Rat Natural Killer Research Articles

A family of highly diverse human and mouse genes structurally links leukocyte FcR, gp42 and PECAM-1.

A group of genes encoding proteins structurally related to the leukocyte Fc receptors (FcRs) and termed the IFGP family was identified in human and mouse. Sequences of four human and two mouse cDNAs predict proteins differing by domain composition. One of the mouse cDNAs encodes a secreted protein, which, in addition to four immunoglobulin (Ig)-like domains, contains a scavenger receptor superfamily-related domain at the C-terminus. The other cDNAs code for the type I transmembrane proteins with the extracellular parts comprised of one to six Ig-like domains. Five homologous types of the Ig-like domains were defined and each protein was found to have a unique combination of the domain types. The cytoplasmic tails of the transmembrane proteins show different patterns of the tyrosine-based signal motifs. While the human IFGP members appear to be B-cell antigens, the mouse genes have a broader tissue distribution with predominant expression in brain. Sequence comparisons revealed that the IFGP family may be regarded as a phylogenetic link joining the leukocyte FcRs with the rat NK cell-specific gp42 antigen and platelet endothelial cell adhesion molecule-1 (PECAM-1), two mammalian leukocyte receptors whose close relatives were not found previously. It is suggested that FcRs, the IFGP proteins and gp42 have arisen by a series of duplications from a common ancestor receptor comprised of five Ig-like domains. The organization of the human genes shows that the IFGP family evolved through differential gain and loss of exons due to recombination and/or mutation accumulation in the duplicated copies.

Characterisation of Two Serine Protease Inhibitors Expressed in the Pituitary Gland

Ser ine p rotease in hibitors (serpins) are a family of structurally related proteins that play key roles in the regulation of proteolytic homeostasis. We have isolated a novel intracellular serpin, termed raPIT5a, from the rat pituitary gland. Northern blot analysis indicated raPIT5a mRNA expression in a range of tissues, including the adrenal gland and the brain. In situ hybridisation histochemistry revealed raPIT5a mRNA expression in specific cell populations in the rat pituitary gland, adrenal gland, and pancreas. Based on sequence similarities to other intracellular serpins, we predicted raPIT5a may inhibit the pro-apoptotic serine protease granzyme B. We confirmed this experimentally by identification of a stable inhibitory complex between granzyme B and raPIT5a. To determine whether granzyme B or granzyme B-related enzymes were expressed in the rat pituitary gland, we performed PCR using primers predicted to amplify granzyme B and two other published granzyme sequences. We identified rat natural killer protease-1 (RNKP-1), the rat homologue of granzyme B, and a novel putative serine protease highly similar to granzyme-like protein III (GLP III), which we termed GLP IIIa. These data suggest raPIT5a may regulate apoptosis in the pituitary by inhibition of granzyme B or GLP IIIa, or members of the caspase enzyme family which have similar substrate specificity. We have also identified expression of a second serpin, called neuroserpin, in pituitary tissue and found that it alters the morphology of the AtT20 corticotrope cell line, presumably through changes in cell adhesion. These results identify new roles for serpins in pituitary cell function.

The athymic nude rat: an animal experimental model to reveal novel aspects of innate immune responses?

Athymic nude rats resemble nude mice in their lack of a normal thymus and functionally mature T cells. They have been useful in the study of mechanisms of tumor growth or graft rejection in immunocompromised hosts since they can accept major histocompatibility complex (MHC) mismatched organ allografts or xenografts for several months and because a number of tumor cell lines of human and rodent origin grow well in these rats. Injection of a few helper T (Th) cells from euthymic littermate rats partly restores the pool of mature T cells as well as full immunocompetence to reject organ allografts and has helped to reveal some of the cell interactions necessary for rejection to occur In contrast, immunologically naive athymic nude rats of certain strains, acutely reject allografts consisting of lymphocytes or bone marrow cells, which is due to the presence of alloreactive natural killer cells. These cells can recognize and kill MHC incompatible hematopoietic cells through the recognition of both mismatches within the classical (RT1.A) and nonclassical (RT1.C/E) MHC class I regions with a repertoire of inhibitory and activating killer lectin-like receptors (KLR) for MHC-I molecules, encoded by the Ly-49 portion of the rat natural killer cell gene complex (NKC). Some of these receptors have been identified and molecularly cloned and show similarities with NK receptors identified in the mouse. Other leukocytes in nude rats, such as dendritic cells, may also contribute to specific innate immune responses in the absence of mature T cells. Nude rats develop T-like cells expressing CD3 and T-cell receptor (TCR) with increasing age. Though their phenotype in peripheral lymphatic tissues resembles that of normal T cells, consisting mainly of CD4+ or CD8+ cells, they lack alloreactivity in vivo and their TCR repertoire is more of an oligoclonal nature. Their contribution to allograft rejection in T-cell-reconstituted rats is therefore questionable, and their role in innate immune response in these rats still enigmatic.

