Pattern of cytokines and pharmacomodulation in sepsis induced by cecal ligation and puncture compared with that induced by endotoxin

The production of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 and their pharmacomodulation were evaluated in a model of polymicrobial sepsis induced in mice by cecal ligation and puncture (CLP) and were compared with the effects of endotoxin (lipopolysaccharide [LPS]) treatment. LPS levels rose as early as 1 h after CLP and increased further after 2 and 21 h. TNF-alpha was detectable in serum, spleen, liver, and lungs during the first 4 h, with a peak 2 h after CLP. IL-1 beta was measurable in serum after 24 h, and levels increased significantly in spleen and liver 4 and 8 h after CLP. IL-6 levels increased significantly in serum throughout the first 16 h after CLP. These cytokines were detectable after LPS injection, with kinetics similar to those after CLP but at a significantly higher level. To cast more light on the differences between these two animal models of septic shock, we studied the effects of different reference drugs. Pretreatment with dexamethasone (DEX); ibuprofen (IBU), an inhibitor of cyclooxygenase; and NG-nitro-L-arginine, an inhibitor of nitric oxide synthase, significantly reduced survival, while chlorpromazine (CPZ) and TNF did not affect it. Only the antibiotics and pentoxifylline significantly increased survival in mice with CLP. However, CPZ and DEX protected the mice from LPS mortality. On inhibiting TNF-alpha with DEX, CPZ, or pentoxifylline, survival was reduced, unchanged, and increased, respectively, and on increasing TNF-alpha with IBU and TNF, survival was decreased or unchanged, respectively, suggesting that the modulation of this cytokine does not play a significant role in sepsis induced by CLP, unlike treatment with LPS. The negative effects of IBU and N(G)-nitro-L-arginine suggest a protective role by prostaglandins and nitric oxide in sepsis induced by CLP.     

Vasoactive intestinal polypeptide (VIP) innervation of rat spleen, thymus, and lymph nodes

In the thymus, VIP-positive (+) fibers were found in the capsular/septal system, cortex, and medulla. In the spleen, VIP+ nerves coursed along large arteries and central arterioles, and in the white pulp, venous/trabecular system, and red pulp. Splenic VIP innervation was more robust in Long-Evans hooded rats than in Fischer 344 rats. VIP+ nerves in mesenteric lymph nodes were found in the cortex, and along the cortical vasculature and medullary cords. No VIP innervation was observed in popliteal lymph nodes. Immunocytes also were VIP+, suggesting that both neural and cellular synthesis of VIP contributes to VIP concentration in lymphoid organs. Surgical sympathectomy did not alter splenic or thymic VIP content, respectively, and VIP innervation of these organs was not altered, suggesting an origin for VIP+ nerves other than the sympathetic nervous system.     

Hemangiosarcoma of liver and spleen treated by hepatic artery ligation, intraportal infusion chemotherapy, and splenectomy

Hepatic artery ligation is useful as a palliation of irressectable hepatic tumors, but does not always produce a satisfactory result. Hepatic tumor with high vascularity is expected to respond more favorably. In this context, primary liver cell carcinoma and carcinoid tumor or leiomyosarcoma of the liver have been satistfactorily treated by hepatic artery ligation. A case is presented of hemangiosarcoma of theliver and spleen treated effectively by hepatic artery ligation, splenectomy, and postoperative intraportal infusion of 5-fluorouracil, as indicated by the regression of hepatic tumors on postoperative scanning and arteriograpms.     

Entamoeba histolytica induces host cell death in amebic liver abscess by a non-Fas-dependent, non-tumor necrosis factor alpha-dependent pathway of apoptosis

Amebic liver abscess is characterized by extensive areas of dead hepatocytes that form cavities surrounded by a thin rim of inflammatory cells and few Entamoeba histolytica trophozoites. E. histolytica produces pore-forming proteins and proteinases, but how trophozoites actually kill host cells has been unclear. Here, we report that E. histolytica induces apoptosis in both inflammatory cells and hepatocytes in a severe combined immunodeficient (SCID) mouse model of amebic liver abscess. By studying infection in C57/BL6.lpr and C57/BL6.gld mice, we found that E. histolytica-induced apoptosis does not require the Fas/Fas ligand pathway of apoptosis, and by using mice with a targeted deletion of the tumor necrosis factor receptor I gene, we have shown that E. histolytica-induced apoptosis is not mediated by tumor necrosis factor alpha. Our data indicate that apoptosis plays a prominent role in the host cell death seen in amebic liver abscess in a mouse model of disease and suggest that E. histolytica induces cell death without using two common pathways for apoptosis.     

