Irma Lemaire

Stimulation of interleukin 2 and interferon gamma production by leukotriene B4 in human lymphocyte cultures

Interleukin 2 (IL-2) and interferon-gamma (IFN) production by human lymphocytes was significantly enhanced in the presence of leukotriene B4 (LTB4) and indomethacin. Depletion of the T8+ suppressor cell subset of human T cells resulted in enhanced lymphokine production and further augmentation by LTB4. These findings suggest that LTB4 can regulate immune responses through its modulation of lymphokine production.

Possible muscarinic regulation of catecholamine secretion mediated by cyclic GMP in isolated bovine adrenal chromaffin cells

Catecholamine secretion and cyclic GMP levels were measured in chromaffin cells isolated from bovine adrenal medulla. Acetylcholine (ACh) and nicotine, but not muscarine, induced 8- to 10-fold increases in catecholamine secretion, with respective ED50 values of 10 and 2 M. Cyclic GMP levels were also increased from 3- to 5-fold in the presence of ACh, and this stimulation was mimicked by muscarine but not by nicotine. Half-maximum stimulations of cyclic GMP levels with ACh and muscarine were observed at 0.1 and 0.3 M respectively. The order of potency of various cholinergic drugs for cyclic GMP stimulation was as follows: ACh > oxotremorine > methacholine > muscarine > carbamylcholine > furthretonium > arecholine > bethanechol. Pilocarpine, McN-A-343, and AHR-602 were inactive at concentrations between 10−8 and 10−3 M. Isobutylmethylxanthine (1 mM), a specific phosphodiesterase inhibitor, caused a 7-fold increase in cyclic GMP and potentiated 3-fold the stimulation of cyclic GMP by ACh. The nicotine-induced catecholamine secretion was inhibited 19 and 33 per cent by the co-stimulation of the muscarinic receptor with 0.2 and 0.5 M ACh, respectively. Isobutylmethylxanthine (1 mM) also caused a 44 per cent inhibition of nicotine-induced catecholamine secretion, and its effect was additive to that of ACh. Atropine (0.1 M) selectively abolished the inhibition caused by ACh. Similar inhibitions were also obtained in the presence of exogenous dibutyryl cyclic GMP or 8-bromo cyclic GMP. These data indicate that the nicotinic stimulation of catecholamine secretion from bovine adrenal chromaffin cells may be regulated by cyclic GMP via the stimulation of a muscarinic receptor.

Stimulation of interleukin-1 and -6 production in alveolar macrophages by the neotropical liana, Uncaria tomentosa (Una de Gato)

Two extracts of different collections of the traditional medicine uña de gato (Uncaria tomentosa) from Peru were characterized by High Pressure Liquid Chromatography as containing approximately 6 mg/g total oxindole content prior to studies with alveolar macrophages. The plant preparations greatly stimulated IL-1 and IL-6 production by rat macrophages in a dose dependent manner in the range of 0.025-0.1 mg/ml. They were also able to enhance IL-1 and -6 in lipopolysaccharide-stimulated macrophages. The results suggest a strong immunostimulant action of this plant.

Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites

Abstract: In isolated bovine adrenal chromaffin cells, β‐endorphin, dynorphin, and levorphanol caused a dose‐dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 μM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD= 0.59 nM). β‐Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5‐I2Tyr1]‐β‐endorphin, [3,5‐I2Tyr1]‐dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 of 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with β‐endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh‐evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 μM, respectively). Inhibition of CA release was also obtained with naloxone (ID50= 14 μM) and naltrexone (ID50 > 10−4 M), two classical antagonists of opiate receptors, and this effect was additive to that of β‐endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.

P2X7 receptor drives osteoclast fusion by increasing the extracellular adenosine concentration

