IFN-gamma production and degranulation are differentially regulated in response to stimulation in murine natural killer cells.

Activation of natural killer (NK) cells is induced via receptors like NKG2D, NKR-P1C and NKp46. This activation is balanced by interactions with inhibitory receptors. NK cell activation can lead to cytotoxicity mediated via polarized exocytosis of secretory lysosomes (degranulation) and interferon (IFN)-gamma production. We studied cell surface mobilization of a molecule present in secretory lysosomes, CD107a (LAMP-1), to monitor the relationship between degranulation of NK cells and their production of IFN-gamma at the single cell level. A comparison of responses in naive mouse NK cells and NK cells pre-activated with the type I interferon-inducer tilorone demonstrated a dramatic influence of pre-activation, allowing potent degranulation and IFN-gamma responses to NKG2D mediated stimulation that were not observed with naive NK cells. Degranulation and IFN-gamma production were performed by overlapping NK cell populations with generally higher frequencies of degranulating than IFN-gamma producing NK cells. An NK cell subset analysis based on expression of Mac-1 and CD27 revealed that immature NK cells (Mac-1(lo) CD27(hi)) are preferentially degranulating, Mac-1(hi) CD27(hi) cells perform both effector functions efficiently, while the most mature (Mac-1(hi) CD27(lo)) NK cells display reduced degranulation but with maintained IFN-gamma production.

Low number of H-2Dd-negative haematopoietic cells in mixed bone marrow chimeras convey in vivo tolerance to H-2Dd-negative cells but fail to prevent resistance to H-2Dd-negative leukaemia.

Natural killer (NK) cells kill cells lacking self major histocompatibility complex (MHC) class I. This missing self reactivity is beneficial in haploidentical bone marrow transplantations to cure leukaemia, in which donor-derived NK cells reject MHC disparate leukaemia cells and prevent relapse. To understand the role of NK cells in transplantation, we have studied NK cell tolerance in mice receiving mixed bone marrow transplants with limiting number of the MHC disparate component. Using an MHC class I (Dd) transgenic mouse model, we generated bone marrow chimeras carrying mixtures of Dd-positive and -negative cells. NK reactivity against Dd-negative cells (missing self) was assayed by outgrowth of lymphoma cells, stability of the chimerism in vivo and killing of Concanavalin A blasts in vitro. Up to 20% Dd-negative haematopoietic cells reduced, but did not abrogate, rejection of Dd-negative tumours and killing of Dd-negative T-cell blasts. In contrast, the ratios between Dd-positive and -negative cells were stable in vivo, suggesting tolerance to normal cells. Our data suggest that NK cell tolerance to normal cells and tumours in mixed MHC environments is differentially regulated, tolerance to normal cells being more easily induced. These results are important in relation to the role of NK cells in antileukaemic reactions after bone marrow transplantation.

cis-Bis(dimethyl sulfoxide-S)dinitratopalladium(II) and cis-dinitratobis(1,4-oxathiane-S)palladium(II).

The Pd atom in each of the two title compounds, [Pd(NO(3))(2)(C(2)H(6)OS)(2)], (I), and [Pd(NO(3))(2)(C(4)H(8)OS)(2)], (II), coordinates two O atoms from two nitrate ligands and two S atoms from dimethyl sulfoxide (dmso) and thioxane (systematic name: 1,4-oxathiane) ligands in a pseudo-square-planar cis-geometry. In the dmso complex, the distances to palladium are Pd-O 2.067 (2) and 2.072 (2) A, and Pd-S 2.2307 (11) and 2.2530 (8) A. The corresponding distances in the thioxane complex are Pd-O 2.053 (3) and 2.076 (2) A, and Pd-S 2.2595 (9) and 2.2627 (11) A. Both compounds may be regarded as dimers with an inversion centre, where one of the coordinating nitrate O atoms in one molecule also interacts with the Pd atom in the adjacent molecule, with Pd-O distances of 2.849 (9) and 3.31 (3) A in (I) and (II), respectively.

Acquisition of external major histocompatibility complex class I molecules by natural killer cells expressing inhibitory Ly49 receptors.