Immune functions in beluga whales (Delphinapterus leucas): evaluation of mitogen-induced blastic transformation of lymphocytes from peripheral blood, spleen and thymus

A quantitative assay was developed to evaluate mitogen-induced lymphoblastic transformation in beluga whales (Delphinapterus leucas) using peripheral blood mononuclear cells, splenocytes and thymocytes. Optimal concentrations of four different mitogens (Con-A, PHA, LPS and PWM) were determined with the use of standard curves. Addition of human recombinant IL-2 (rhIL-2) after 48 h in culture with the different mitogens suggests that Con-A, PHA and PWM, but not LPS, stimulate T cells in belugas, as they do in other animal species. The addition of 2-mercaptoethanol did not enhance significantly the proliferation of cells stimulated by Con-A, PHA and LPS, while it did with the cells stimulated by PWM and those cultured without mitogen. The proliferative response of cells was suppressed when the culture medium was supplemented by beluga serum instead of fetal calf serum. This assay will be useful to assess the status of the immune functions in different populations of beluga whales as well for further in vitro immunotoxicological experiments.     

2',2'-Difluorodeoxycytidine (gemcitabine) induces apoptosis in myeloma cell lines resistant to steroids and 2-chlorodeoxyadenosine (2-CdA)

The paucity of effective cytotoxic agents for the treatment of steroid resistant multiple myeloma explains the ongoing search for alternative substances for chemotherapy of this disease. In the present study, the purine antagonist 2-chlorodeoxyadenosine (2-CdA, cladribine) and the pyrimidine antagonist 2',2'-difluorodeoxycytidine (gemcitabine) were tested on four myeloma cell lines (i.e., U 266, OPM 2, RPMI 8226, IM 9), one plasma cell leukemia cell line (HS Sultan) and a myeloid control cell line (HL 60), all of which are resistant to 10-6 M dexamethasone. Gemcitabine has been found to be promising in the chemotherapy of other tumors with low proliferative activity, but its effectiveness against myeloma cells has not been analyzed so far. In our tests, gemcitabine induced a significant degree of apoptosis in all cell lines investigated. After incubation for 48 h with 10 microM gemcitabine, the median numbers of apoptotic cells were in the range of 45% in the OPM 2 and 79% in the U 266 cell line. All of the investigated cell lines were responsive to concentrations of 10 microM gemcitabine even after an exposure of only 30 min, three of them (U 266, HS Sultan, IM 9) also responded to a concentration of 10 nM. Higher concentrations and longer exposure times were necessary to suppress the growth of normal hematopoietic bone marrow progenitor cells. In contrast to gemcitabine, standard concentrations of 2-CdA (i.e., 30 and 300 nM) failed to induce a significant degree of apoptosis in the cell lines investigated but inhibited the growth of myeloid progenitor cells. The results suggest that gemcitabine induces apoptosis in myeloma and plasma cell leukemia lines resistant to steroids and 2-CdA. The fact that tumor cell apoptosis was achieved at concentrations clinically achievable and tolerable, which at the same time do not inhibit the growth of normal CFU-GM progenitor cells, favors the initiation of phase I trials with this drug for the treatment of multiple myeloma.     

PYK2 in osteoclasts is an adhesion kinase, localized in the sealing zone, activated by ligation of alpha(v)beta3 integrin, and phosphorylated by src kinase