Target cell induced activation of NK cells in vitro: cytokine production and enhancement of cytotoxic function.

This study examines the effect of fixed AK-5 tumour cells on rat NK cells. Co-culture of NK cells with fixed tumour cells augmented the cytotoxicity of NK cells against NK-sensitive targets, YAC-1 and AK-5, and induced the secretion of IFN-gamma by NK cells. Antibody against IFN-gamma suppressed the anti-tumour activity of NK cells, whereas the addition of T cells during co-culture enhanced this activity. However, macrophages and B cells had no significant effect when present during co-culture with NK cells. All the inducible cytotoxicity was contained within the NK (CD161+) and NKT (CD3+, CD161+) subsets of lymphocytes. However, in the presence of T cells, the cytolytic potential of NKT cells was higher than that of NK cells alone. The augmentation of cytotoxic activity of NK cells by AK-5 cells in presence of T cells was dependent on IL-2 and IFN-gamma secretion. NK cell activation was blocked by specific antibodies to IL-2 and IFN-gamma in the presence of T cells. Interaction between fixed AK-5 cells with NK and T cell populations induced the expression of Fas-L and perforin in NK cells. These data demonstrate that fixed AK-5 cells initiated cytokine synthesis by NK cells, and the enhanced cytotoxic activity in the presence of T cells was induced as a consequence of the products secreted by activated T lymphocytes. The present observations reflect the possible interactions taking place in vivo after the transplantation of AK-5 tumour in animals. They also suggest direct activation of NK cells after their interaction with the tumour cells.

RETRACTED: Toward an Optimal Oligosaccharide Ligand for Rat Natural Killer Cell Activation Receptor NKR-P1

Aminosugars have a good affinity for the NKR-P1A protein, the major activating receptor at the surface of rat natural killer cells. We have systematically investigated the structural requirements of the recombinant soluble dimeric form of the receptor for its optimal carbohydrate ligands. While N-acetylD-mannosamine was the best neutral monosaccharide ligand, its participation in the context of an extended oligosaccharide sequence was equally important. The IC(50) value for the GalNAcbeta1 --> ManNAc disaccharide was nearly 10(-10) M with a further possible increase depending on the type of the glycosidic linkage and the aglycon nature. From the point of view of its availability, stability, and affinity for the receptor and a potential in vivo use, these studies are pivotal for the design of an oligosaccharide or glycomimetics suitable for further clustering into the multivalent glycodendrimers.

Rat natural killer cell receptor systems and recognition of MHC class I molecules.

Rat natural killer (NK) cells recognize MHC-I molecules encoded by both the classical (RT1-A) and non-classical (RT1-C/E/M) MHC class I (MHC-I) regions. We have identified a receptor, the STOK2 antigen, which belongs to the Ly-49 family of killer cell lectin-like receptors, and we have localized the gene encoding it to the rat natural killer cell gene complex. We have also shown that it inhibits NK cytotoxicity when recognizing its cognate MHC-I ligand RT1-A1c on a target cell. This is the first inhibitory Ly-49-MHC-I interaction identified in the rat and highlights the great similarity between rat and mouse Ly-49 receptors and their MHC ligands. However, the mode of rat NK-cell recognition of target cells indicates that positive recognition of allo-MHC determinants, especially those encoded by the RT1-C/E/M region, is a prevalent feature. NK cells recruited to the peritoneum as a consequence of alloimmunization display positive recognition of allodeterminants. In one case, NK cells activated in this way have been shown to be specific for the immunizing, non-classical class I molecule RT1-Eu. These findings show that allospecific NK cells sometimes show features reminiscent of the adaptive immune response.

