dedicated to Prof. Dr. E. Mutschler on the occasion of his 60th birthday.Supported by Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen, Projekt-Nr. 40008786.     

Effects of PAF and PAF antagonists on the shape of venous endothelial cellsin vitro

Three platelet-activating factor (PAF) antagonists were tested for their ability to prevent or reduce PAF-induced shape changes of large vein endothelial cellsin vitro. BN52021 had a significant protective action at concentrations of 1 M and 0.1 M, but at 100 M had a damaging effect of its own. CV3988 (0.1 M and 1 M) and L652, 731 (20 M) did not reduce the responses to PAF, and at higher concentrations (CV3988 10 M and 100 M, L652, 731 100 M) both compounds alone caused significant changes of shape. BN52021 (0.1 M) was also effective against leukotriene (LT) C₄, at 1 M against bradykinin and LTE₄, and at 10 M against LTD₄ and the calcium ionophore A23187. BN52021 (10 M) was ineffective against shape changes induced by histamine, prostaglandin (PG) E₂ and lysophosphatidylcholine (LPC). Neither indomethacin (100 M) nor verapamil (20 M) altered the response to PAF.Using electron spin resonance (ESR) spectrometry it was shown that the damaging effects of LPC and CV3988 may be due partly to their detergent properties. It is suggested that the mechanism by which PAF alters the shape of large vein endothelial cells is primarily receptor mediated.     

Length-dependent effects of osmotic compression on skinned rabbit psoas muscle fibers

The goal of this study was to characterize the interrelationship between sarcomere length and interfilament spacing in the control of Ca²⁺ sensitivity in skinned rabbit psoas muscle fibers. Measurements were made at sarcomere lengths 2.0, 2.7 and 3.4 m. At 2.7 m the fiber width was reduced by 17% relative to that at 2.0 m and the pCa₅₀ for force development was increased by 0.3 pCa units. In the presence of 5% Dextran T-500 the fiber width at sarcomere length 2.0 m was also decreased by 17% and the Ca²⁺ sensitivity was increased to the same value as at 2.7 m. In contrast, at sarcomere length 2.7 m the addition of as much as 10% Dextran T-500 had no effect on Ca²⁺ sensitivity. At sarcomere length 3.4 m there was an additional 7% compression and the Ca²⁺ sensitivity was increased slightly (0.1 pCa units) relative to that at 2.7 m. However at 3.4 m the addition of 5% Dextran T-500 caused the Ca²⁺ sensitivity to decrease to the level seen at 2.0 m. Given that the skinning process causes a swelling of the filament lattice it is evident that the relationship between sarcomere length and Ca²⁺ sensitivity observed in skinned fibers may not always be applicable to intact fibers. These data are consistent with measurements of Ca²⁺ in intact fibers which indicate that there might be a decline in Ca²⁺ sensitivity at long sarcomere lengths.     

A pharmacologic study on the histamine releasing effect of atracurium and other muscle relaxants in rat isolated ileum

In this study, the effects of histamine, antihistamines (terfenadine and mepyramine), 5-hydroxytryptamine, and muscle relaxants, atracurium, vecuronium and gallamine, on the tone and contractility of rat ileum were studied and compared in vitro.The aim of the present investigation was to measure, pharmacologically, the histamine releasing effect of muscle relaxants, e.g. atracurium, vecuronium and gallamine, by comparing their contractile response in the absence and presence of antihistamines and comparing their mechanical responses with those produced by histamine and 5-hydroxytryptamine (5-HT).The results showed that the antihistamines, triludan (terfenadine) and mepyramine produced opposite effects in rat ileum. Terfenadine (0.1–20 M) produced concentration-dependent contractions in the rat ileum, whereas mepyramine (0.1–10 M) relaxed the muscle, e.g. by 1.2 g tension. Atracurium (0.5–500 M), vecuronium (0.2–200 M), and gallamine (0.1–7.0 M) produced marked contractions (1.5–4.0 g tension) in rat ileum, and these contractions were markedly reduced by mepyramine (1.3 M) or terfenadine (5 M), implicating histamine release in the generation of these contractions. However, there was some residual contraction which was not blocked by mepyramine, but by 5-HT antagonist, methysergide (1 M), indicating that a mechanism other than histamine release may be responsible for the residual contraction, i.e. release of other mediators such as 5-HT, prostaglandins, or calcium. 5-HT (0.5–500 M) and histamine (0.5–500 M) produced contractions in the rat ileum, but 5-HT was more effective than histamine in producing these contractions. Similarly, gall amine was more effective than atracurium and vecuronium in contracting the rat ileum. Since very high concentrations of muscle relaxants were used, it is suggested that in clinical concentrations, the histamine releasing effect of muscle relaxants was minimal, except that of gallamine, which may release histamine event at very low concentrations. The results are discussed in terms of pharmacologic and immunologic implications of drug reactions at the rat intestinal smooth muscle.     

Inhibitory effect of phloretin on histamine release from isolated rat mast cells

The ability of the flavonoid phloretin to inhibit histamine release from rat mast cells varied considerably with the releasing agent investigated. The response to the combination of the ionophore A23187 and the phorbol ester TPA and to suboptimal concentrations of the ionophore (0.5 M) was potently inhibited (IC₅₀ about 5 M), whereas phloretin was less potent against responses to the ionophore (1 M) IC₅₀ of 17 M), to antigen alone and in combination with TPA (IC₅₀ of 30–50 M), to TPA in the absence of calcium (IC₅₀ of 50 M) and to compound 48/80 in the absence and presence of calcium (IC₅₀ of 60–90 M). The inhibition by phloretin at concentrations above 10M was partly counteracted by glucose (5 mM) indicating effects on oxidative metabolism. The flavonoid quercetin was equally potent in inhibiting histamine release induced by antigen, the ionophore at different concentrations and in combination with TPA (IC₅₀ of 20M). Although not conclusive, the results are consistent with an inhibition of protein kinase C by phloretin at concentrations below 10 M. At higher concentrations unspecific actions become apparent and phloretin therefore seems to be of limited utility as a probe for signal-pathways in cell responses.