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V Kaever

Publications and source records attributed to V Kaever.

At least 37 records · Page 2Linked to original sources
PubMed1994

Transient expression of prostaglandin endoperoxide synthase-2 during mouse macrophage activation.

We investigated regulation of macrophage prostaglandin production during activation by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). An in vitro model was established using the mouse macrophage-like cell line RAW 264.7. Cells were cultivated in the presence of IFN-gamma and LPS for up to 48 h and changes in the secretion of nitric oxide (NO.) and tumor necrosis factor alpha (TNF-alpha) were observed as activation markers. Under these conditions a prompt and strong increase in PGE2 production was found in the first 8 h followed by nearly constant generation of PGE2 during the next 40 h. In contrast, the activity of prostaglandin endoperoxide synthase (PGHS), measured as PGE2 production of microsomal protein fractions, was also increased, but reached a clear maximum at 24 h. Recently a second form of PGHS was cloned (PGHS-2) and specific antibodies and mRNA probes for both isoforms are available. PGHS-2 enzyme was expressed maximally after 24 h of activation whereas PGHS-1 was not influenced. In the presence of IFN-gamma and LPS, PGHS-2 mRNA expression reached a maximum at 8 h but PGHS-1 mRNA was not induced during the whole time period. These data indicate that changes in PG synthesis following macrophage activation are due to regulation of PGHS-2 expression.

Animals↗
PubMed1994

Inhibition of lymphocyte proliferation by the anti-arthritic drug sinomenine.

The effect on lymphocyte proliferation of sinomenine, a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum was investigated in vitro using mouse spleen cells and human peripheral blood mononuclear cells. It could be demonstrated that sinomenine markedly inhibited [3H]thymidine incorporation in mouse spleen cells activated with concanavalin A (IC50 = 400 microM) or by two-way mixed lymphocyte culture (IC50 = 60 microM) and also in human peripheral blood mononuclear cells activated with phytohemagglutinin, 12-O-tetradecanoylphorbol-13-acetate plus ionomycin, or mixed lymphocyte culture (IC50 ranging from 34 to 129 microM). Time kinetic experiments revealed that sinomenine was effective only when added within the first 48 h after the onset of mixed lymphocyte culture, which lasted for 5 days. Inhibition of lymphocyte proliferation by sinomenine was reversible. Accordingly, the drug showed no direct cytotoxicity in our cellular systems and had no inhibitory effect on the proliferation of the cytokine-independent growth of the human leukaemic T-cell lymphoblast cell line Jurkat. It can be considered that these anti-proliferative effects are part of the anti-inflammatory and anti-arthritic mechanisms of sinomenine obvious in clinical trials.

Animals↗
PubMed1994

Human peritoneal mesothelial cell prostaglandin synthesis: induction of cyclooxygenase mRNA by peritoneal macrophage-derived cytokines.

Increasing evidence suggests that the mesothelial cell contributes to the control of inflammation in both the normal and inflamed peritoneal cavity. The present study examines the regulation of prostaglandin production by human peritoneal mesothelial cells (HPMC) following stimulation with peritoneal macrophage-conditioned medium and the cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). IL-1 beta and TNF-alpha stimulated significant release of prostaglandin above background levels in a time and dose dependent manner. Stimulation of HPMC with IL-1 beta (500 pg/ml) or TNF-alpha (100 pg/ml) for 24 hours resulted in the release of 24.5 +/- 4.3 (N = 11) (z = 3.40, P < 0.001 vs. control) and 19.4 +/- 4.5 (N = 10; z = 3.29, P < 0.001 vs. control) pg 6-keto-PGF1 a/micrograms cellular protein, respectively. Pretreatment of HPMC with dexamethasone (10(-6) to 10(-9) M) inhibited both constitutive and cytokine stimulated prostaglandin synthesis in a dose dependent manner. Both PMø-CM and PMø-S.epiCM stimulated 6-keto-PGF1 alpha and PGE2 synthesis by HPMC in a time and dose dependent manner (PMø-S.epiCM >> PMø-CM). Co-incubation of HPMC with PMø-S.epiCM in the presence of anti-IL-1 beta and/or anti-TNF-alpha antibody, interleukin-1 receptor antagonist or soluble TNF receptor (TNF p75) significantly reduced the capacity of these supernatants to stimulate prostaglandin synthesis. Exposure of HPMC to cytokines or PMø-S.epiCM resulted in the time dependent increase in the levels of both Cox-1 and Cox-2 mRNA as assessed by RT/PCR analysis with the greatest increase being seen for Cox-2. These data demonstrate specific stimulation of eicosanoid metabolism in HPMC by peritoneal macrophage derived cytokines, indicating the possible importance of these mediators in the activation of intraperitoneal prostaglandin synthesis. HPMC prostaglandins might act as important pro/anti-inflammatory mediators contributing to a cytokine network in the peritoneal cavity during CAPD peritonitis.

