Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators' interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-alpha. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL.

A systematic study was undertaken to characterize the role of APO 2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (APO2L/TRAIL) and Fas ligand (FasL) together with the expression of several anti- or proapoptotic proteins in the down-regulation of normal human T cell responses. We have observed for the first time that the higher sensitivity of normal human T cell blasts to apoptosis and activation-induced cell death (AICD) as compared with naive T cells correlates with the increased expression of Bcl-x short (Bcl-x(S)) and Bim. T cell blasts die in the absence of interleukin 2 (IL-2) with no additional effect of death receptor ligation. In the presence of IL-2, recombinant APO2L/TRAIL or cytotoxic anti-Fas monoclonal antibodies induce rather inhibition of IL-2-dependent growth and not cell death on normal human T cell blasts. This observation is of physiological relevance, as supernatants from T cell blasts, pulse-stimulated with phytohemagglutinin (PHA) or through CD3 or CD59 ligation and containing bioactive APO2L/TRAIL and/or FasL expressed on microvesicles or direct CD3 or CD59 ligation, had the same effect. Cell death was only observed in the presence of cycloheximide or after a pulse through CD3 or CD59, correlating with a net reduction in cellular Fas-associated death domain-like IL-1 beta-converting enzyme-inhibitory protein long (c-FLIPL) and c-FLIPS expression. We also show that death receptor and free radical generation contribute, at least partially, to AICD induced by PHA and also to the inhibition of IL-2-dependent cell growth by CD3 or CD59 ligation. Finally, we have also shown that T cell blasts surviving PHA-induced AICD are memory CD44(high) cells with increased c-FLIPS and Bcl-x(L) expression.

The time-course of changes in the levels of albumin, alpha-fetoprotein (AFP), alpha(1)-protease inhibitor (alpha(1)-antitrypsin), alpha(1)-acid glycoprotein, fetuin, haptoglobin, transferrin, IgG and the major acute-phase protein (Pig-MAP) in the blood sera of pigs during the first days and weeks of life was investigated by quantitative radial immunodiffusion. The serum of newborn pigs before suckling was characterised by a very low concentration of total proteins (approximately 25 mg mL(-1)), low levels of albumin and transferrin and the lack of immunoglobulins. In contrast, alpha(1)-acid glycoprotein and fetuin are present at high levels (approximately 12 and 5 mg mL(-1) respectively). The results of the present study show that the piglets undergo a very rapid metabolic maturation with regard to serum proteins, evolving from a characteristic 'fetal' pattern to an 'adult' one. We have paid special attention to the evolution of haptoglobin and Pig-MAP, which are two important acute-phase proteins in pigs. The evolution of serum levels of these proteins suggests that piglets must overcome a moderate acute-phase situation during the first week of life.

We have undertaken a study to characterize the lipolytic pathway responsible for the generation of free fatty acids (FFA) during Fas/CD95-induced apoptosis in Jurkat cells. It was initially shown that the cellular lipid fraction that suffered the major quantitative decrease during Fas-induced apoptosis was that of phosphatidylcholine (PC). In addition, the secretion of palmitic acid-derived FFA was largely prevented by D609, an inhibitor of PC-specific phospholipase C (PC-PLC) and also by the diacylglycerol lipase (DAGL) inhibitor RHC-80267, suggesting that the secretion of these FFA during Fas-induced apoptosis is mediated by the generation of DAG by a PC-PLC activity and, sequentially, by a 1-DAGL activity which generates the FFA from its sn-1 position. The endocannabinoid 2-arachidonoyl glycerol (2-AG) should be generated as a sub-product of this pathway, but it did not accumulate inside the cells nor was secreted into the supernatant. Interestingly, the complete inhibition of free AA secretion during Fas-induced apoptosis was only achieved by using the AA trifluoromethylketone, which not only inhibits all types of phospholipase-A(2) (PLA(2)) activities, but also the described lytic activities on 2-AG. Using a combination of RHC-80267 and the iPLA(2)-specific inhibitor bromoenol lactone, it was shown that the DAGL pathway also cooperates with iPLA(2) in the generation of free arachidonate.