C1qRp elicits a Ca++ response in rat NK cells but does not influence NK-mediated cytotoxicity.

The cell surface receptor C1qRp (receptor for C1q, regulating phagocytosis) present on macrophages and neutrophils, is presumed to stimulate phagocytosis in these cells. However, C1qRp is also present on natural killer (NK) cells, and in these cells its physiological function is not currently known. We have investigated putative functions of this cell surface molecule in rat NK cells with the aid of two novel monoclonal antibodies (MoAb) LOV3 and LOV8 against rat C1qRp. NK cells are known to be potent cytotoxic effector cells, both through specific recognition of ligands on a target cell and killing of antibody-coated target cells (antibody-dependent cellular cytotoxicity, ADCC). NK cells prestimulated with MoAbs LOV3 or LOV8 did not exhibit altered ADCC. Furthermore, the addition of MoAb LOV3 or LOV8 to cytotoxic cultures of NK cells and Fc-receptor positive tumour cells did not affect killing in a redirected killing assay, indicating that the receptor did not influence NK cytotoxicity. However, this is the first paper to show that an intracellular Ca++-response is induced in rat NK cells upon stimulation of C1qRp with LOV3 and LOV8. The response induced by the antibodies was only minimally reduced in the presence of EGTA, indicating that most of the response is owing to the Ca++ mobilization from intracellular calcium stores.

Molecular and pharmacological characterization of the murine tachykinin NK 3 receptor

Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK 3 receptor from mouse brain. The murine NK 3 receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK 3 receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK 3 receptor and human NK 3 receptor, although substance P and neurokinin A were more potent stimulators of Ca 2+ mobilization in murine NK 3 receptor cells. NK 3 receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK 3 receptor compared to human NK 3 receptor, and about 5- to 10-fold reduced potency in the murine NK 3 receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK 3 receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.

Synthesis of chitooligomer-based glycoconjugates and their binding to the rat natural killer cell activation receptor NKR-P1.

NKR-P1 protein is an important activating receptor at the surface of the rat natural killer cells. GlcNAc and chitooligomers were identified as strong activation ligands in vitro and in vivo. Their clustering brings about increase of their affinity to the NKR-P1 by 3-6 orders. Here we describe novel methodology for preparation of neoglycoproteins based on BSA carrying the chitooligomers (n = 2-5). Further on we developed novel methodology of the coupling of glycosylamines via aromatic-SCN activated linker both to protein or synthetic cores. Inhibition studies of chitooligomer glycoconjugates with the NKR-P1 receptor show that our neoglycoproteins are very strong ligands with high binding affinity (-log IC(50) = 13-15). In analogy with our previous observations with GlcNAc clustered on protein or PAMAM backbones the synthetic chitooligomer clusters should provide considerably better ligands in the in vivo antitumor treatment.

Molecular Cloning of Rat NK Target Structure —The Possibility of CD44 Involvement in NK Cell‐Mediated Lysis

The nature of target molecules of natural killer (NK) cell-mediated lysis remains to be elucidated. As we previously reported, mAb 109 recognizes one of the tumor-associated antigens, designated as 109 antigen (Ag), expressed on the cell surface of rat fibrosarcomas W31 and W14, which are transformants of WFB (rat fetal fibroblast cell line) with H-ras oncogene. 109Ag was thought to be a target structure of NK cells since mAb 109 inhibited NK cell-mediated lysis against W31 and W14. Here, we demonstrate by molecular cloning that 109Ag is identical to rat CD44. Immunoprecipitation and immunoblotting studies also showed that mAb 109 and anti-rat CD44 mAb OX-50 recognize the same protein of W31 cell lysates with an 86 kDa molecular size. CD44 was suggested to be a target structure of NK cell-mediated lysis; however, rat CD44 cDNA transfection alone into CD44 null cell lines did not result in up-regulation of target cell susceptibility to NK cell-mediated lysis. Our results therefore indicated that CD44 may play a crucial role as one of the target structures in our rat fibrosarcoma system though the cell surface expression of CD44 alone does not affect NK susceptibility of the target cells.

