Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats

Staphylococcus aureus is a gram-positive bacterium that produces several enterotoxins, which are responsible for most part of pathological conditions associated to staphylococcal infections, including lung inflammation. This study aimed to investigate the underlying inflammatory mechanisms involved in leukocyte recruitment in rats exposed to staphylococcal enterotoxin B (SEB). Rats were anesthetized with pentobarbital sodium and intratracheally injected with either SEB or sterile phosphate-buffered saline (PBS, 0.4 ml). Airways exposition to SEB (7.5-250 ng/trachea) caused a dose- and time-dependent neutrophil accumulation in BAL fluid, the maximal effects of which were observed at 4 h post-SEB exposure (250 ng/trachea). Eosinophils were virtually absent in BAL fluid, whereas mononuclear cell counts increased only at 24 h post-SEB. Significant elevations of granulocytes in bone marrow (mature and immature forms) and peripheral blood have also been detected. In BAL fluid, marked elevations in the levels of lipid mediators (LTB(4) and PGE(2)) and cytokines (TNF-alpha, IL-6 and IL-10) were observed after SEB instillation. The SEB-induced neutrophil accumulation in BAL fluid was reduced by pretreatment with dexamethasone (0.5 mg/kg), the COX-2 inhibitor celecoxib (3 mg/kg), the selective iNOS inhibitor compound 1400 W (5 mg/kg) and the lipoxygenase inhibitor AA-861 (200 microg/kg). In separate experiments carried out with rat isolated peripheral neutrophils, SEB failed to induce neutrophil adhesion to serum-coated plates and chemotaxis. In conclusion, rat airways exposition to SEB causes a neutrophil-dependent lung inflammation at 4 h as result of the release of proinflammatory (NO, PGE(2), LTB(4), TNF-alpha, IL-6) and anti-inflammatory mediators (IL-10).     

The effect of sesquiterpene lactones on the release of human neutrophil elastase

Sesquiterpene lactones (SLs) are natural products responsible for the anti-inflammatory activity of a variety of medicinal plants, mainly from the Asteraceae family. Here, we investigated whether they also influence the process of exocytosis of pro-inflammatory enzymes, such as the human neutrophil elastase (HNE). Altogether, eight structurally different SLs from the eudesmanolide, guaianolide, pseudoguaianolide, and germacranolide type were studied. Neutrophils were isolated from fresh human blood. After pre-incubation with different concentrations of the respective SL and cytochalasin B, the exocytosis of elastase was initiated either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Inhibition of HNE release was measured by p-nitroaniline formation. The SLs exhibited an inhibitory effect on elastase release from neutrophils challenged either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Concentration-response curves were recorded and the IC(50) values ranged from 2 to 30 microM. Studies on isolated HNE showed that a selective direct inhibition on HNE can be excluded. Interestingly, the inhibitory activity did not correlate with the number of alpha,beta-unsaturated carbonyl functions. The structure-activity relationship and the molecular mechanism are discussed.     

The lathyrus toxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and homocysteic acid sensitize CA1 pyramidal neurons to cystine and L-2-amino-6-phosphonohexanoic acid

A brief exposure of hippocampal slices to L-quisqualic acid (QUIS) sensitizes CA1 pyramidal neurons 30- to 250-fold to depolarization by certain excitatory amino acids analogues, e.g., L-2-amino-6-phosphonohexanoic acid (L-AP6), and by the endogenous compound, L-cystine. This phenomenon has been termed QUIS sensitization. A mechanism similar to that previously described for QUIS neurotoxicity has been proposed to describe QUIS sensitization. Specifically, QUIS has been shown to be sequestered into GABAergic interneurons by the System x(c)(-) and subsequently released by heteroexchange with cystine or L-AP6, resulting in activation of non-NMDA receptors. We now report two additional neurotoxins, the Lathyrus excitotoxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and the endogenous compound, L-homocysteic acid (HCA), sensitize CA1 hippocampal neurons >50-fold to L-AP6 and >10-fold to cystine in a manner similar to QUIS. While the cystine- or L-AP6-mediated depolarization can be inhibited by the non-NMDA receptor antagonist CNQX in ODAP- or QUIS-sensitized slices, the NMDA antagonist D-AP5 inhibits depolarization by cystine or L-AP6 in HCA-sensitized slices. Thus, HCA is the first identified NMDA agonist that induces phosphonate or cystine sensitization. Like QUIS sensitization, the sensitization evoked by either ODAP or HCA can be reversed by a subsequent exposure to 2 mM alpha-aminoadipic acid. Finally, we have demonstrated that there is a correlation between the potency of inducers for triggering phosphonate or cystine sensitivity and their affinities for System x(c)(-) and either the non-NMDA or NMDA receptor. Thus, the results of this study support our previous model of QUIS sensitization and have important implications for the mechanisms of neurotoxicity, neurolathyrism and hyperhomocystinemia.     