Tumor cells have developed multiple mechanisms to evade control by the immune system. Tumoral cells expressing Fas ligand (FasL) have been proposed to "counterattack" against activated antitumoral effector immune cells, although some authors have indicated that FasL is not expressed on the surface of the same tumors, such in the case of melanoma cells. However, other factors could be implicated, such as the balance of soluble versus membrane-bound forms or the secretion of death ligands on the surface of microvesicles, as described previously by our group in human T cells. In the present study, we analyzed the expression and secretion of FasL and APO2 ligand (APO2L)/TRAIL in the human melanoma cell line MelJuSo. We have observed the expression of preformed FasL and APO2L/TRAIL in these cells, their secretion associated with microvesicles upon melanoma activation with PHA or with alpha-melanocyte stimulating hormone (alpha-MSH), and the toxicity of these microvesicles against normal human T cell blasts. We have also observed that the mechanism of secretion of FasL and APO2L/TRAIL from melanoma cells is depending both on microtubules and actin filaments. From these data, it can be concluded that the MelJuSo melanoma cell line has the possibility to "counterattack" against activated immune effector cells. However, the in vivo outcome seems more complex since it has been also described that FasL expressed in tumors has a proinflammatory effect.

ABSTRACT We have isolated from calf serum a protein with an apparent M r of 120,000. The protein was detected by using antibodies against major acute-phase protein in pigs with acute inflammation. The amino acid sequence of an internal fragment revealed that this protein is the bovine counterpart of ITIH4, the heavy chain 4 of the inter-alpha-trypsin inhibitor family. The response of this protein in the sera was determined for animals during experimental bacterial and viral infections. In the bacterial model, animals were inoculated with a mixture of Actinomyces pyogenes , Fusobacterium necrophorum , and Peptostreptococcus indolicus to induce an acute-phase reaction. All animals developed moderate to severe clinical mastitis and exhibited remarkable increases in ITIH4 concentration in serum (from 3 to 12 times the initial values, peaking at 48 to 72 h after infection) that correlated with the severity of the disease. Animals with experimental infections with bovine respiratory syncytial virus (BRSV) also showed increases in ITIH4 concentration (from two- to fivefold), which peaked at around 7 to 8 days after inoculation. Generally, no response was seen after a second infection of the same animals with the virus. Because of the significant induction of the protein in the animals in the mastitis and BRSV infection models, we can conclude that ITIH4 is a new positive acute-phase protein in cattle.

Pig apolipoprotein (apo) A-IV cDNA was cloned, characterized and compared to the human ortholog. Mature porcine apo A-IV consists of 362 amino acids and displays a 75.6% sequence identity with human protein. Pig apo A-IV is the smallest reported mammalian apo A-IV because it lacks the repeated motifs of glutamine and glutamic acid at the carboxyl terminus. A phylogenic tree of apo A-IV mammalian proteins reveals that porcine apo A-IV is more closely related to humans and primates than to rodents. This protein is highly hydrophobic and is mainly associated with lipoproteins.

Apoptosis induced by cells from the immune system is frequently associated with an increase in the ceramide content of target cells, due to the activation of sphingomyelinases (SMase). Some studies have also reported the release of saturated and monounsaturated free fatty acids (FFA) from apoptotic cells. However, the possible relationship between these lipid biochemistry events has not been characterized. We have analysed for the first time the release of FFA triggered by tumor necrosis factor-alpha (TNF-alpha), Fas/CD95 or the perforin/granzyme system of cytotoxic T lymphocytes (CTL) and their relationship to intracellular ceramide generation. TNF-alpha- and Fas-induced apoptosis are associated with both intracellular ceramide generation from sphingomyelin (SM) and release of palmitic-derived FFA, with similar kinetics. Intracellular SMase activation and FFA release from target cells during Fas-induced apoptosis are much more rapid and efficient if Fas-based cytotoxicity is exerted by alloantigenic CTL. In the case of perforin/granzyme-based cytotoxicity exerted by CTL, intracellular ceramide generation and FFA release from target cells seem to depend on the type of lysis induction used. Importantly, the correlation between intracellular SMase activation and the release of palmitic acid-derived FFA from target cells has been observed in all types of cytotoxicity assayed. In addition, exogenous natural ceramide induces the rapid release of the same FFA, well before any apoptotic sign is detected, and FFA release during Fas-induced apoptosis is inhibited in SM-depleted cells by chronic fumonisin-B(1) treatment. These results demonstrate a novel connection between the release of palmitic acid-derived FFA and intracellular ceramide accumulation during apoptosis induction.