Characterization and molecular cloning of rat C1qRp, a receptor on NK cells.

Here we report the generation of monoclonal antibodies (mAb) LOV3 and LOV8 to a 110-130-kDa membrane glycoprotein expressed by rat NK cells. This NK surface molecule was identified by eucaryotic expression cloning as the structural orthologue of the phagocytosis-stimulating receptor for complement factor C1q and mannose-binding lectin on human macrophages, C1qRp. Rat C1qRp is a monomeric type I integral membrane protein consisting of 643 amino acids with an N-terminal lectin-like domain, five epidermal growth factor-like domains, a transmembrane domain and a 45-residue cytoplasmic domain. It is encoded by a single gene on rat chromosome 3q41-q42 and is 67% and 87.5% identical at the amino acid level to human and mouse C1qRp, respectively. Rat C1qRp is expressed by resting and by activated NK cells, on subpopulations of NKR-P1(+) T cells (NK/T cells), dendritic cells, macrophages and granulocytes, but not by B cells or NKR-P1(-) T cells. Expression of this innate immune receptor is therefore not restricted to hematopoietic cells of the myeloid lineage, but is also expressed on subsets of cells of lymphoid origin. The mAb did not affect the cytotoxic function of NK cells, and C1qRp on NK cells may have functions not related to NK killing.

Independent endocytosis of the NK 1 and NK 3 tachykinin receptors in neurons of the rat myenteric plexus

In the myenteric plexus of rat ileum, NK 1 and NK 3 receptors are co-located almost exclusively on neurons of a single population. This study compares endocytosis of NK 1 and NK 3 receptors in these neurons. In the absence of agonist, 26.2±2.8% of NK 1 receptor and 29.1±1.1% of NK 3 receptor was located in the cytoplasm of the neurons; the remaining receptor was on the surface. The tachykinin neurotransmitters, substance P (10 pM–10 μM) and neurokinin A (10 pM–100 μM), both induced concentration-dependent endocytosis of NK 1 and NK 3 receptors. The selective NK 1 receptor agonist, [Sar 9,Met(O 2) 11]-substance P (1 μM), induced endocytosis of NK 1 receptor (64.2±1.5% in cytoplasm) but not NK 3 receptor (32.9±5.0%). The NK 1 receptor endocytosis was reduced by the selective NK 1 receptor antagonist, CP-99994 (100 nM), but not by the selective NK 3 receptor antagonist, SR-142801 (1 μM). The selective NK 3 receptor agonist, senktide (10 nM), induced endocytosis of NK 3 receptor (61.2±5.4%) but not NK 1 receptor (34.0±4.5%). The NK 3 receptor endocytosis was blocked by SR-142801 but not by CP-99994. We also investigated the effects of monensin, which generally blocks recycling of endocytosed receptor. In the absence or presence of exogenous agonist, monensin caused a build-up of NK 1 receptor, but not NK 3 receptor, in the cytoplasm of neurons. The results demonstrate independent, agonist-induced endocytosis of NK 1 and NK 3 receptors in neurons of the myenteric plexus of rat ileum and suggest that the mechanisms of recycling of NK 1 and NK 3 receptors differ.