Murine natural killer (NK) cells express inhibitory Ly49 receptors specific for major histocompatibility complex (MHC) class I molecules. We report that during interactions with cells in the environment, NK cells acquired MHC class I ligands from surrounding cells in a Ly49-specific fashion and displayed them at the cell surface. Ligand acquisition sometimes reached 20% of the MHC class I expression on surrounding cells, involved transfer of the entire MHC class I protein to the NK cell, and was independent of whether or not the NK cell expressed the MHC class I ligand itself. We also present indirect evidence for spontaneous MHC class I acquisition in vivo, as well as describe an in vitro coculture system with transfected cells in which the same phenomenon occurred. Functional studies in the latter model showed that uptake of H-2D(d) by Ly49A+ NK cells was accompanied by a partial inactivation of cytotoxic activity in the NK cell, as tested against H-2D(d)-negative target cells. In addition, ligand acquisition did not abrogate the ability of Ly49A+ NK cells to receive inhibitory signals from external H-2D(d) molecules. This study is the first to describe ligand acquisition by NK cells, which parallels recently described phenomena in T and B cells.

Cis- and trans-influences in [PtCl2(SPh2)2].

Both cis- and trans-dichlorobis(diphenyl sulfide)platinum(II), [PtCl(2)(C(12)H(10)S)(2)], crystallize as mononuclear pseudo-square-planar complexes. In the cis compound, the Pt-Cl distances are 2.295 (2) and 2.319 (2) A, and the Pt-S distances are 2.280 (2) and 2.283 (2) A. In the trans compound, Pt is located on a centre of inversion and the Pt-Cl and Pt-S distances are 2.2786 (15) and 2.3002 (12) A, respectively.

Differential effects on T cell and NK cell development by tissue-specific expression of H-2D(d) transgene.

The effect of tissue-specific expression of the MHC class I molecule H-2D(d) on T cell and NK cell specificity was studied in transgenic mice expressing the H-2D(d) gene under the control of the mouse metallothionein-I promoter. MTD mice expressed high amounts of H-2D(d) in the liver, intestine and testis, but only minute amounts in the thymus, spleen and kidney. Zinc administration resulted in a 1.5- and 8.5-fold increase in H-2D(d) expression in the liver and the intestine, respectively, but did not affect expression in the other organs tested. T cell tolerance developed towards H-2D(d) in MTD mice, even in the absence of zinc. In contrast, NK cell-mediated natural resistance against lymphoma grafts was not seen in MTD mice, despite zinc administration. NK cells in MTD mice also failed to develop self tolerance to H-2D(d). The lack of functional effects did not result from inability of NK cells in MTD mice to interact with H-2D(d), as down-regulation of Ly49A receptor expression was observed on liver NK cells in MTD mice. Our data reveal a difference between T cells and NK cells in their requirements for MHC class I molecules in specificity development.

External and internal calibration of the MHC class I-specific receptor Ly49A on murine natural killer cells.

Expression of the H-2Dd-specific inhibitory receptor Ly49A on murine NK cells is subject to MHC class I-dependent modulation in vivo. As a result, NK cells in H-2Dd-transgenic mice express low cell surface levels of Ly49A, whereas NK cells from nontransgenic C57BL/6 (B6) mice express high levels. The purpose of this study was to assess the role of MHC class I molecules on the NK cell itself vs those on surrounding cells in this calibration and to test whether the Ly49A levels are subject to regulation in mature NK cells also. Analysis of transgenic mice with mosaic expression of an H-2Dd/Ld transgene showed that MHC class I molecules on surrounding cells (external ligands) and on the NK cell itself (internal ligands) played distinct roles in the determination of Ly49A levels. External ligands were involved in down-regulation of Ly49A levels in vivo, whereas internal ligands kept the down-regulated levels of Ly49A low upon NK cell activation in vitro. Furthermore, in an experimental system based on adoptive transfer of spleen cells, receptor down-regulation of Ly49A occurred as a rapid adaptation process in mature NK cells after interaction with the H-2Dd ligand in vivo. This suggests that Ly49 levels are not fixed but can be changed in mature NK cells when they are exposed to a changed MHC class I environment.