Fas/CD95 is a type-I membrane glycoprotein, which induces apoptotic cell death when ligated by its physiological ligand. We generated previously hyperproliferative sublines derived from the human Tcell leukemia Jurkat, Jurkat-ws and Jurkat-hp, which lost Fas/CD95 surface expression. We have now observed that the total amount of Fas protein is similar in the sublines and in the parental cells, indicating that in the sublines Fas remains in an intracellular compartment. We have found that the protein is directed toward lysosomes in the sublines, where it is degraded. This defect in the secretory pathway correlates with loss of polyunsaturated fatty acids from cellular lipids, and with the lack of expression of endophilin-I and CtBP/BARS, enzymes that regulate vesicle fission by catalyzing the acylation of arachidonate into lysophosphatidic acid. In addition, great multillamer bodies, which contained acid phosphatase activity, absent in the parental Jurkat cells, were observed by transmission electron microscopy in the sublines.

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments ∼500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments approximately 500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.

Jurkat cells and the derived TCR / CD3-defective subline, J.RT3.T3.5 undergo activation induced cell death (AICD) when stimulated with phytohemagglutinin (PHA). Since J.RT3.T3.5 cells do not express antigen receptor, we searched for the molecules that could be ligated by PHA and induce AICD in this cell line. We show here that the glycosylphosphatidylinositol linked CD59 molecule is expressed at the surface of Jurkat and J.RT3.T3.5 cells, and when cross-linked by specific antibodies can induce cell death. The toxicity of supernatants from PHA-stimulated Jurkat or J.RT3.T3.5 cells was prevented by a combination of the blocking anti-Fas mAb SM1 / 23 and anti-APO2L / TRAIL mAb 5C2. However, toxicity of supernatants from anti-CD59 stimulated cells was specifically prevented by the anti-APO2L blocking antibody. Anti-CD59 cross-linking induced AICD also in normal human T cell blasts, which secreted toxic molecules into the supernatant. The toxicity of these supernatants on Jurkat cells was fully prevented by the anti-APO2L blocking antibody, showing that CD59 crosslinking induces the preferential release of APO2L also in normal T cells. The possible physiological and / or pathological consequences of this observation are discussed.

Doxorubicin induces caspase-3 activation and apoptosis in Jurkat cells but inhibition of this enzyme did not prevent cell death, suggesting that another caspase(s) is critically implicated. Western blot analysis of cell extracts indicated that caspases 2, 3, 4, 6, 7, 8, 9, and 10 were activated by doxorubicin. Cotreatment of cells with the caspase inhibitors Ac-DEVD-cho, Z-VDVAD-fmk, Z-IETD-fmk, and Z-LEHD-fmk alone or in combination, or overexpression of CrmA, prevented many morphological features of apoptosis but not loss of mitochondrial membrane potential (ΔΨm), phospatidilserine exposure, and cell death. Western blot analysis of cells treated with doxorubicin in the presence of inhibitors allowed elucidation of the sequential order of caspase activation. Z-IETD-fmk or Z-LEHD-fmk, which inhibit caspase-9 activity, blocked the activation of all caspases studied, lamin B degradation, and the development of apoptotic morphology, but not cell death. All morphological and biochemical features of apoptosis, as well as cell death, were prevented by cotreatment of cells with the general caspase inhibitor Z-VAD-fmk or by overexpression of Bcl-2. Doxorubicin cytotoxicity was also blocked by the protein synthesis inhibitor cycloheximide. Delayed addition of Z-VAD-fmk after doxorubicin treatment, but prior to the appearance of cells displaying a low ΔΨm, prevented cell death. These results, taken together, suggest that the key mediator of doxorubicin-induced apoptosis in Jurkat cells may be an inducible, Z-VAD-sensitive caspase (caspase-X), which would cause ΔΨm loss, release of apoptogenic factors from mitochondria, and cell death.

