Prognostic significance of activated cytotoxic T-lymphocytes in primary nodal diffuse large B-cell lymphomas.

Clinical outcome in diffuse large B-cell lymphoma (DLBCL) remains unpredictable, despite the identification of clinical prognostic parameters. Here, we investigated in pretreatment biopsies of 70 patients with DLBCL whether numbers of activated cytotoxic T-lymphocytes (CTLs), as determined by the percentage of CD3-positive lymphocytes with granzyme B (GrB) expression, have similar prognostic value as found earlier in Hodgkin's lymphoma and anaplastic large-cell lymphoma and whether loss of major histocompatibility complex (MHC)-I molecules or expression of the GrB antagonist protease inhibitor 9 (PI9) may explain immune escape from CTL-mediated cell death. Independent of the International Prognostic Index (IPI), the presence of >/=15% activated CTLs was strongly associated with failure to reach complete remission, with a poor progression-free and overall survival time. Downregulation of MHC-I light- and/or heavy-chain expression was found in 41% of interpretable cases and in 19 of 56 interpretable cases PI9 expression was detected. We conclude that a high percentage of activated CTLs is a strong, IPI independent, indicator for an unfavorable clinical outcome in patients with primary nodal DLBCL. Although in part of DLBCL expression of PI9 and loss of MHC-I expression was found, providing a possible immune-escape mechanism in these cases, no correlation with clinical outcome was found.

Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells.

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis.

The granzyme B inhibitor, protease inhibitor 9, is mainly expressed by dendritic cells and at immune-privileged sites.

Granzyme B is released from CTLs and NK cells and an important mediator of CTL/NK-induced apoptosis in target cells. The human intracellular serpin proteinase inhibitor (PI)9 is the only human protein able to inhibit the activity of granzyme B. As a first step to elucidate the physiological role of PI9, PI9 protein expression in various human tissues was studied. A mAb directed against human PI9 was developed, which specifically stained PI9-transfected COS-7 cells, and was used for immunohistochemistry. Both in primary lymphoid organs and in inflammatory infiltrates, PI9 was present in different subsets of dendritic cells. Also T-lymphocytes in primary and organ-associated lymphoid tissues were PI9 positive. Endothelial cells of small vessels in most organs tested as well as the endothelial layer of large veins and arteries showed strong PI9 staining. Surprisingly, high PI9 protein expression was also found at immune-privileged sites like the placenta, the testis, the ovary, and the eye. These data fit with the hypothesis that PI9 is expressed at sites where degranulation of CTL or NK cells is potentially deleterious.

Cloning and expression of CTP:phosphoethanolamine cytidylyltransferase cDNA from rat liver.

CTP:phosphoethanolamine cytidylyltransferase (ET) is a key regulatory enzyme in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. As a first step in the elucidation of the structure-function relationship and the regulation of ET, an ET cDNA was cloned from rat liver. The cloned cDNA encodes a protein of 404 amino acid residues with a calculated molecular mass of 45.2 kDa. The deduced amino acid sequence is very similar to that of human ET (89% identity). Furthermore, it shows less, but significant, similarity to yeast ET as well as to other cytidylyltransferases, including rat CTP:phosphocholine cytidylyltransferase and Bacillus subtilis glycerol-3-phosphate cytidylyltransferase. Like human and yeast ET, rat ET has a large repetitive internal sequence in the N- and C-terminal halves of the protein. Both parts of the repeat contain the HXGH motif, the most conserved region in the N-terminal active domain of other cytidylyltransferases, indicating the existence of two catalytic domains in ET. The hydropathy profile revealed that rat ET is largely hydrophilic and lacks a hydrophobic stretch long enough to span a bilayer membrane. There was no prediction for an amphipathic alpha-helix. Transfection of COS cells with the cDNA clone resulted in an 11-fold increase in ET activity, corresponding to an increase in the amount of ET protein as detected on a Western blot. Determination of the ET activity during liver development showed a 2. 5-fold increase between day 17 of gestation and birth (day 22) and the amount of ET protein changed accordingly. Northern blot analysis showed that this was accompanied by an increase in the amount of ET mRNA. Between day 17 of gestation and birth, the amount of mRNA in fetal rat liver increased approx. 6-fold, suggesting the regulation of ET at both pretranslational and post-translational levels during rat liver development.

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides.

Phospholipids and sphingolipids are important precursors of lipid-derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross-talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat-2 fibroblasts. Addition of short-chain C6-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 microM C6-ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time-periods (>/= 16 h) to 50 microM C6-ceramide resulted in apoptosis. The structural analogue dihydro-C6-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-choline and CDP-ethanolamine under the influence of C6-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6-ceramide-treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6-ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase.