Accessibility of endothelial and inducible nitric oxide synthase to the intracellular citrulline-arginine regeneration pathway

This study investigates our hypothesis that argininosuccinate synthase (AS), the rate-limiting enzyme for arginine (L-arg) regeneration from citrulline (L-cit), plays a pivotal role in supplying L-arg to endothelial (eNOS), but not inducible (iNOS) nitric oxide synthase, for nitric oxide (NO) production. Transgenic rat blood-brain barrier (TR-BBB) endothelial cells were used as a model to elucidate the accessibility of the L-arg compartments for NOS isozymes. NO production via eNOS or iNOS, with or without alpha-methyl-DL-aspartic acid (MDLA), an AS inhibitor, was measured by a fluorometric method. NO production via eNOS was activated by the calcium ionophore A23187, while via iNOS was induced by cytokines. AS activity was assayed by the amount of argininosuccinate regenerated from radioactive aspartic acid from cell extracts. Upon increased AS activity (5.9-fold) in cells grown in L-arg-free/L-cit-supplemented medium, A23187-activated NO production also significantly increased, however cytokine-induced NO production was not detected. A23187-activated NO production was observed not only in L-arg containing medium, but also L-arg-free and L-arg-free/L-cit-supplemented medium, and was abolished by MDLA regardless of medium type. Cytokine-induced NO production was only observed in L-arg containing medium, not in L-arg-free or L-arg-free/L-cit-supplemented medium, and it was not inhibited by MDLA in the L-arg containing medium. Our results indicate that extracellular L-arg was the only L-arg pool for cytokine-induced NO production and intracellular L-arg regenerated from L-cit via AS pathway was the major L-arg pool for A23187-activated NO production in TR-BBB endothelial cells. Therefore, modulation of AS activity could be a promising strategy to selectively alter NO production via eNOS, but not iNOS.     

Recombinant arginine deiminase as a differential modulator of inducible (iNOS) and endothelial (eNOS) nitric oxide synthetase activity in cultured endothelial cells

Modulation of the extracellular level of arginine, substrate for nitric oxide synthetases, is a promising modality to alleviate certain pathological conditions where excess nitric oxide (NO) is produced. However, complications arise, as only preferential inhibition of the inducible nitric oxide synthetase (iNOS), but not endothelial nitric oxide synthetase (eNOS), is desired for the treatment of NO over-production. We investigated the effect of arginine deprivation mediated by a recombinant arginine deiminase (rADI) on the activity of iNOS and eNOS in an endothelial cell line, TR-BBB. Our results demonstrated that cytokine-induced NO production depends on the extracellular arginine as substrate. However, if sufficient citrulline is present in the medium, A23187-activated NO production by eNOS does not rely on extracellular arginine. Treatment with rADI can markedly inhibit cytokine-induced NO production via iNOS, but not A23187-activated NO production via eNOS. Our results also showed that the decrease of NO production by iNOS could be achieved by depleting arginine from the medium even under the conditions that would up-regulate iNOS expression. Thus, rADI appears to be a novel selective modulator of iNOS activity that may be a used as a tool in the study of pathological disorders where NO over-production plays a key role.     