Alpha1/alpha2 domains of H-2D(d), but not H-2L(d), induce "missing self" reactivity in vivo--no effect of H-2L(d) on protection against NK cells expressing the inhibitory receptor Ly49G2.

Introduction of the MHC class I transgene H-2Dd on C57BL/6 (B6) background conveys NK cell-mediated "missing self" reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2Dd/Ld MHC class I transgenes, and show that the alpha1/alpha2 domains of H-2Dd were necessary and sufficient to induce "missing self" recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the alpha1/alpha2 domains of H-2Ld induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the alpha1/alpha2 domains of H-2Ld on NK cell specificity was surprising, considering that both H-2Ld and H-2Dd have been reported to interact with Ly49G2. Therefore, the role of H-2Ld for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2Dd, but not H-2Ld, abolished killing by sorted Ly49G2+ NK cells, indicating that H-2Ld does not inhibit NK cells via the Ly49G2 receptor.

MHC class I mosaic mice reveal insights into control of Ly49C inhibitory receptor expression in NK cells.

We have analyzed lymphocyte development in natural MHC class I chimeric mice, generated through a transgenic approach in beta2-microglobulin (beta2m)-/- mice. In these mice, MHC class I+ cells coexist with an equal proportion of MHC class I-deficient cells. These MHC class I mosaic mice had normal numbers of CD8+ T cells, which had a target cell specificity similar to that of wild-type mice. Consequently, the mice did not develop any signs of autoimmunity. They also had normal numbers of NK cells. This allowed an examination of the MHC class I influence on the expression of the Ly49C inhibitory receptor on NK cells. This receptor binds to H-2Kb. It is expressed at low levels on NK cells in wild-type mice of the H-2b haplotype, but at markedly higher levels on NK cells in beta2m-/- mice and other strains of mice lacking expression of H-2Kb. Relatively little is known about how MHC class I molecules affect expression of the Ly49 receptors. Through the analysis of the present MHC class I mosaic mice, we demonstrate that the expression levels of Ly49C on NK cells is a consequence not only of MHC class I expression in the environment, but also of the expression of MHC class I molecules by the NK cells themselves. These findings are discussed in relation to the biological role of the calibration of the Ly49 inhibitory receptor expression in relation to self-MHC class I.

Beta2-microglobulin-deficient NK cells show increased sensitivity to MHC class I-mediated inhibition, but self tolerance does not depend upon target cell expression of H-2Kb and Db heavy chains.

Mice lacking beta2-microglobulin (beta2m- mice) express greatly reduced levels of MHC class I molecules, and cells from beta2m- mice are therefore highly sensitive to NK cells. However, NK cells from beta2m- mice fail to kill beta2m- normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of beta2m- NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to beta2m- NK cells, we made the following observations: First, beta2m- NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to beta2m- concanavalin A (Con A)-activated T cell blasts. Secondly, beta2m- NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than beta2m+ NK cells. Thirdly beta2m- NK cells were not only tolerant to beta2m- Con A blasts but also to Con A blasts from H-2Kb-/Db- double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of beta2m- NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in beta2m- mice, which may instead operate through mechanisms involving triggering pathways.

Natural killer cell tolerance in mice with mosaic expression of major histocompatibility complex class I transgene.

We have studied natural killer (NK) cell tolerance in a major histocompatibility complex (MHC) class I transgenic line, DL6, in which the transgene product was expressed on only a fraction of blood cells. In contrast with transgenic mice expressing the same transgene in all cells, NK cells from mosaic mice failed to reject transgene-negative bone marrow or lymphoma grafts. However, they retained the capability to reject cells with a total missing-self phenotype, i.e., cells lacking also wild-type MHC class I molecules. Tolerance against transgene-negative cells was demonstrated also in vitro, and could be broken if transgene-positive spleen cells of mosaic mice were separated from negative cells before, or after 4 d of culture in interleukin-2. The results provide support for selective NK cell tolerance to one particular missing-self phenotype but not to another. We suggest that this tolerance is determined by NK cell interactions with multiple cells in the environment, and that it is dominantly controlled by the presence of cells lacking a specific MHC class I ligand. Furthermore, the tolerant NK cells could be reactivated in vitro, which suggests that the tolerance occurs without deletion of the potentially autoreactive NK cell subset(s), and that it may be dependent upon the continuous presence of tolerizing cells.

