Nitric oxide inhibits exocytosis of cytolytic granules from lymphokine-activated killer cells

Abstract

NO inhibits cytotoxic T lymphocyte killing of target cells, although the precise mechanism is unknown. We hypothesized that NO decreases exocytosis of cytotoxic granules from activated lymphocytes. We now show that NO inhibits lymphokine-activated killer cell killing of K562 target cells. Exogenous and endogenous NO decreases the release of granzyme B, granzyme A, and perforin: all contents of cytotoxic granules. NO inhibits the signal transduction cascade initiated by cross-linking of the T cell receptor that leads to granule exocytosis. In particular, we found that NO decreases the expression of Ras, a critical signaling component within the exocytic pathway. Ectopic expression of Ras prevents NO inhibition of exocytosis. Our data suggest that Ras mediates NO inhibition of lymphocyte cytotoxicity and emphasize that alterations in the cellular redox state may regulate the exocytic signaling pathway.granzyme ͉ inflammation ͉ lymphocyte ͉ mitogen-activated protein kinases ͉ Ras N O plays a complex set of roles in the immune system (1-4). NO is generated from L-arginine by one of three isoforms of NO synthase (NOS): neuronal NOS (NOS1), endothelial NOS (NOS3), and inducible NOS (iNOS or NOS2) (5, 6). NO can act as an innate immune effector, inhibiting the replication of diverse pathogens such as Mycobacterium tuberculosis, Leishmania, and coxsackievirus (7-12). However, NO and the reactive nitrogen intermediates produced by oxidation of NO can be harmful to the host. For example, NO plays a role in LPS-induced hypotension, LPSinduced lung damage, autoimmune vasculitis, autoimmune encephalomyelitis, autoimmune nephritis, and acute allograft rejection (13-21). Furthermore, NO can suppress inflammation and decrease cell injury. For example, NO inhibits vascular inflammation in part by decreasing endothelial exocytosis of factors that would otherwise promote leukocyte adherence to the vessel wall (22). NO also modulates the immune response, inhibiting T lymphocyte proliferation and differentiation, B lymphocyte proliferation and antibody production, and immune cell production of cytokines (12).NO may also be able to modulate inflammation in part by regulating immune cell killing of target cells. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells kill infected cells or tumor cells by several distinct pathways, including activation of the Fas͞Fas ligand pathway and exocytosis of cytolytic granules. These cytolytic granules contain perforin, serine proteases (including granzymes), and other effector molecules that promote death of target cells. When a T cell or NK cell recognizes its target, an immunological synapse is formed, a cluster of signaling, adhesion, and cytoskeletal proteins that includes the T cell receptor or a NK cell receptor, tyrosine kinases such as Lck and ZAP-70, and adaptor proteins such as LAT (linker of T cell activation) and . This cluster of signaling molecules activates downstream pathways, including Ras, Raf, MAPK͞ERK kinase (MEK), and ERK. The microtubule organizing center directs the...

A novel immunomodulatory mechanism of ribavirin in suppressing natural killer cell function

“…NO-mediated cell death is prominent among the many paths the immune system uses to kill target cells [28,35]. While some studies suggest that NO blocks activation of NK and cytotoxic T lymphocyte cells [36][37][38], another report suggests that NO has no effect on lymphokine activated killer (LAK) cells [39]. Some more studies however showed that NO increases LAK activity A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16 tetrahydrobiopterin alone did not influence NK cell lytic capacity treatment with NO alone significantly suppressed cytotoxicity of NK cells.…”

Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1α

“…Although our studies are based on in vitro studies with an exogenous NO donor and do not directly address the importance of endogenously produced NO within inflamed airways, a number of studies have demonstrated that the use of 500 M DETA-NO adequately mimics NO production and NO-mediated effects by e.g. activated macrophages (36,40), suggesting that our studies are relevant for in vivo inflammatory conditions. Therefore, our data demonstrate a pathophysiological role for increased airway NO under conditions of inflammation and highlight mechanisms by which increased expression and activity of NOS2 could contribute to airway epithelial injury and disrepair/remodeling during chronic airway inflammatory disease.…”

“…Recent studies in other cell systems have shown that NO is capable of reducing ERK1/2 phosphorylation, which was related to down-regulation of Ras expression (40) or to induction of the MAPK phosphatase MKP-1 (41,42). We therefore explored changes in either Ras or MKP-1 expression in association with NO-mediated ERK1/2 dephosphorylation.…”

H2S Increases Survival during Sepsis: Protective Effect of CHOP Inhibition

“…Tissue was homogenized in RIPA buffer with protease and phosphatase inhibitors and protein content determined. Immunoblotting was performed as previously described (25). Briefly, proteins were separated on a gradient SDS-PAGE, transferred to a PVDF membrane and blocked with 5% milk buffer.…”