Osteoclast activation is initiated by adhesion to the bone surface, followed by cytoskeletal rearrangement, the formation of the sealing zone, and a polarized ruffled membrane. This study shows that PYK2/CAKbeta/RAFTK, a cytoplasmic kinase related to the focal adhesion kinase, is highly expressed in rat osteoclasts in vivo. Using murine osteoclast-like cells (OCLs) or their mononuclear precursors (pOCs), generated in a coculture of bone marrow and osteoblastic MB1.8 cells, we show: (a) tyrosine phosphorylation of PYK2 upon ligation of beta3 integrins or adhesion of pOCs to serum, vitronectin, osteopontin, or fibronectin but not to laminin or collagen; (b) coimmunoprecipitation of PYK2 and c-Src from OCLs; (c) PYK2 binding to the SH2 domains of Src; (d) marked reduction in tyrosine phosphorylation and kinase activity of PYK2 in OCLs derived from Src (-/-) mice, which do not form actin rings and do not resorb bone; (e) PYK2 phosphorylation by exogeneous c-Src; (f) translocation of PYK2 to the Triton X-100 insoluble cytoskeletal fraction upon adhesion; (g) localization of PYK2 in podosomes and the ring-like structures in OCLs plated on glass and in the sealing zone in OCLs plated on bone; and (h) activation of PYK2, in the presence of MB1.8 cells, parallels the formation of sealing zones and pit resorption in vitro and is reduced by echistatin or calcitonin and cytochalasin D. Taken together, these findings suggest that Src-dependent tyrosine phosphorylation of PYK2 is involved in the adhesion-induced formation of the sealing zone, required for osteoclastic bone resorption.     

Competitive inhibition of choline phosphotransferase by geranylgeraniol and farnesol inhibits phosphatidylcholine synthesis and induces apoptosis in human lung adenocarcinoma A549 cells

We have previously shown that, among various isoprenoids, farnesol and geranylgeraniol specifically induced actin fiber disorganization, growth inhibition, and apoptosis in human lung adenocarcinoma A549 cells (Miquel, K., Pradines, A., and Favre, G. (1996) Biochem. Biophys. Res. Commun. 225, 869-876). Here we demonstrate that isoprenoid-induced apoptosis was preceded by an arrest in G0/G1 phase. The isoprenoid effects were independent of protein prenylation and of mitogen-activated protein kinase activity. Moreover, geranylgeraniol and farnesol induced a rapid inhibition of phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by choline phosphotransferase and not at the level of CTP:phosphocholine cytidylyltransferase, the key enzyme of the pathway. Inhibition of choline phosphotransferase was confirmed by in vitro assays on microsomal fractions, which clearly showed that the isoprenoids acted by competitive inhibition with the diacylglycerol binding. Exogenous phosphatidylcholine addition prevented all the biological effects of the isoprenoids, including actin fiber disorganization and apoptosis, suggesting that inhibition of phosphatidylcholine biosynthesis might be the primary event of the isoprenoid action. These data demonstrate the molecular mechanism of geranylgeraniol and farnesol effects and suggest that the mevalonate pathway, leading notably to prenylated proteins, might be linked to the control of cell proliferation through the regulation of phosphatidylcholine biosynthesis.     

Tigabine hydrochloride, an inhibitor of gamma-aminobutyric acid (GABA) uptake, induces cortical depolarizations in vitro

The effect of the gamma-aminobutyric acid uptake inhibitor tiagabine hydrochloride was studied on electrical responses in cortical wedges prepared from 20-30 day-old, audiogenic seizure-prone DBA/2 mice. Perfusion of tiagabine (50 microM) for 15 min, evoked large, slow depolarizations with a frequency of 6-8/h which persisted for 4-5 h. The GABA(A) receptor antagonists, bicuculline (10 microM) and picrotoxin (100 microM), inhibited established depolarizations. These depolarizations were also calcium-dependent and blocked by tetrodotoxin. The non-opioid antitussive, dextromethorphan, which has been shown to inhibit glutamate release, irreversibly blocked the depolarizations. Conversely, 4-aminopyridine (50 microM), a potassium channel antagonist, markedly potentiated the responses. The NMDA receptor antagonist, 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, had no effect on the depolarizations at concentrations up to 100 microM but the AMPA/kainate receptor antagonist, 6,7-dinitroquinoxaline-2.3-dione at high concentrations (100 and 200 microM), reversibly decreased the frequency without affecting the amplitude. It is concluded that the tiagabine-induced depolarizations in this in vitro preparation were initiated through GABA(A) receptors leading, possibly, to a release of excitatory amino acids.     