Forskolin-induced up-regulation and functional supersensitivity of dopamine D2long receptors expressed by Ltk- cells.

Mouse fibroblast Ltk- cells, stably expressing the human dopamine D2long receptor, were grown in the presence of forskolin (100 microM) for 4 or 16 h. The 16 h treatment resulted in a significant up-regulation of the dopamine D2long receptors by 43-96% as measured with [3H]raclopride with no change in the Kd value. A significant increase in the maximal inhibition of acute forskolin-stimulated cAMP accumulation by dopamine (0.1 nM-3 microM) was found both at 4 and 16 h. No such D2long-receptor-coupled response to dopamine could be detected in wild-type, untransfected, Ltk- cells with or without forskolin treatment. Furthermore, basal cAMP levels as well as the maximal response to acute forskolin stimulation decreased in the D2long receptor expressing cells with the treatment, by 33% and 23% respectively. The results indicate that persistent maintenance of high cAMP levels in transfected Ltk- cells may lead to adaptive quantitative and functional changes of the dopamine D2long receptor reminiscent of receptor supersensitivity induced by chronic antagonist treatment in vivo where the receptor targeted is inhibitorily coupled to adenylyl cyclase, as is the D2long receptor. This may provide a model for studying mechanisms underlying dopamine D2 receptor up-regulation and receptor supersensitivity, not readily induced in cell lines.

Time course of regional flow distribution following reperfusion of the isolated ischaemic rat heart.

1. The regional distribution of flow was studied at different times after the onset of reperfusion in isolated rat heart preparations. The hearts were submitted to 30 min of global ischaemia followed by 60 min of reperfusion. Microspheres labelled with various nuclides were added to the perfusate before ischaemia and 1, 5, 20, and 60 min after the onset of reperfusion. 2. One minute after the start of reperfusion, the flow to the left ventricular inner layer was restricted to 0.5 +/- 0.2 mL/min per g (2-3% of the pre-ischaemic flow). In this segment, the perfusion remained at the same low level during the entire reperfusion period studied. At the onset of reperfusion the flow to the outer layer of the left ventricle was 4.8 +/- 1.7 mL/min per g (37% of the pre-ischaemic flow), and 3.0 +/- 1.3 mL/min per g (27% of the pre-ischaemic value) to the free wall of the right ventricle. The flow was progressively reduced in the outer layer of the left ventricle in the course of reperfusion. After 60 min of reperfusion the flow to the left ventricular outer layer was 2.5 +/- 0.9 mL/min per g (19% of pre-ischaemic flow when compared with the onset of reperfusion [P less than 0.05]). 3. It is concluded that a 'no-reflow' condition develops very early during reperfusion and becomes more marked during this period.

Recombinant human extracellular superoxide dismutase reduces concentration of oxygen free radicals in the reperfused rat heart.

STUDY OBJECTIVE: The aim of the study was to determine whether recombinant human extracellular superoxide dismutase type C (rh-EC-SOD C) has antioxidant effects similar to those of the previously studied Cu-Zn superoxide dismutase. DESIGN: Electron paramagnetic resonance spectroscopy was used for quantification of free radical generation during reperfusion of postischaemic isolated rat hearts. The hearts were perfused with Krebs-Henseleit buffer and then subjected to 15 min global ischaemia. N-tert-butyl-alpha-phenylnitrone (PBN) was added as a spin trapping agent to the perfusate. EXPERIMENTAL MATERIAL: 18 hearts from Sprague-Dawley rats (280-400 g) were used. MEASUREMENTS AND MAIN RESULTS: Reperfusion resulted in a burst of radical formation. In presence of rh-EC-SOD C plus catalase, the PBN spin adduct concentration was only 18% (p less than 0.05) of the value in the control group. With bovine Cu-Zn superoxide dismutase plus catalase added to the buffer the corresponding spin adduct formation was 44% of control (NS = v rh-EC-SOD C). CONCLUSIONS: In isolated rat heart, rh-EC-SOD C reduces free radical concentrations at least to the same extent as Cu-Zn superoxide dismutase.