The objective of this study was to investigate, using a pulse-chase technique, the different incorporation of (1-(14)C) n -9, n -6 and n 3 fatty acids into hepatoma lipids and their secretion to the culture medium. Docosahexaenoic acid (DHA) accumulated preferentially into the triacylglycerol while arachidonic acid (AA) did into the phospholipid fraction. DHA was poorly secreted to the culture medium whereas AA was secreted to a large extent. The fatty acids were initially esterified mainly into phosphatidylcholine and phosphatidylethanolamine. During the 24 h chase, a general shift from phosphatidylcholine to phosphatidylethanolamine was observed. Linoleic acid was esterified in cardiolipin to a much greater extent than any other fatty acid and it was not converted to more polyunsaturated fatty acids. The supplementation of the culture medium with polyunsaturated fatty acids had no inhibitory effect on the growth of the hepatoma cells, in marked contrast to observations made in other tumoral cells. The reasons for the resistance of the hepatoma cells to polyunsaturated fatty acid toxicity, including the possible antioxidant effect of linoleic acid accumulation in cardiolipin, are also discussed.

The acute‐phase expression of pig MAP (major acute‐phase protein)/ITIH4 (inter‐α‐trypsin inhibitor heavy chain 4) and haptoglobin were analysed in primary cultures of isolated pig hepatocytes in response to recombinant human (rh) cytokines: tumor necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1), interleukin‐6 (IL‐6), as well as to bacterial lipopolysaccharide (LPS). Analysis of pig MAP/ITIH4 and haptoglobin mRNAs was carried out by RT‐PCR amplification. Secreted proteins from the cytokine‐treated hepatocytes were quantified by immunochemical techniques. Time‐course and dose–response experiments show that pig MAP/ITIH4 and haptoglobin belong to the type II acute‐phase proteins, as they are specifically induced by rhIL‐6 and not by rhTNF‐α or rhIL‐1. Stimulation of cultured pig hepatocytes with rhIL‐6 for 48 h at doses of 1000 U·mL −1 showed a fourfold to fivefold increase in pig MAP/ITIH4 concentration in the medium, while the concentration of haptoglobin only increased twofold. A similar increase in the concentration of pig MAP/ITIH4 was also observed in media of LPS‐treated hepatocytes with the simultaneous generation of IL‐6 by the Kupfer cells present in the cultures. Albumin secretion decreased after stimulation with doses of 100 or 1000 U·mL −1 rhTNF‐α, rhIL‐1 or rhIL‐6. Therefore, it can be concluded that pig MAP/ITIH4 behaves as a major acute‐phase protein produced by porcine hepatocytes under the effect of inflammatory cytokines.

A prominent tyrosine phosphorylated protein of 85 kDa (p85) was detected in highly proliferative sublines derived from the Jurkat T cell leukemia. We undertook a study to characterize the identity of this protein and its possible role in the hyperproliferative phenotypes observed. Using immunoblot and immunoprecipitation techniques, this protein was characterized as the p85 regulatory subunit of phosphatidylinositol 3-kinase. Cell proliferation and p85 tyrosine phosphorylation was not affected by tyrphostin AG-490, an inhibitor of Jak kinases, wortmannin or LY294002, inhibitors of the activity of the catalytic phosphatidylinositol 3-kinase subunit. Herbimycin-A and PPI, inhibitors of src-like protein tyrosine kinases, and genistein, a general tyrosine kinase inhibitor, inhibited p85 tyrosine phosphorylation and induced cell death in the sublines. PD98059, an inhibitor of Mek, inhibited cell growth of the sublines, but not that of the parental cells. It was concluded that tyrosine phosphorylation of p85 is associated with highly proliferative tumoral phenotypes, at least in T cell leukemias, independent of the phosphatidylinositol 3-kinase activity of the catalytic subunit.