Base Sequence↗
PubMed1994

Does the reamer type influence the degree of lung dysfunction after femoral nailing following severe trauma? An animal study.

In multiple trauma patients with lung contusion, pulmonary complications have been reported that were attributed to intramedullary stabilization of the femur. The reaming procedure of the medullary canal is thought to play a major role. We investigated whether different types of reamers might exert different amounts of fat mobilization into the vascular system and different degrees of pulmonary dysfunction. Adult female Merino sheep were submitted to hemorrhagic shock (2 h, 50 mm Hg) and a unilateral lung contusion; in addition, a lung lymph fistula was created. Pulmonary capillary permeability, central venous triglyceride levels, 11-dehydro-thromboxane B2 (dh-TXB2) levels, and pulmonary artery pressure were determined. After recovery, animals were randomly assigned to intramedullary femoral nailing using several types of reamers: group A, AO reamer (n = 8); group B, Biomet reamer (n = 7); group H, Howmedica reamer (n = 6); group C, controls, no reaming (n = 4). Intramedullary reaming caused a significant (p < 0.05) increase in pulmonary artery pressure in groups A and B; dh-TXB2 levels increased in all groups. Statistically significant (p < 0.05) pulmonary capillary permeability damage was measured in group A only. Intramedullary femoral nailing can cause transient pulmonary hemodynamic and mediator effects as well as increased pulmonary capillary permeability. In the present study, this effect was evident in group A reamer systems only, which may be due to reamer construction.

Animals↗
PubMed1994

Impairment of macrophage eicosanoid and nitric oxide production by an alkaloid from Sinomenium acutum.

The effects of sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methyl- 9 alpha,13 alpha,14 alpha-morphinan-6-one), a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum, on different macrophage capacities were investigated in vitro using resident mouse peritoneal macrophages and the macrophage-like cell line RAW 264.7. Sinomenine markedly decreased prostaglandin E2 and leukotriene C4 synthesis of macrophages stimulated by zymosan or calcium ionophore and also significantly inhibited the nitric oxide production of RAW 264.7 cells activated by interferon-gamma/lipopolysaccharide. It can be considered that these effects are part of the analgesic, anti-inflammatory, and antirheumatic mechanisms of sinomenine.

Alkaloids↗
PubMed1993

Differentiation-associated expression of prostaglandin G/H synthase in monocytic cells.

Significant progress in the investigation of the regulation of prostanoid formation has recently been made by cloning a second gene coding for prostaglandin G/H synthase (PGHS; EC 1.14.99.1). In this study we examined the expression of the two PGHS isoforms during phorbol ester induced monocytic differentiation of human myeloid leukemia cells (U937). Murine and ovine PGHS-1 probes hybridized to 2.8- and 5.5-kb mRNA species, whereas the murine PGHS-2 probe hybridized to a 5.3-kb species. Western blot analysis using antisera to mouse PGHS-1 and to a synthetic peptide derived from a mouse PGHS-2-specific region revealed a band of 70 kDa for PGHS-1 and a doublet of about 85 kDa for PGHS-2. Unlike PGHS-2, which was not expressed in U937 control cells, both PGHS-1 protein and mRNA were detected in untreated U937 cells. TPA strongly induced PGHS-2 protein and also increased the amount of PGHS-1 protein. Correspondingly, a marked induction of PGHS-2 mRNA was found, but virtually no change in the expression of the PGHS-1 2.8-kb mRNA occurred. The induction of both PGHS isoforms turned out to be dexamethasone-sensitive. The suppression of PGHS-2 induction was more pronounced. These results suggest that both PGHS-1 and to a larger extent PGHS-2 contribute to the upregulation of prostanoid synthesis during monocytic differentiation.

Animals↗
PubMed1993

Effects of cyclosporin and FK-506 on glomerular mesangial cells. Evidence for direct inhibition of thromboxane synthase by low cyclosporin concentrations.