The bisphosphonate incadronate (YM175) causes apoptosis of human myeloma cells in vitro by inhibiting the mevalonate pathway

It has recently been suggested that bisphosphonates may have direct antitumor effects in vivo, in addition to their therapeutic antiresorptive properties. Bisphosphonates can inhibit proliferation and cause apoptosis in human myeloma cells in vitro. In macrophages, bisphosphonate-induced apoptosis was recently found to be a result of inhibition of the mevalonate (MVA) pathway. The aim of this study was to determine whether bisphosphonates also affect human myeloma cells in vitro by inhibiting the MVA pathway. Incadronate and mevastatin (a known inhibitor of the MVA pathway) caused apoptosis in JJN-3 myeloma cells and inhibited cell proliferation. Geranylgeraniol and farnesol prevented incadronate-induced apoptosis and had a partial effect on cell cycle arrest. MVA and geranylgeraniol prevented mevastatin-induced apoptosis and inhibition of proliferation and completely prevented the effect of mevastatin on the cell cycle. These observations demonstrate that incadronate-induced apoptosis in human myeloma cells in vitro is the result of inhibition of the MVA pathway.     

Cytochrome c induces caspase-dependent apoptosis in intact hematopoietic cells and overrides apoptosis suppression mediated by bcl-2, growth factor signaling, MAP-kinase-kinase, and malignant change

It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)-dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3-dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control.     

5-Azacytidine (5Az) induces apoptosis in PC12 cells: a model for 5Az-induced apoptosis in developing neuronal cells

Our previous in vivo and in vitro studies showed that 5-azacytidine (5Az), a cytidine analog, induced apoptosis in developing neuronal cells in mice. To develop a system in which the precise molecular mechanism of 5Az-induced apoptosis in developing neuronal cells could be elucidated, we carried out the present study with PC12 cells. These cells are derived from a rat pheochromocytoma and extrude neurites in response to nerve growth factor (NGF). Light microscopy showed dose-dependent pyknotic and karyorrhectic changes in undifferentiated PC12 cells. Although they were less sensitive to 5Az, NGF-treated differentiated cells showed the same changes. Analysis by the TUNEL method (an in situ method for the detection of apoptosis) showed positive signals in the pyknotic and fragmented nuclei of these cells. Transmission electron microscopy revealed margination, segmentation, and condensation of nuclear chromatin, cell body shrinkage, and cytoplasmic vacuolization. Scanning electron microscopy demonstrated bleb formation on the cell surface. These pathomorphological changes are typical of apoptosis. 5Az seemed to affect cells that were in the proliferative stage; when the cells were terminally differentiated, their sensitivity to 5Az appeared to decline. PC12 cells could be used as a pathomorphological and biochemical model for studies of 5Az-induced neuronal cell apoptosis at the molecular and genetic level.     

7-Hydroxystaurosporine (UCN-01) induces apoptosis in human colon carcinoma and leukemia cells independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

CD95 (Fas/APO-1) induces ceramide formation and apoptosis in the absence of a functional acid sphingomyelinase

CD95 is a potent inducer of apoptosis. It activates the caspase cascade, but also induces ceramide (Cer) production, reportedly involving acid sphingomyelinase (aSMase) activity. A role for Cer as a second messenger for apoptosis induction was proposed, based on the finding that synthetic Cer analogues can induce cell death. We have tested whether aSMase is required for 1) apoptosis induction and 2) Cer production by CD95. For this purpose, we have used cultured Niemann-Pick disease (NPD) lymphoid cells with a defined mutation (R600H) in the aSMase protein. Despite their inherited deficiency of aSMase, we found that these cells readily undergo apoptosis upon CD95 stimulation. After retrovirus-mediated gene transfer of the aSMase cDNA, the transduced (i.e. "corrected") NPD cells showed neither increased levels of apoptosis nor altered kinetics of caspase-8 and caspase-3 activation and apoptosis induction as compared with empty vector-transduced cells. The slow sustained elevation of Cer levels in response to CD95, which we have previously documented for Jurkat T cells (Tepper, A. D., Boesen-de Cock, J. G. R., de Vries, E., Borst, J., and van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 24308-24312), was similarly found in NPD cells. Moreover, the kinetics of Cer formation remained unaffected after aSMase transduction. These results indicate that this Cer does not result from aSMase activity. We conclude that aSMase is not required for and does not facilitate CD95-mediated apoptosis and that it is not responsible for the late Cer response.     