Molecular characterization of the rat NK cell receptor 2B4

2B4 (CD244) is a cell surface glycoprotein of the immunoglobulin superfamily involved in the regulation of natural killer and T lymphocyte function. It is the high affinity counter-receptor for CD48. In mouse and human NK cells, crosslinking of 2B4 with a specific monoclonal antibody or with CD48 can trigger cell-mediated cytotoxicity, IFN-γ secretion, phosphoinositol turnover and NK cell invasiveness. Recent reports of defective 2B4 signaling and NK cell function in X-linked lymphoproliferative syndrome suggest that this may contribute to the progression of this human disease. Here we describe the molecular characterization of the rat 2B4 gene. The cDNA encodes a protein of 395 amino acid residues that contain two Ig domains in the extracellular region and three unique tyrosine motifs (TxYxxV/I/A) in the cytoplasmic region. The predicted protein has 81 and 68% similarity with mouse 2B4 and human 2B4, respectively. Additionally, it has 94 and 89% similarity at the protein level with the recently reported rat 2B4 related genes, r2B4R-tm and r2B4R-se respectively. Northern blot analysis indicated the presence of multiple transcripts in rat LAK cells and RNK-16 cells. Immunoprecipitation and deglycosylation studies showed that rat 2B4 is glycosylated to similar extent as that of mouse and human 2B4. The cloning of r2B4 in the light of the availability of rat NK cell lines should facilitate in vitro and in vivo experiments to decipher the functional role of 2B4 in NK cell biology.

Ly-49D transfected NK cells show reduced interleukin-10 production in response to H-2d and increased lytic activity: implication by interaction using class I MHC alloantigen+ target cells in pregnancy.

Mating of CBA/J (H-2k) with DBA/2 (H-2d) males leads to a high rate of spontaneous resorption (about 40%), which is not seen in other mating combinations, such as CBA/J X BALB/c. The activation of natural killer cells (NK cells) seems to be a key mechanism for the maternal-fetal intolerance in allogeneic pregnancy, and recurrent spontaneous abortion. The effect of expression of the NK cell activating receptor Ly49D recognizing BALB/c or DBA/2 class I MHC was investigated. Intracellular interleukin (IL)-100 production was detected and target cell survival rates were calculated after 22 hr coincubation of rat NK cells transfected, or not. with a murine Ly49D receptor, with either male BALB/c or male DBA/2 splenocytes, by using flow cytometry. Ly49D negative rat NK cells produced 13.7% more IL-10 than Ly49D positive rat NK cells, and more splenocytes were killed by Ly49D transfected rat NK cells (survival rate 2.45%) than by Ly49D negative rat NK cells (survival rate 4.36%). After physiological stimulation with BALB/c or DBA/2 splenocytes, rat NK cells are able to synthesize IL-10. Recognition of mouse splenocyte major histocompatibility complex (MHC) by Ly49D mice receptor decreased IL-10 production. The observed increase in killing activity might be a result of this phenomenon. NK cell activation via the Ly49D receptor might play an important role in pregnancy failure, but cannot explain why CBA/J X DBA/2 matings are abortion prone, and CBA/J X BALB/c matings are abortion resistant.

Analyzing nonspecific cytotoxic cells in fish.

The nonspecific cytotoxic cell (NCC) is believed to be the evolutionary precursor of the mammalian natural killer (NK) cell. Evidence for a phylogenetic linkage between NCCs of teleost fish and NK cells from higher vertebrates was first obtained functionally; that is, NCCs lyse many of the same target cells as human (K562), mouse (YAC-1) and rat NK cells (,). The best parallels between NK and NCC are found in the target cell recognition of NCC vs adherent lymphokine (interleukin-2 [IL-2]) activated killer cells (ALAK). Both ALAK and NCC kill promiscuously. Their wide range of targets include many different histological types of allogeneic tumor cells, xenogeneic tumor targets, protozoan parasites, as well as certain virus transformed cells (,). Beyond the observations that both types of cells kill the same targets, two other properties link NK cells with NCC.