The cellular sources or molecular mechanisms responsible for the derangement of vasoactive prostanoid levels during immunosuppressive cyclosporin (CSA) therapy have not been defined. Using cultured rat glomerular mesangial cells (MC), the cytostatic, cytotoxic and prostanoid synthesis modulating effects of CSA and FK-506 have been measured and compared with the immunosuppressive action of these drugs. Both, CSA and FK-506 inhibited proliferation of MC at similar doses (IC50 approximately 1 microgram.ml-1). Lymphoproliferation was suppressed with IC50s of 50 ng.ml-1 and < 1 ng.ml-1, respectively. In contrast, and unlike FK-506, CSA caused mesangiolysis (IC50 = 4.5 micrograms.ml-1) and concentration dependently inhibited the interleukin-1 beta (IL-1 beta) stimulated mesangial cell release of TXB2 at nanomolar doses (IC50 = 50 ng.ml-1). In kinetic experiments (6-48 h), CSA 1 ng.ml-1 partially and 1 microgram.ml-1 completely abolished the IL-1 beta augmented mesangial secretion TXB2 at all the time points tested. Both, low and high doses of CSA reduced PGE2 release by only 20-40% and then not until at least 24 h of incubation. Measuring enzymatic capacity of membrane fractions of MC to generate TXB2 or PGE2 from added arachidonic acid (10(-5) M), CSA (0.1-1000 ng.ml-1) caused a dose dependent reduction in cyclooxygenase (COX)/thromboxane synthase activity up to 76%, while PGE2 synthesis (COX/prostaglandin synthase) was decreased by 34%. Immunoblots with a specific COX-1 antiserum revealed that COX-1 protein expression of MC was not affected by CSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Heterogeneity in the mobilization of cytoplasmic calcium by human polymorphonuclear leukocytes in response to fMLP, C5a and IL-8/NAP-1.

The visible excitation and emission wave-lengths of the recently developed fluorescent Ca2+ indicator fluo-3 permit analysis of the intracellular Ca2+ concentration, [Ca2+]i, in flow cytometry with a 488-nm argon laser. The role of [Ca2+]i in human polymorphonuclear leukocyte heterogeneity was investigated in response to formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and interleukin 8/neutrophil attractant/activation protein 1 (IL-8/NAP-1) by flow cytometry. The [Ca2+]i changes in different subpopulations within a heterogeneous cell suspension were resolved upon stimulation with fMLP. Using an anti-CD16 phycoerythrin-conjugated antibody and fluo-3 simultaneously, neutrophils affected and nonaffected in Ca2+ mobilization were distinguished in two patients suffering from glycogen storage disease type 1b. In normal neutrophils, a different time course of Ca2+ mobilization of neutrophil subpopulations immediately after stimulation with fMLP was detected. In addition, after stimulation with a low concentration of IL-8/NAP-1 (10(-10) M) two subsets of neutrophils appeared; one of them showed an increase in [Ca2+]i, while the other did not. These results indicate heterogeneity in the neutrophil signal transduction process involved in Ca2+ mobilization. Therefore, flow cytometric analyses can resolve changes in single-cell [Ca2+]i distribution patterns, which is important for the understanding of [Ca2+]i in neutrophil heterogeneous activation processes.

Aniline Compounds↗
PubMed1992

Simultaneous detection of whole blood chemiluminescence in microtitre plates.

The measurement of reactive oxygen species provides a simple method for monitoring the degree of activation of leukocytes in various disorders, and for determining the effects of drugs on this activation. The present report describes the determination of luminol- or lucigenin-amplified chemiluminescence of whole blood in a microtitre plate assay with a 96-well luminometer (HAMAMATSU MTP reader). Using heparinized venous human blood from healthy donors, optimal chemiluminescence intensities were determined at a blood dilution of 1/100 in a total volume of 0.25 ml of Hank's balanced salt solution, containing 0.4 mmol/l luminol as enhancer and either opsonized zymosan (1 milligram) or the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (10(-6) mol/l), as stimuli. The in vitro effects of nordihydroguaiaretic acid, diphenylene iodonium, and diclofenac were tested. After preincubation of the diluted whole blood with these drugs for 15 min, the zymosan-stimulated chemiluminescence was diminished in all cases. The specific NADPH oxidase inhibitor, diphenylene iodonium, was most effective (half maximal inhibition at 1.5 x 10(-8) mol/l), whereas higher concentrations of the antioxidant, nordihydroguaiaretic acid (1.6 x 10(-6) mol/l), or the nonsteroidal antiinflammatory drug, diclofenac (about 10(-5) mol/l), were needed to achieve half maximal inhibition. In addition to its usefulness in the rapid screening of drug effects this assay system seems to be very beneficial for the clinical diagnosis of congenital disorders. Furthermore, it is suited as an effective and simple method for the continuous determination of the phagocyte functional state in patients in pathophysiological situations and during therapy.

Acridines↗
PubMed1992

Cleavage of phosphatidylcholine: an additional mechanism for stimulation of macrophage eicosanoid synthesis?

