Reversal of human neutrophil survival by leukotriene B(4) receptor blockade and 5-lipoxygenase and 5-lipoxygenase activating protein inhibitors

Persistent neutrophilia is a feature of chronic obstructive pulmonary disease (COPD). Leukotriene synthesis inhibitors and cysteinyl leukotriene receptor antagonists have shown efficacy in the treatment of asthma. Antagonism of leukotriene (LT)B(4) receptors is being considered as a mode of treating COPD. We examined the capacity for inhibition of leukotriene synthesis and LTB(4) receptor antagonism to reduce survival of neutrophils from patients with COPD and those from normal subjects. The basal apoptosis level of these cells was 55.4 +/- 2.4% (mean +/- SEM) of total cells. Separate exposure to lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), dexamethasone (DEX), and LTB(4) increased neutrophil survival (p < 0. 001). The LTB(4) receptor antagonist SB201146 abolished LPS-induced survival in a concentration-dependent manner (10 pmol to 0.1 microM), with an IC(50) of 1.9 nM. Combined exposure to SB201146 and to the cysteinyl leukotriene antagonist SKF104353 did not have a greater effect on survival than did exposure to SB201146 alone. Inhibition of 5-lipoxygenase (5-LO) with BWA4C and of 5-LO-activating protein (FLAP) with MK886 abolished GM-CSF- and DEX-induced neutrophil survival. BWA4C and MK886 abolished GM-CSF- induced neotrophil survival in a concentration-dependent manner (1 nM to 10 microM), with IC(50) values of 182.0 nM and 63.1 nM, respectively. These findings demonstrate reversal of LPS-, GM-CSF-, and DEX-induced neutrophil survival by LTB(4) receptor antagonism and inhibitors of 5-LO and FLAP. They also suggest a potential additional antiinflammatory mode of action of these compounds through reduction of cell survival.     

Expression of 5-lipoxygenase in human colorectal cancer

INTRODUCTION Evidence from epidemiological and animal studies suggests that a high-fat consumption is associated with an increased incidence and growth of tumors at several specific organ sites, including breast, pancreas, colon, and prostate[1]. In this …     

Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.     

Cytoplasmic immunoglobulins in chronic lymphocytic leukemia B cells

It is generally assumed that chronic lymphocytic leukemia of B cell origin (B-CLL) is characterized by the presence of surface membrane immunoglobulins (SmIg) and by the absence of cytoplasmic immunoglobulins (CyIg). In a variable number of cases SmIg are not detectable because of their low density on the cellular surface. Because a constant presence of CyIg in 20 subjects suffering from B-CLL has been reported recently, we reexamined 15 SmIg-negative and 10 SmIg- positive B-CLL patients by SmIg and CyIg determinations. We used a direct immunofluorescence method on peripheral blood mononuclear cells for the detection of SmIg and, after fixation, for CyIg. CyIg were detectable in 24 out of 25 cases, with a fluorescence intensity ranging from weak to moderate. The existence of frequent negative results for CyIg determination in B-CLL reported in the literature probably depends on the low sensitivity of the method used. We conclude that CyIg determination is useful in phenotyping every B-CLL patient, especially SmIg-negative ones.     

Antigen receptor nonresponsiveness in chronic lymphocytic leukemia B cells

B chronic lymphocytic leukemia (B-CLL) are clonal populations of mIgM+ or mIgM+/mIgD+ CD5+ B cells that appear to be arrested in the follicular mantle-zone B-cell stage. Functional analyses have shown two groups of B-CLL that can be distinguished based on their capacity to proliferate in response to B-cell antigen receptor complex (BCR) cross- linking. To investigate the molecular basis for this phenomenon, we have analyzed both architecture and functional properties of BCR complexes on these two groups of B-CLL. Both groups were found to express structurally similar BCR. However, protein tyrosine kinase (PTK) activity associated with and specific for BCR constituents was strongly diminished in nonresponsive B-CLL. Moreover, the PTK-dependent assembly of Shc/Grb2 complexes, which may couple the BCR to p21ras, was absent in these B-CLL. Finally, of all PTKs tested, the expression of PTK syk was found to be considerably lower in nonresponsive B-CLL. Thus, absence of mitogenic responses upon BCR cross-linking in particular B-CLL was found to be strictly correlated with diminished induction of BCR-associated PTK activity and lower levels of PTK syk. Because nonresponsive B-CLL closely resembles tolerant autoreactive B cells both functionally and biochemically, distinction between B-CLL with respect to functional properties in vitro may be determined by differences in antigen encounter in vivo.     

Reversible, calcium-dependent membrane association of human leukocyte 5-lipoxygenase.