Urinary DNA as an indicator of nephrotoxicity caused by endotoxin and gentamicin in mice

Extracellular DNA is a non-specific marker of cell death. Urinary DNA, as an indicator of nephrotoxicity, was investigated in endotoxin/gentamicin-injected mice. In mice injected both with endotoxin (15 mg/kg) and gentamicin (80 mg/kg), urinary DNA concentration was markedly increased for several days; in contrast, there was at most a slight and transient excretion of DNA in mice receiving gentamicin or endotoxin alone. Plasma DNA concentrations increased for 24-48 h in endotoxin-injected mice, then decreased rapidly. Mice injected with gentamicin and endotoxin showed widespread and severe kidney lesions with tubular cell necrosis and intraluminal casts while mice receiving gentamicin or endotoxin alone showed at most few and mild lesions. In mice receiving lower doses of endotoxin (5-10 mg/kg) and 80 mg/kg gentamicin, urinary DNA peaked at 72-96 h, at a time when plasma DNA had returned to normal concentrations. Maximal urinary DNA concentrations depended upon endotoxin dose. In conclusion, urinary DNA is a marker of definite cell death occurring in the urinary tract and could represent a new indicator of nephrotoxicity in clinical and experimental situations.     

Rho-regulated signals induce apoptosis in vitro and in vivo by a p53-independent, but Bcl2 dependent pathway

Rho proteins are a branch of GTPases that belongs to the Ras superfamily which are critical elements of signal transduction pathways leading to a variety of cellular responses. This family of small GTPases has been involved in diverse biological functions such as cytoskeleton organization, cell growth and transformation, cell motility, migration, metastasis, and responses to stress. We report that several human Rho proteins including Rho A, Rho C and Rac 1, are capable of inducing apoptosis in different cell systems like murine NIH3T3 fibroblasts and the human erythroleukemia K562 cell line. Since K562 cells are devoid of p53, apoptosis induced by Rho in this system is independent of p53. Rho-dependent apoptosis is mediated by the generation of ceramides, and it is drastically inhibited by ectopic expression of Bcl2, both under in vitro and in vivo conditions. Furthermore, the human oncogenes vav and ost that have been shown to function as guanine exchange factors for Rho proteins, were also able to induce apoptosis under similar conditions. Finally, we also report that the levels of endogenous Rho proteins are increased when U937 myeloid leukemia cells are exposed to apoptosis-inducing conditions such as TNF alpha treatment. Furthermore, TNF alpha-induced apoptosis in these cells is inhibited by expression of a dominant negative mutant of Rac 1 but it is not affected by a similar mutant of Rho A. These results suggest that Rho proteins play an important role in the physiological regulation of the apoptotic response to stress-inducing agents.     

Inhibition of (Na/K)-ATPase by NFE induces an increase in mechanical activity of perfused guinea-pig heart

The effect of 1-(5-nitro-2-furyl)-2-(phenylsulfonyl)-2-(furylcarbonyl)- ethylene (NFE) on stimulation of (Na/K)-ATPase by sodium and potassium ions was tested in isolated, partially purified sarcolemmal preparation from guinea-pig hearts. NFE inhibited competitively the stimulation of the enzyme by increasing concentrations of potassium. This inhibition was characterized by a significant (p < 0.001) increase of the K0.5 value, a considerable decrease of the Hill's cooperativity constant n as well as by an insignificant diminution of the Vmax value. Contrary to the effect on stimulation by potassium, NFE inhibited non-competitively the stimulation of the ATPase by sodium ions with a significant (p < 0.001) depression of Vmax but without any considerable effect on the K0.5 and n values. These results indicated that NFE may interact with the molecule of (Na/K)-ATPase in a locus close to or identical with the potassium binding site of the enzyme, i.e., in a similar mode as it was well documented for ouabain. This possibility was strongly supported by the finding that NFE administered at the concentration of 0.1 mumol/l in the perfusion medium increased significantly (p < 0.01) the mechanical activity of isolated perfused guinea-pig heart (Langendorff preparation). Nevertheless, it also caused some adverse effects such as a slight increase in coronary flow resistance and in heart rate as well as in the left ventricular end-diastolic pressure.(ABSTRACT TRUNCATED AT 250 WORDS)