The serum concentration of the inter-alpha trypsin inhibitor heavy chain 4 protein (ITIH4) increases (from 1.4-3 times) in male patients suffering of different acute-phase processes (myocardial infarction, unstable angina or programmed surgery). The concentration of C-reactive protein (CRP) in these samples ranged from 15 mu g/ml to 133 mu g/ml, Using the hepatocarcinoma HepG2 cell line we have observed upregulation of ITIH4 mRNA expression upon dose-response treatments with interleukin-6 (IL-6). This effect correlates with the increase of radiolabeled ITIH4 in the cellular media of S-35-labeled HepG2 cells treated with the cytokine. A similar effect was observed for haptoglobin mRNA, used as a control for acute-phase protein expression. IL-I beta, although upregulating the expression of alpha(1)-acid glycoprotein in these cells, did not induce any effect in the expression of ITIH4. No changes were observed after TNF-alpha treatments. The results presented here indicate that ITIH4 is a type II acute-phase protein in humans, (C) 1999 Academia Press.

Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100-200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (</=1 h) after PHA stimulation, well before the cell enters apoptosis. FasL- and APO2L-containing vesicles are also present in supernatants from PHA-activated fresh human PBMC. These observations provide the basis for a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation.

Alpha-fetoprotein (AFP) is a major globulin of embryonic plasma and a physiological carrier of unesterified fatty acids. In the present work, we have characterized the interaction of AFP and albumin, a major serum protein of adult mammals which presents numerous biochemical analogies with AFP, with the plasma membrane of the rat Morris 7777 hepatoma cells. Time course analysis of the uptake of AFP and albumin by these cells showed a saturable profile at 4 degrees C and 37 degrees C. Saturable binding of 125I-AFP or 125I-albumin were observed when the concentration of these proteins increased (ranging from 0.3 to 4.5 microM). The Hill and Scatchard analysis revealed the existence of binding sites in the surface of hepatoma cells, with a k'd = 2.2 x 10(-6) M (2.9 x 10(6) sites/cell) in the case of AFP and a k'd = 4.5 x 10(-6) M (3.9 x 10(6) sites/cell) in the case of albumin. 125I-AFP and 125I-albumin bound to the cells were completely displaced in the presence of a 200-fold excess of unlabeled AFP or albumin, respectively, suggesting that these interactions were specific. We have observed crossed competition between AFP and albumin for their respective binding sites; no such crossed competition was observed when an excess of unlabeled transferrin was added. Pulse-chase experiments showed that about 50% of the AFP and 75% of the albumin taken up by the cells were released undegraded into the medium after 1 h. Cytochemical studies performed with covalent conjugates of AFP, albumin and transferrin with horseradish peroxidase have shown that AFP and albumin entered the cells via a vesicular system. This intracellular pathway is different from that of transferrin, a plasma protein whose internalization mediated by specific receptors via coated pits has been reported in other cells. The results presented here suggest that AFP and albumin interact with sites in the membrane of hepatoma cells, probably physically related, and then they are transported inside the cells by a mechanism different from that described for transferrin.

Freshly isolated fetal hepatocytes transformed 4.3, 8.5 and 19.2 pmol/min/10(6) cells of stearic, linoleic and alpha-linolenic acids, respectively, complexed to albumin or alpha-fetoprotein (AFP), to more unsaturated derivatives. Thus, fetal hepatocytes displayed high elongase and delta9, delta6, delta5-desaturase activities, as well as an ability to synthesize hexaene derivatives. Desaturase activities decreased when the time of culture of fetal hepatocytes (previous to incubation with the substrate) was prolonged, being practically undetectable after 24 h of culture. However, the rate of fatty acid uptake remained nearly constant. When AFP was used as the carrier the amount of hexaene fatty acid derivatives of alpha-linolenic acid recovered in cells was reduced up to 50% by albumin. This effect was associated with an increase of radioactivity found in the culture medium of hepatocytes incubated with AFP compared to albumin. Both observations taken together could be explained by an efflux of hexaene derivatives from cells caused by AFP.