Cleavage of phosphatidylcholine (PtdChol) as putative mechanism leading to enhanced eicosanoid synthesis was investigated in mouse peritoneal macrophages. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to intact [3H]choline-labelled cells did not enhance radioactivity release above control levels. Membrane-bound PtdChol-specific phospholipase (PL) activity was then measured in a cell-free assay using [3H]choline-labelled membranes as endogenous substrate. Preincubation of macrophages with TPA increased PL activity in a time course resembling the one observed for protein kinase C (PKC) activation. Diacylglycerol (DAG) species derived from PtdChol might therefore serve as additional stimulator of rapid macrophage eicosanoid synthesis via PKC.

Animals↗
PubMed1991

Functional expression of NADPH oxidase components (alpha- and beta-subunits of cytochrome b558 and 45-kDa flavoprotein) by intrinsic human glomerular mesangial cells.

Recently, we have shown that human glomerular mesangial cells (HMCs) release oxygen radicals from the plasma membrane in response to cytokines. Now we have used diphenylene iodonium, a covalent binding inhibitor of activated 45-kDa flavoprotein, in neutrophils radiolabeled with 125I and could identify a 45-kDa protein band in a separated HMC plasma membrane fraction. Low temperature difference spectroscopy showed a peak absorbance at 428 and 558 nm. Direct potentiometry of HMC membranes (-340 to -160 mV) showed the presence of a low potential cytochrome (76 pmol/mg to HMC membrane protein) identified as cytochrome b558. In slot blots, mouse monoclonal antibody (mAb) 7D5, specific for the extracellular domain of the alpha-subunit, showed a positive reaction with HMCs. In Western blots, mAb 449, directed against the cytoplasmic epitope of the alpha-subunit, identified a 23-kDa protein; and mAb 48, raised against the large (beta) subunit of cytochrome b558 of human neutrophils (Verhoeven, A. J., Bolscher, B. G. J. M., Meerhof, L. J., van Zwieten, R., Keijer, J., Weening, R. S., and Roos, D. (1989) Blood 73, 1686-1694), detected a smear between 75 and 100 kDa in denatured HMC membrane protein. These data determined with HMCs, suggest for the first time the expression of three essential components of NADPH:O2- oxidoreductase in mesenchymal cells.

Antibodies, Monoclonal↗
PubMed1991

Cholera toxin-mediated inhibition of signalling in Jurkat cells is followed by, but not due to a loss of T cell receptor complex.

Cholera toxin treatment of the human T cell lymphoma Jurkat resulted in inhibition of signalling via the T cell antigen receptor complex (TcR/CD3-complex). Cholera toxin specifically ADP-ribosylated the alpha-subunit of the stimulatory G-protein of the adenylate cyclase (Gs alpha), no other proteins were modified in the intact cells. ADP-ribosylation of Gs alpha and its subsequent activation led to an increase of the cyclic AMP level and in addition, to a drastic reduction of the cell-surface density of the TcR/CD3-complex. Recently, we demonstrated that the effect of cholera toxin at the receptor level is not due to an increased cAMP level (4). As inhibition of signalling is also not cAMP-mediated (8), we examined whether the modulation of the TcR/CD3-complex could be the reason for the interruption of the signalling cascade. Analyzing the time courses of the multiple cholera toxin effects in Jurkat cells at 37 degrees C, the following sequence was found: ADP-ribosylation of Gs alpha--increase of cyclic AMP level--inhibition of signalling via the TcR/CD3-complex--decrease of cell-surface density of the TcR/CD3-complex. Treatment of Jurkat cells at 20 degrees C with cholera toxin resulted in an increase of cyclic AMP and inhibition of signal transduction, while no decrease of TcR/CD3-complex density could be observed. These data imply that receptor loss from the cell-surface is not causative for the inhibition of signalling. More likely, activation of Gs uncouples signal transduction in Jurkat cells via the TcR, which by a so far unknown mechanism is followed by a loss of the receptor from the cell surface.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

The ratio of macrophage prostaglandin and leukotriene synthesis is determined by the intracellular free calcium level.

