Jayasree Nath

Modulation of human neutrophil inflammatory responses by nitric oxide: studies in unprimed and LPS-primed cells.

Because nitric oxide (NO) can act both as a regulatory and as a toxic molecule, we have studied N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLF) ‐stimulated responses of human neutrophils (PMNs) during various conditions of NO modulation in unprimed and bacterial lipopolysaccharide (LPS) ‐primed cells. Effects of various NO modulators were assessed on stimulated superoxide (O2 ‐) generation, granule exocytosis, homotypic aggregation, and rises in intracellular free Ca2+ ([Ca2+]i). Significant differences in the effects of various NO modulators on inflammatory responses of PMNs kept in stirred suspension versus those kept under static and/or adherent conditions, were observed. L‐arginine, the physiological substrate for NO synthase (NOS), and NG‐nitro‐L‐arginine methyl ester, an inhibitor of NOS, both caused a 40‐50% inhibition of LPS‐induced priming of O2 ‐ generation in PMNs in stirred suspension, but not in LPS‐primed PMNs under static or adherent conditions. The NO donors, sodium nitroprusside and S‐nitroso‐N‐acetylpenicillamine, completely abrogated the LPS‐induced priming of 02 ‐ generation in PMNs in suspension, while causing only a 40‐50% inhibition in PMNs under static or adherent conditions. The Ca2+ ionophore, A23187, prevented the LPS‐induced priming of 02 ‐generation without affecting 02 ‐ generation in unprimed PMNs. LPS priming of PMNs induced about a twofold increase in fMLF‐stimulated homotypic aggregation, exocytosis of secondary granules, and rises in [Ca2+]i. In related studies, we also provide definitive evidence for enzymatic formation of NO in human PMNs and demonstrate a significant decrease in NO levels in LPS‐primed PMNs. Taken together, these findings indicate that NO modulates PMN inflammatory responses and plays a protective role in priming and activation processes of inflammatory PMNs. J. Leukoc. Biol. 62: 805–816; 1997.

Interactive role of nitric oxide and superoxide anion in neutrophil-mediated endothelial cell injury.

This study addresses the interactive role of nitric oxide (NO) and reactive oxygen intermediates (ROI) by direct quantitation of NO and superoxide (O2 –) in human neutrophil (PMN)‐endothelial cell (EC) coculture during PMN‐mediated EC injury. The results directly demonstrate an inverse correlation between NO and ROI levels in PMN‐EC coculture, which significantly alters the PMN‐EC adhesion and PMN‐mediated EC killing. N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLF)‐stimulated PMN adhesion to cytokine‐treated EC was decreased (>25%) in the presence of S‐nitroso‐N‐penicillamine, a NO donor. NO also inhibited EC killing by stimulated PMN, suggesting its cytoprotective role. In addition, a significant decrease in NO levels was observed in the PMN‐EC coculture compared with the EC cultured alone (422.45 ± 35.76 vs. 800.79 ±41.69 pmol). The reduced NO levels were restored by the addition of superoxide dismutase, a scavenger of O2 –, suggesting that PMN‐derived O2 – is involved in the neutralization of NO in the coculture. The results indicate an inverse correlation between NO and O2 – in PMN‐EC interactions and suggest the need for a critical balance between these two radicals in the regulation of PMN‐mediated tissue injury. J. Leukoc. Biol. 64: 185–191; 1998.

Packed blood cells stored in AS-5 become proinflammatory during storage

BACKGROUND: Studies have shown that packed blood cells (PBCs) stored in AS‐1 (Adsol, Baxter) and AS‐3 (Nutricel, Medsep Corp.) accumulate proinflammatory substances, which may contribute to increased complications from allogeneic blood transfusion. This study assessed whether supernates from PBCs stored in AS‐5 (Optisol, Terumo Corp.) prime neutrophils (PMNs), activate platelets (PLTs), and accumulate proinflammatory cytokines and PMN granule constituents.

Effect and localization of trifluralin in Plasmodium falciparum gametocytes: an electron microscopic study.

Trifluralin, a herbicide which is known to bind to plant and algal tubulin, induced ultrastructural changes in the microtubules of the mature Plasmodium falciparum gametocytes in vitro. Trifluralin treatment led to disassembly of the well ordered subpellicular microtubules, whereas it had no effect on microtubules of human platelets or of rat neuronal cells in vitro. The disassembled microtubules showed fragmented large tubular structures, which frequently were associated with the pellicular membranes. Electron microscopic autoradiography showed radioactive trifluralin associated with the microtubule fragments. These results provide evidence that trifluralin selectively binds to microtubules in malaria parasites and causes disruption of their structure.

Effects of Nitrogen Dioxide on the Expression of Intercellular Adhesion Molecule-1, Neutrophil Adhesion, and Cytotoxicity: Studies in Human Bronchial Epithelial Cells