Dietary pectin lowers sphingomyelin concentration in VLDL and raises hepatic sphingomyelinase activity in rats.

There is evidence that cholesterol and sphingomyelin metabolism are interrelated, and thus the hypothesis tested was that dietary pectin, because it can alter hepatic cholesterol metabolism, would also alter hepatic sphingomyelin metabolism. For that purpose, 4-wk-old female Wistar rats were fed a diet without or with pectin (20 g/100 g) up to 21 d. In accordance with previous work, pectin consumption caused a significant (P < 0.001) reduction in hepatic (65%), whole plasma (37%), and VLDL (80%) cholesterol levels. Pectin also significantly reduced VLDL sphingomyelin concentrations (57%), but raised the amount of sphingomyelin in the high density lipoproteins (HDL)-2 fractions (58%), so that the level of sphingomyelin in whole plasma remained unaffected. Pectin did not affect the sphingomyelin concentration in the liver. Pectin consumption did not affect the hepatic sphingomyelin synthesizing enzymes, serine palmitoyltransferase, phosphatidylcholine:ceramide phosphocholine transferase, or phosphatidylethanolamine:ceramide phosphoethanolamine transferase. In contrast, dietary pectin activated both lysosomal (28%) and plasma membrane (26%) sphingomyelinase and thus may have enhanced sphingomyelin degradation. An attempt was made to describe the effects of dietary pectin on sphingomyelin metabolism in terms of altered fluxes through liver and plasma, with whole liver and whole plasma concentrations of sphingomyelin remaining unaffected.

Channelling of intermediates in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine in mammalian cells.

Previous studies with electropermeabilized cells have suggested the occurrence of metabolic compartmentation and Ca2+-dependent channeling of intermediates of phosphatidylcholine (PC) biosynthesis in C6 rat glioma cells. With a more accessible permeabilization technique, we investigated whether this is a more general phenomenon also occurring in other cell types and whether channeling is involved in phosphatidylethanolamine (PE) synthesis as well. C6 rat glioma cells, C3H10T12 fibroblasts and rat hepatocytes were permeabilized with Staphylococcus aureus alpha-toxin, and the incorporation of the radiolabelled precursors choline, phosphocholine (P-choline), ethanolamine and phosphoethanolamine (P-EA) into PC and PE were measured both at high and low Ca2+ concentrations. In glioma cells, permeabilization at high Ca2+ concentration did not affect [14C]choline or [14C]P-choline incorporation into PC. However, reduction of free Ca2+ in the medium from 1.8 mM to <1 nM resulted in a dramatic increase in [14C]P-choline incorporation into permeabilized cells, whereas [14C]choline incorporation remained unaffected. Also, in fibroblasts, reduction of extracellular Ca2+ increased [14C]P-choline and [14C]P-EA incorporation into PC and PE respectively. In hepatocytes, a combination of alpha-toxin and low Ca2+ concentration severely impaired [14C]choline incorporation into PC. Therefore, alpha-toxin-permeabilized hepatocytes are not a good model in which to study channeling of intermediates in PC biosynthesis. In conclusion, our results indicate that channeling is involved in PC synthesis in glioma cells and fibroblasts. PE synthesis in fibroblasts is also at least partly dependent on channeling.

Reversible translocation of CTP:phosphocholine cytidylyltransferase from cytosol to membranes in the adult bovine liver around parturition.

The key regulatory enzyme of phosphatidylcholine (PC) synthesis, CTP:phosphocholine cytidylyltransferase (CT), is known to be activated in vitro by translocation from soluble to particulate fractions of the cell. In the present study the periparturient cow was chosen as a model to investigate whether translocation of CT can contribute to the regulation of PC synthesis in vivo. Between parturition and 1.5 weeks post-partum, the cytosolic CT activity in the liver of the adult animal decreased 1.9-fold, and this correlated with a 1.8-fold increase in microsomal CT activity. At that time, microsomal CT activity started to decline again whereas the cytosolic activity rose concomitantly until both activities reached their pre-partum values at 8 weeks post-partum. The activities of soluble and membrane-bound CTP:phosphoethanolamine cytidylyltransferase (ET), the analogous enzyme in the CDP-ethanolamine pathway, did not change significantly throughout this period. Whereas hepatic PC concentrations declined until about 2 weeks post-partum and thereafter gradually returned to pre-partum levels, the PC levels in very-low-density-lipoproteins, started to rise 2 weeks after the partus reaching a maximum of 219% of the original value at 8 weeks post-partum. These results strongly suggest that there is a reversible redistribution of CT between cytosol and membranes in a physiologically relevant animal model, supporting the concept that translocation of CT is occurring in vivo.