A selective plasmin inhibitor, trans-aminomethylcyclohexanecarbonyl-L-(O-picolyl)tyrosine-octylamide (YO-2), induces thymocyte apoptosis

The treatment of rat thymocytes with YO-2, a novel inhibitor of plasmin, resulted in an increase in DNA fragmentation. DNA fragmentation was also induced by another YO compounds such as YO-0, -3, -4 and -5. These YO compounds are the inhibitor of plasmin activity. On the other hand, YO-1, -6 and -8 that hardly inhibit plasmin activity had no effect on DNA fragmentation. Analysis of fragmented DNA from thymocytes treated with YO-2 by agarose gel electrophoresis revealed that the compound caused internucleosomal DNA fragmentation. In addition, judging from a laser scanning microscopy, annexin V-positive and propidium iodide-negative cells were increased by the treatment of the cells with the compound. Moreover, chromatin condensation was observed in thymocytes treated with the compound. These results demonstrated that YO-2 induces thymocyte apoptosis. There seemed to be some correlation between the apoptosis induced by YO compounds and their plasmin inhibitory effect. However, because the other protease inhibitors including pepstatin A, leupeptin, AEBSF, DFP and E-64-d did not affect DNA fragmentation, YO compounds are likely to have unique mechanism on plasmin or to show the effect on the other plasmin-like proteases. The plasmin inhibitory activity may have an important role in YO-2-induced apoptosis. Furthermore, the stimulations of caspase-8, -9 and -3-like activities were observed in thymocytes treated with YO-2. These results suggest that YO-2 induces thymocyte apoptosis via activation of caspase cascade.     

STIM1 but not STIM2 is an essential regulator of Ca influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells

Extracellular Ca²⁺ entry, primarily mediated through store-operated Ca²⁺ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca²⁺ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca²⁺ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca²⁺ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca²⁺ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca²⁺ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca²⁺ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development.     

Role of sodium in intracellular calcium elevation and leukotriene B4 formation by receptor-mediated activation of human neutrophils

The role of Na(+) and Na(+) exchangers in intracellular Ca(2+) elevation and leukotriene B(4) (LTBs) formation was investigated in granulocyte macrophage colony-stimulating factor (GM-CSF)-primed, fMLP-stimulated human neutrophils. Isotonic substitution of extracellular Na(+) with N-methyl-D-glucamine(+) (NMDG(+)) resulted in over 85% inhibition of the LTBs generation observed (from 14.1+/-0.9pmol/10(6) neutrophils to 1.7+/-1.0pmol/10(6) neutrophils at 0.3 microM fMLP). Isotonic substitution of Na(+) with NMDG(+) also induced a significant inhibition of fMLP-induced rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) (from 2.17- to 0.78-fold increase over basal levels). Pretreatment with an inhibitor of the Na(+)/Ca(2+) exchanger (benzamil) did not inhibit either [Ca(2+)](i) rise or LTBs production, indicating that the observed effects of extracellular Na(+)-deprivation were unrelated to the Na(+)/Ca(2+) exchanger in receptor-mediated Ca(2+) influx, as previously hypothesized. LTBs production by thapsigargin-activated neutrophils was not affected by Na(+) depletion, but was totally abolished in the presence of EGTA, suggesting that store depletion-driven extracellular Ca(2+) influx is required for leukotriene synthesis and that this process is independent of Na(+)-deprivation. Exposure to Na(+)-free medium for the time of GM-CSF priming led to a significant decrease of intracellular pH values, suggesting a role of the Na(+)/H(+) exchanger in intracellular Na(+) depletion. Reducing the time of Na(+)-deprivation totally reversed the observed effect on LTBs production, resulting in enhanced, rather than inhibited, formation of LTBs. These results indicate that LTBs generation and [Ca(2+)](i) rise in human neutrophils primed by GM-CSF and stimulated with fMLP is dependent on intracellular Na(+) concentration, and, at variance with previously published results, unrelated to the Ca(2+) influx through the Na(+)/Ca(2+) exchanger.