Nitrogen Dioxide (NO2) is a product of high-temperature combustion and an environmental oxidant of concern. We have recently reported that early changes in NO2-exposed human bronchial epithelial cells are causally linked to increased generation of proinflammatory mediators, such as nitric oxide/nitrite and cytokines like interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-8. The objective of the present in vitro study was to further delineate the cellular mechanisms of NO2-mediated toxicity, and to define the nature of cell death that ensues upon exposure of normal human bronchial epithelial (NHBE) cells to a brief high dose of NO2. Our results demonstrate that the NHBE cells undergo apoptotic cell death during the early post-NO2 period, but this is independent of any significant increase in caspase-3 activity. However, necrotic cell death was more prevalent at later time intervals. Interestingly, an increased expression of HO-1, a redox-sensitive stress protein, was observed in NO2-exposed NHBE cells at 24 h. Since neutrophils (PMNs) play an active role in acute lung inflammation and resultant oxidative injury, we also investigated changes in human PMN–NHBE cell interactions. As compared to normal cells, increased adhesion of PMNs to NO2-exposed cells was observed, which resulted in an increased NHBE cell death. The latter was also increased in the presence of IL-8 and TNF-α + interferon (IFN)-γ, which correlated with upregulation of intercellular adhesion molecule-1 (ICAM-1). Our results confirmed an involvement of nitric oxide (NO) in NO2-induced cytotoxicity. By using NO synthase inhibitors such as L-NAME and 3-aminoguanidine (AG), a significant decrease in cell death, PMN adhesion, and ICAM-1 expression was observed. These findings indicate a role for the L-arginine/NO synthase pathway in the observed NO2-mediated toxicity in NHBE cells. Therapeutic strategies aimed at controlling excess generation of NO and/or inflammatory cytokines may be useful in alleviating NO2-mediated adverse effects on the bronchial epithelium.

Anti-inflammatory effects of U74389F in a rat model of intestinal ischemia/reperfusion injury.

OBJECTIVE To investigate the role of eicosanoid generation and neutrophilic infiltration in the protective effects of U74389F against ischemia/reperfusion injury in the small intestines of rats. DESIGN Prospective, randomized, controlled study. SETTING University research laboratory. SUBJECTS Adult, male Sprague-Dawley rats weighing between 200 and 300 g. INTERVENTIONS Groups (5-8) of rats treated with U74389F or vehicle were subjected to a sham operation and 30 mins of ischemia by occlusion of the superior mesenteric artery or 30 mins of ischemia followed by 60 or 120 mins of reperfusion. U74389F (2.5 mg/kg i.v.) or vehicle (citrate buffer) were slowly injected 2 mins before ischemia. MEASUREMENTS AND MAIN RESULTS Ischemia significantly (p < .05) increased mucosal injury (0 [normal] to 5) in both U74389F and untreated rats. In contrast, U74389F significantly (p < .05) attenuated the severity of injury after reperfusion. In vehicle-treated rats, ischemia/reperfusion significantly reduced villus height in both U74389F and untreated groups. However, the surface epithelial layer was intact in the U74389F but not in the vehicle-treated group. In addition, compared with the vehicle-treated group, U74389F significantly reduced neutrophil infiltration and prevented the increase in leukotriene B4 and prostaglandin E2 in response to ischemia and reperfusion. CONCLUSIONS This study demonstrates that the mechanism of U74389F against mesenteric ischemia/reperfusion includes a delay and reduction of neutrophilic infiltrate, an inhibition of leukotriene B4 production, and a facilitation of mucosal restitution.

Novel structure in the pellicular complex of Plasmodium falciparum gametocytes.

ABSTRACT. Using transmission electron microscopy, transverse dense bands were found to be associated with subpellicular microtubules and inner membraner in Plasmodium falciparum gametocytes. These dense bands may act as supportive structures to maintain the parallel arrangement of the microtubules, and/or to connect them to the inner membranes.

Temperature-dependent inhibition of fmet-leu-phe-stimulated superoxide generation by C-I and H-7 in human neutrophils

Contrary to previous reports by other investigators, protein kinase inhibitors C-I and H-7 (at 10(-6)M) caused a significant inhibition of fmet-leu-phe-stimulated superoxide (O2-) generation in human neutrophils. The observed inhibition of O2- production was a highly temperature-sensitive event and occurred only when C-I or H-7 was added to neutrophils at 37 degrees C. When the temperature at which C-I or H-7 added to neutrophils was varied between 16 degrees C to 37 degrees C, no significant inhibition of fmet-leu-phe-stimulated O2- production by C-I or H-7 was observed even at 35 degrees C. However, when added at 37 degrees C, both the maximal rate and the final extent of fmet-leu-phe induced O2- production were significantly inhibited (greater than 50%) by 10(-6)M C-I or H-7. A relatively weaker protein kinase C antagonist, HA1004, was not inhibitory under identical experimental conditions. In contrast, inhibition of the PMA-induced O2- generation by C-I or H-7 was not found to be similarly temperature-dependent. These results indicate that some temperature-dependent cellular event(s) is critically involved in the observed inhibition of fmet-leu-phe-induced O2- generation by C-I or H-7, and suggest a role for protein kinase C in the signal transduction process.

Neutrophil‐Endothelial Cell Interactions: Inverse Correlation between Nitric Oxide and Superoxide Anionsa

Nitric oxide ( ⋅ NO), a free-radical gas produced by many different cell types, has diverse effects in biological systems, which may be either beneficial or detrimental, depending on the amounts of ⋅ NO generated and also on the nature of the immediate microenvironment where ⋅ NO is synthesized and released. 1–4 Interaction of neutrophils (PMN) with endothelial cells (EC) is of central importance in the regulation of acute inflammatory response. PMN-mediated cell, tissue or organ injury forms the basis of many pathophysiologic conditions, and the vascular endothelium is an important target of oxidant-induced injuries in a variety of inflammatory conditions. 5 Although an excess production of ⋅ NO in the presence of superoxide anions ( ⋅ O 2 − ) is known to have cytotoxic effects, 6 ⋅ NO is also implicated in the protection of injury to target cells or tissues, during cellular events that are associated with production of reactive oxygen intermediates (ROI). The interactive role of ROI and ⋅ NO is particularly relevant in the context of inflammatory phagocytes, which generate large amounts of ROI during diapedesis and infiltration to sites of infection, inflammation and/or injury. 3,4