The induction of eicosanoid synthesis in various cell types by different physiological stimuli is dependent on an increase in the intracellular calcium level and stimulation of the protein kinase C (PKC). In a model system this can be mimicked by using calcium ionophores and direct PKC activators. In mouse peritoneal macrophages calcium ionophores induced the formation of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4). A synergistic enhancement of both eicosanoids could be achieved by simultaneous addition of the calcium ionophore A23187 together with a suboptimal dose of the direct protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Low concentrations of the ionophore, resulting in only marginally increased intracellular calcium levels, led to a more than additive prostaglandin E2 production in combination with TPA. Higher concentrations of A23187 together with TPA favoured LTC4 synthesis, whereas PGE2 levels at the same time were even diminished. This observed shift from prostaglandin to leukotriene formation was amplified by simultaneous addition of indomethacin. Manganese as inhibitor of the A23187-induced calcium influx decreased PGE2 synthesis. On the other hand, in the presence of manganese LTC4 production was also inhibited at high concentrations of A23187 but elevated in the absence or at low doses of A23187. Our data provide evidence that in macrophages the ratio of cyclooxygenase and lipoxygenase products caused by mediators, acting via the phospholipase C or D/PKC signal transduction pathway, is regulated by the extent of the intracellular calcium increase.

Animals↗
PubMed1990

Altered arachidonic acid metabolism during differentiation of the human monoblastoid cell line U937.

The human cell line U937 was used as a model for differentiation along the mononuclear phagocyte lineage. Following treatment with the phorbol ester TPA, PGE2 and TxB2 secretion was induced 50-100-fold, and both PGF2 alpha and PGI2 levels became detectable in the supernatant of TPA-differentiated U937 cells. The content of the prostaglandin precursor, arachidonic acid, remained unchanged in the cellular phospholipids of undifferentiated and TPA-differentiated U937 cells. Of the enzymes involved in the availability and metabolism of arachidonic acid, phospholipase A2 activity was increased 2-fold in the membranes of TPA-differentiated U937 cells, whereas lysophosphatide acyltransferase activity remained unaltered. Cyclooxygenase activity, however, was enhanced 5-10-fold, which was due to enhanced expression of the enzyme as demonstrated by dot-blot analysis. The data suggest that the capacity to secrete prostaglandins is acquired during differentiation with TPA and results mainly from an increased cyclooxygenase activity. Despite the capacity of TPA-differentiated U937 cells to synthesize prostaglandins, none of the known monocytic stimuli further stimulated prostaglandin secretion in TPA-differentiated U937 cells. Generation of leukotrienes appears to represent a later state in the differentiation along the monocyte-macrophage lineage, since neither LTB4 nor cysteinyl-leukotrienes were detectable in the supernatants of either undifferentiated or TPA-differentiated U937 cells.

Arachidonic Acid↗
PubMed1990

The G protein coupling T cell antigen receptor/CD3-complex and phospholipase C in the human T cell lymphoma Jurkat is not a target for cholera toxin.

Intact Jurkat cells could be stimulated by monoclonal antibodies against the Tcell antigen receptor complex (OKT3 directed against the CD3 complex, BMA031 directed against constant framework epitopes in the alpha/beta heterodimer). The accumulation of inositol phosphates was inhibited by prior incubation of the cells with cholera holotoxin. The inhibitory effect of cholera toxin (CT) was not cAMP mediated because forskolin (a direct activator of adenylate cyclase) did not mimic the inhibitory effect. When measuring phospholipase C (PLC) in a cell-free assay system by using [3H]inositol-labeled membranes, the enzyme could be stimulated by the poorly hydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate (GTP gamma S). Both anti-receptor antibodies augmented the GTP gamma S stimulatory effect, while the antibodies alone had no stimulatory capacity. In membranes from CT-pretreated cells, whereas the antibodies lost their stimulatory effect on PLC as in untreated cells, whereas the antibodies lost their stimulatory capacity in the presence of GTP gamma S. These data imply that CT exerts its inhibitory effect on signaling by acting at the receptor level while the PLC regulating G protein is not a target for CT-mediated alterations. This assumption is supported by the finding that in intact Jurkat cells CT, which ADP ribosylated only the alpha-subunit of the stimulatory G protein of the adenylate cyclase, led to a loss of the T cell antigen receptor complex from the cell surface as demonstrated by a decrease of receptor density using flow cytometry analysis. Receptor loss could not be achieved by forskolin treatment or incubation of the cells with the binding subunit of the toxin alone.

Adenosine Diphosphate Ribose↗
PubMed1990

Human glomerular mesangial cells inactivate leukotriene B4 by reduction into dihydro-leukotriene B4 metabolites.

Due to its potent chemotactic properties leukotriene B4 is an important mediator of inflammatory reactions. Cultured human kidney mesangial cells converted exogenously added leukotriene B4 efficiently into three different more lipophilic metabolites, two of them probably representing dihydro-leukotriene B4 isomers. This represents an alternative metabolic pathway, in contrast to leukotriene B4 omega-oxidation found in human polymorphonuclear leukocytes. Both dihydro-leukotriene B4 isomers had nearly completely lost their ability to induce leukocyte chemotaxis as compared to leukotriene B4.

Cells, Cultured↗