Defects in bone homeostasis are a major health problem. Osteoclast differentiation and activation have a crucial role in bone remodeling in health and disease. Osteoclasts are bone‐resorbing cells derived from mononuclear phagocyte progenitors. The key event in osteoclast formation is fusion of mononucleate precursors to form mature multinucleated osteclasts. Here we provide evidence of an absolute requirement for the P2X7 receptor, ATP release, and adenosine signaling in human osteoclast formation, as shown by the following findings: macrophage‐colony stimulating factor/receptor activator for nuclear fac‐tor‐κB ligand (M‐CSF/RANKL)‐stimulated fusion of human monocytes is fully prevented by an anti‐P2X7 mAb, by specific P2X7 pharmacological antagonists, or by inhibition of CD39/NTPDase;fusion‐competent monocytes release ATP via the P2X7 receptor; accelerated degradation of released ATP by addition of either apyrase or hexokinase strongly increases fusion; removal of extracellular adenosine by adenosine deaminase blocks, while addition of exogenous adenosine strongly potentiates, fusion; and pharmacologic stimulation of the adenosine A2A receptor increases, while selective A2A blockade inhibits, fusion. These results show that the purinergic axis plays a crucial and as yet undescribed role in osteoclast formation and reconcile previous evidence advocating a keyrole for either ATP or adenosine receptors in multinucleated giant cell formation.—Pellegatti, P., Falzoni, S., Donvito, G., Lemaire, I., Di Virgilio, F. P2X7 receptor drives osteoclast fusion by increasing the extracellular adenosine concentration. FASEB J. 25, 1264–1274 (2011). www.fasebj.org

Asbestos-induced lung injury in the sheep model: The initial alveolitis

In order to study the cellular and biochemical changes in early asbestosis, three groups of sheep were repeatedly exposed to intratracheal instillations of either saline (controls), low doses of UICC chrysotile asbestos (LD), or high doses of the fibers (HD) until an alveolitis was observed in all HD sheep during the twelfth month of exposure. All sheep were studied bimonthly by transbronchial lung biopsy (LB), bronchoalveolar lavage (BAL), pulmonary function tests (PFT), and chest roentgenograms (CXR). While LBs of the HD sheep demonstrated large accumulations of monocyte-macrophages in the alveolar and interstitial spaces, those of controls and LD sheep did not. In BAL, there was no difference in total and differential cell counts between groups, but the BAL lymphocyte proliferative capacity was clearly depressed in all asbestos-exposed sheep. In the BAL supernatant, total proteins (mainly albumin, beta + gamma globulins) and lactate dehydrogenase were significantly elevated in the HD group only. This alveolitis was associated with a fall in vital capacity, lung compliance, diffusing capacity, and arterial PO2. Abnormalities on CXR appeared 3 months later. Thus, the cellular and biochemical features of early asbestosis are clearly distinct from those reported in idiopathic pulmonary fibrosis.

Involvement of the Purinergic P2X7 Receptor in the Formation of Multinucleated Giant Cells

Multinucleated giant cells (MGC), a hallmark of chronic inflammatory reactions, remain an enigma of cell biology. There is evidence implicating the purinergic P2X7 receptor in the fusion process leading to MGC. To investigate this, we used HEK 293 cells stably transfected with either 1) the full-length rat P2X7 receptor (P2X7 cells), 2) a rat P2X7 receptor lacking the C-terminal domain (P2X7TC), or 3) a mock vector, and rat alveolar macrophages (MA) expressing the native receptor. P2X7 cells cultured in serum-free medium formed increased numbers of MGC and displayed a higher fusion index compared with mock transfectants. Stimulation of P2X7 pore-forming activity in P2X7 cells by polymyxin B (PMB) further increased significantly the formation of MGC. Conversely, blockers of P2X-receptors including oxidized ATP, brilliant blue G, and pyridoxal phosphate-6-azophenyl-2′-4′-disulfonic acid inhibited significantly MGC formation in both unstimulated and PMB-stimulated P2X7-transfected cells. In contrast, cells transfected with the truncated P2X7TC were devoid of pore-forming activity, did not respond to PMB stimulation, and failed to form enhanced numbers of MGC, thus behaving as mock transfectants. As found for P2X7-transfected cells, PMB also potentiated dose-dependently the formation of multinucleated MA by rat alveolar MA. Pretreatment with oxidized ATP abrogated the PMB stimulatory effects. Together, these data demonstrate unequivocally the participation of P2X7 receptor in the process of MGC formation. Our study also provides evidence suggesting that stimulation of the P2X7 receptor pathway in MA may mediate increased formation of MGC during chronic inflammatory reactions.

Molecular mechanisms of cyclic AMP action: a genetic approach.