CTP:phosphoethanolamine cytidylyltransferase.

CTP:phosphoethanolamine cytidylyltransferase (ET) catalyzes the conversion of phosphoethanolamine into CDP-ethanolamine. Immunogold electron microscopy studies have demonstrated that, in hepatocytes, ET is localized predominantly in areas of the cytoplasm that are rich in rough endoplasmic reticulum (RER). Within these areas the enzyme shows a bimodal distribution between the cisternae of the RER and the cytosolic space. Studies on the substrate specificity of ET have shown that it can utilize both CTP and dCTP as substrates, but not other trinucleotides. In addition, the enzyme shows a very pronounced specificity for phosphoethanolamine. Under most conditions ET contributes significantly to the overall regulation of the CDP-ethanolamine pathway. Reversible binding of the enzyme to the endoplasmic reticulum could potentially play a key-role in metabolic channeling of phosphatidylethanolamine synthesis. ET has been purified from rat liver. Convincing evidence has been provided that ET and CTP:phosphocholine cytidylyltransferase (CT), the analogous enzyme in the CDP-choline pathway, are separate activities that reside on different proteins. The gene coding for yeast ET has been cloned. The deduced amino acid sequence contained a region in the N-terminal half with significant similarities to the conserved catalytic domain of both yeast and rat CT. The human cDNA for ET was also cloned recently. The predicted amino acid sequence of human ET shows a high degree of similarity (36% identity) to that of yeast ET, but the human protein is longer than the yeast protein, especially at the C-terminal region. Interestingly, both yeast and human ET have a large repetitive sequence in their N-terminal and C-terminal half.

Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues.

To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues.

Growth advantage of human leiomyoma cells compared to normal smooth-muscle cells due to enhanced sensitivity toward insulin-like growth factor I.

Human uterine leiomyomas exhibit increased IGF-I binding compared to myometrium, while both tissues show IGF-I gene expression. In this study we have examined the functional importance of these findings by testing the presence of IGF-I in 15 leiomyoma biopsies and in 18 myometrium biopsies and the capacity of smooth-muscle cells cultured from these tissues to react to IGF-I. The mean IGF-I peptide concentration in leiomyomas was 3 times higher than in myometrium. This resulted from increased IGF-I uptake in leiomyomas rather than from increased synthesis, as these tissues contain higher concentrations of type-I IGF receptors, as detected by immunohistochemistry, and equal levels of IGF-I mRNA. Blocking IGF-I transport with cytochalasin-B and with the type-I IGF receptor blocking antibody alpha IR3 in cultured cells induced decreased immunostaining intensity for IGF-I in most myometrium and leiomyoma cultures, indicating that the detected IGF-I is internalized. Depending on the culture conditions, IGF-I administration yielded increased survival or a higher proliferation rate in leiomyoma cultures than in myometrium cultures, indicating the increased importance of exogenous IGF-I for the growth of transformed smooth-muscle cells. We conclude that the increased concentrations of type-I IGF receptors in leiomyoma compared to myometrial smooth-muscle cells are functional with respect to the enhanced internalization of IGF-I and that they provide these tumor cells with a growth advantage compared to their normal counterparts.

Identification of novel phosphorylation sites in the beta-subunit of translation initiation factor eIF-2.

Initiation factor eIF-2 (a trimer of subunits alpha, beta and gamma) attaches the initiator Met-tRNA to the ribosome during the initiation of translation in eukaryotic cells. Both the alpha and beta subunits can be phosphorylated although the sites in the beta-subunit have not previously been fully identified. Here we identify the sites at which eIF-2 beta is phosphorylated in vitro by three well-characterised protein kinases, casein kinase-2 (which phosphorylates serine residues-2 and -67), protein kinase C (serine-13) and cAMP-dependent protein kinase (serine-218). This constitutes an essential prerequisite for studying the phosphorylation of eIF-2 beta in vivo. Indeed, we present evidence that at least one of these sites (serine-67) is phosphorylated in reticulocytes. The major kinase activity against eIF-2 beta in reticulocyte lysates appears in CK-2 and protein phosphatase-2A is the principal enzyme responsible for dephosphorylation of eIF-2 beta phosphorylated by this kinase.