Natural Killing of Xenogeneic Cells Mediated by the Mouse Ly-49D Receptor

AbstractNK lymphocytes lyse certain xenogeneic cells without prior sensitization. The receptors by which NK cells recognize xenogeneic targets are largely uncharacterized but have been postulated to possess broad specificity against ubiquitous target ligands. However, previous studies suggest that mouse NK cells recognize xenogeneic targets in a strain-specific manner, implicating finely tuned, complex receptor systems in NK xenorecognition. We speculated that mouse Ly-49D, an activating NK receptor for the MHC I ligand, H2-Dd, might display public specificities for xenogeneic target structures. To test this hypothesis, we examined the lysis of xenogeneic targets by mouse Ly-49D transfectants of the rat NK cell line RNK-16 (RNK.Ly-49D). Of the xenogeneic tumor targets tested, RNK.Ly-49D, but not untransfected RNK-16, preferentially lysed tumor cells derived from Chinese hamsters and lymphoblast targets from rats. Ly-49D-dependent recognition of Chinese hamster cells was independent of target N-linked glycosylation. Mouse Ly-49D also specifically stimulated the natural killing of lymphoblast targets derived from wild-type and MHC-congenic rats of the RT1lv1 and RT1l haplotypes, but not of the RT1c, RT1u, RT1av1, or RT1n haplotypes. These studies demonstrate that Ly-49D can specifically mediate cytotoxicity against xenogeneic cells, and they suggest that Ly-49D may recognize xenogeneic MHC-encoded ligands.

The Regulatory Role of CD45 on Rat NK Cells in Target Cell Lysis

To investigate the role of CD45 in rat NK cell function, we developed new mAbs directed against rat CD45. mAb ANK12 binds to a high molecular isoform of CD45 and mAb ANK74 binds to the common part on all known CD45 isoforms, as has been described for the anti-rat CD45 mAb OX1. The ability of these mAbs to affect NK cell-mediated lysis was tested using the Fc receptor-positive target cell line P815. mAb ANK12 was found to significantly enhance the lysis of P815, whereas ANK74 and the anti-CD45 mAb OX1 did not. In addition, cross-linking of the CD45 isoform by ANK12 induced tyrosine phosphorylation of specific proteins in NK cells. Subsequently, the involvement of CD45 in the negative signaling after "self" MHC class I recognition by rat NK cells was investigated. The anti-CD45 mAbs were found to affect NK cell-mediated lysis of syngeneic tumor cell lines, depending upon the expression level of MHC class I on target cells. mAbs ANK74 and OX1 only inhibited lysis of the syngeneic tumor cell lines that expressed low levels of MHC class I. Furthermore, both mAbs caused an inhibition of NK cell-mediated lysis of these tumor cell lines when MHC class I molecules on the tumor cell lines were masked by an Ab. These results suggest that CD45 regulates the inhibitory signal pathway after self MHC class I recognition, supposedly by dephosphorylation of proteins.

Uterine natural killer cells are targets for a trophoblast cell-specific cytokine, prolactin-like protein A.

PRL-like protein A (PLP-A) is a member of the PRL family expressed in trophoblast cells coincident with establishment of the chorioallantoic placenta. The purpose of this investigation was to identify targets for PLP-A. Using an alkaline phosphatase-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of decidua from pregnant and pseudopregnant rats. These observations are supported by the codistribution of PLP-A targets with cells expressing the rat NK cell surface marker, gp42, the absence of PLP-A binding in conceptuses from NK cell-deficient tg epsilon26 mice, and the specific interaction of PLP-A with a rat NK cell line, RNK-16. We have further demonstrated that PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK lymphocytes.

Uterine Natural Killer Cells Are Targets for a Trophoblast Cell-Specific Cytokine, Prolactin-Like Protein A

PRL-like protein A (PLP-A) is a member of the PRL family expressed in trophoblast cells coincident with establishment of the chorioallantoic placenta. The purpose of this investigation was to identify targets for PLP-A. Using an alkaline phosphatase-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of decidua from pregnant and pseudopregnant rats. These observations are supported by the codistribution of PLP-A targets with cells expressing the rat NK cell surface marker, gp42, the absence of PLP-A binding in conceptuses from NK cell-deficient tgε26 mice, and the specific interaction of PLP-A with a rat NK cell line, RNK-16. We have further demonstrated that PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK lymphocytes.

Interleukin-2–Activated Rat Natural Killer Cells Express Inducible Nitric Oxide Synthase That Contributes to Cytotoxic Function and Interferon-γ Production

Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2–activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2–activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2–activated NK cells. Moreover, IL-2–induced interferon-γ production appears also to be dependent, at least in part, on iNOS induction.