Publisher Summary Cells can be obtained from a metazoan source and proliferate indefinitely in tissue culture. Under these conditions, they resemble free-living microorganisms. In the studies presented in this chapter, such an approach has resulted in the isolation, from a cloned wild-type population, of mutants with alterations in cyclic adenosine monophosphate (cAMP)-related functions. Random spontaneous genetic variation produces similar rare subpopulations that are mutant in virtually any biological function not essential for cell viability. The low frequency of such events makes it necessary to devise a means to isolate mutants efficiently. In the experiments represented in the chapter, this task was relatively straightforward because of the lethal effect of cAMP on S49 cells. In some cases, mutation leads to alterations of protein function because of an alteration in the sequence of DNA coding for structural genes. In other instances, variants can arise from mutation of regulatory genes or even by mechanisms not generally considered genetic, for example, modulation of gene expression that occurs in embryonic development and differentiation. A single genetic event can affect the basal activity of adenylatecyclase and the stimulation of the enzyme by cholera toxin, prostaglandin El, isoproterenol, and sodium fluoride. The results conclude that a single class of molecules is involved in multiple adenylate cyclase-receptor systems in the same cell.

M-CSF and GM-CSF promote alveolar macrophage differentiation into multinucleated giant cells with distinct phenotypes.

Multinucleated giant cells (MGC) are a hallmark of granulomatous reactions but the mechanisms that regulate their formation are unknown. To address this issue, we cultured resident alveolar macrophages (AM) from rat lung and examined the effects of defined cytokines on AM differentiation and MGC formation. The presence of MGC was found after 3 days in culture with maximal numbers obtained at 7 days and thereafter (up to 21 days). Macrophage colony‐stimulating factor (M‐CSF) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) (25–75 U/mL) stimulated the formation of MGC (up to 4‐fold), whereas interleukin (IL) ‐3, IL‐10, and interferon‐γ (IFN‐γ) had no stimulatory effect. Interestingly, MGC with distinct phenotypes were observed in AM cultures: (1) spherical MGC with 3–16 nuclei, dense cytoplasm, and lower expression of β3 integrin (Type 1) and (2) irregular MGC with 3–30 nuclei, thin and vacuolated cytoplasm, and higher expression of βsintegrin (Type 2). Furthermore, the actions of M‐CSF and GM‐CSF on AM were found to he different. GM‐CSF promoted, in AM cultures, the appearance of an elongated fibroblastoid phenotype and stimulated mostly the formation of Type 2 MGC. In contrast, M‐CSF did not cause significant change in the general morphology of regular AM but stimulated the appearance of both Type 1 and Type 2 MGC. Reverse transcriptase‐polymerase chain reaction analysis demonstrated that, under these conditions, M‐CSF induced GM‐CSF gene expression in AM. In addition, neutralizing antibodies against M‐CSF selectively decreased the formation of Type 1 MGC, whereas neutralizing anti‐GM‐CSF inhibited Type 2 formation. These data suggest that M‐CSF promotes AM differentiation into Type 1 MGC, whereas GM‐CSF stimulates the formation of Type 2 and that M‐CSF and GM‐CSF may selectively regulate in an autocrine fashion AM differentiation into distinct MGC. J. Leukoc. Biol. 60: 509–518; 1996.

An assessment of the fibrogenic potential of very short 4T30 chrysotile by intratracheal instillation in rats.

Three groups of five rats each received, respectively, a single intratracheal instillation of saline (control), 5 mg of UICC chrysotile B asbestos, and 5 mg of a preparation of very short chrysotile fibers (4T30, 100% less than 8 micron) isolated by a sedimentation procedure. At various intervals after the treatment (1 to 60 days), assessment of lung morphology was performed on each animal. Although the two types of chrysotile fibers have similar chemical composition, structure, and surface charge, the lung tissue reaction differed considerably. Lungs of animals exposed to UICC chrysotile B showed significant pathological alterations as early as 7 days following treatment. The lesions were localized in and around terminal bronchioles and consisted of inflammatory cells, fibroblasts and collagen deposition which distorted and obstructed small airways. Reaction to very short 4T30 chrysotile fibers was quite distinct. Seven days after treatment, lungs of these animals showed alveolar and interstitial accumulation of inflammatory cells. The alveolitis persisted 60 days after treatment and no fibrosis was apparent. It appears that very short 4T30 chrysotile fibers are much less fibrogenic than UICC chrysotile B and that intratracheal instillations in rats may represent a useful mean of rapidly assessing the fibrogenic potential of various dusts. These observations support the concept that fiber length is an important factor for fibrogenicity of asbestos.