Maximal activity of human leukocyte 5-lipoxygenase requires Ca2+, ATP, a microsomal membrane preparation, and two cytosolic stimulatory factors. We report here some effects of Ca2+ on the physical properties of the 5-lipoxygenase. When leukocytes were homogenized in the presence of 2 mM EDTA, 5-lipoxygenase was found to be a soluble enzyme. However, when Ca2+ was added to homogenization buffers at 0-1 mM in excess of EDTA, increasing quantities of the enzyme were recovered in the microsomal membrane fraction (100,000 X g pellet). The membrane-associated enzyme was resolubilized by washing pellet preparations in buffers containing 2 mM EDTA and was partially purified by anion-exchange chromatography. Studies of the stimulatory-factor requirements of the membrane-associated, resolubilized, and partially purified enzyme indicated that one of the cytosolic 5-lipoxygenase stimulatory factors exhibited a reversible, Ca2+-dependent membrane association, similar to that of the enzyme itself. Ca2+ also caused a destabilization of the 5-lipoxygenase. Homogenates prepared in the presence of Ca2+ contained lower total enzyme activity, and retention of activity in these samples over time was also diminished.     

Molecular cloning and amino acid sequence of human 5-lipoxygenase.

5-Lipoxygenase (EC 1.13.11.34), a Ca2+-and ATP-requiring enzyme, catalyzes the first two steps in the biosynthesis of the peptidoleukotrienes and the chemotactic factor leukotriene B4. A cDNA clone corresponding to 5-lipoxygenase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antibody. Additional clones from a human placenta lambda gt11 cDNA library were obtained by plaque hybridization with the 32P-labeled lung cDNA clone. Sequence data obtained from several overlapping clones indicate that the composite cDNAs contain the complete coding region for the enzyme. From the deduced primary structure, 5-lipoxygenase encodes a 673 amino acid protein with a calculated molecular weight of 77,839. Direct analysis of the native protein and its proteolytic fragments confirmed the deduced composition, the amino-terminal amino acid sequence, and the structure of many internal segments. 5-Lipoxygenase has no apparent sequence homology with leukotriene A4 hydrolase or Ca2+ -binding proteins. RNA blot analysis indicated substantial amounts of an mRNA species of approximately equal to 2700 nucleotides in leukocytes, lung, and placenta.     

Analysis of signal transduction in B chronic lymphocytic leukemia cells

We report here experiments on the analysis of cellular signal transduction in a series of patients with chronic B cell disorders (B cell chronic lymphocytic leukemia [B-CLL] and prolymphocytic leukemia). We compared the response of the leukemic cells with primary external signals (interleukin 2 [IL-2] or B cell differentiation factors [BCDF or IL-6]) with their response to secondary inducers (the phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA] or the calcium ionophore A23187) that circumvent the first part of the signal transduction pathway by directly activating the key enzyme protein kinase C. One BCDF was synthesized by mitogen-activated peripheral blood B lymphocytes; a second BCDF was constitutively produced by the human bladder carcinoma cell line T24. Changes in morphology, Tac (IL-2 receptor) expression, RNA synthesis measured by 3H-uridine uptake, and immunoglobulin production tested by enzyme-linked immunosorbent assay were used as parameters of successful signal transduction. TPA alone and TPA plus A23187 (synergistically) effectively initiated differentiation in all the leukemia cases. Neither IL-2 nor BCDF (singly or in combinations) caused equivalent responses. On the other hand, IL-2 and BCDF produced a substantial differentiation effect on normal B lymphocytes. Our data suggest that (a) B-CLL cells are able to respond to direct stimulation of the second messenger pathway (through protein kinase C) but not to the physiological stimuli IL-2 or BCDF; (b) the defect in signal transduction appears to be located upstream of protein kinase C (a possible candidate is a G protein); (c) malignant B cells may spontaneously or after treatment with inducers express the IL- 2 receptor (Tac antigen) in the absence of a functional differentiating response to IL-2; and (d) signs of proliferation/differentiation in B- CLL samples after incubation with IL-2 or BCDF might be due to contamination of the cell populations with residual normal B cells.     

抑制5-脂氧合酶表达对胰腺癌细胞增殖和凋亡的影响

目的 应用RNA干扰(RNAi)技术阻断5-脂氧合酶(5-LOX)的表达,观察其对人胰腺癌细胞SW1990增殖及凋亡的作用.方法 构建4个靶向5-LOX的小干扰RNA(small interference,siRNA)和1个阴性对照siRNA质粒表达载体,采用Lipofectamine TM2000法转染SWl990细胞,RT-PCR和Western blot检测RNAi后SWl990细胞的5-LOX mRNA和蛋白表达,MTT法检测细胞的增殖抑制率,流式细胞仪检测细胞凋亡率.结果 阴性对照和4个序列特异性的siRNA对SWl990细胞5-LOXmRNA表达的抑制率分别为(3.0±1.4)%、(18.8±1.5)%、(53.5±2.3)%、(56.1±2.0)%、(52.4±2.5)%;5-LOX蛋白表达抑制率分别为(4.5±2.0)%、(18.1±2.5)%、(50.4±4.3)%、(48.9±4.4)%、(45.9±4.0)%.转染24 h后癌细胞增殖抑制率分别为(2.1±1.0)%、(5.5±1.3)%、(11.9±1.2)%、(13.4±1.1)%、(13.8±1.3)%.;48 h的增殖抑制率分别为(3.0±1.3)%、(16.0±2.2)%、(25.7±2.5)%、(25.3±3.1)%、(27.2±3.2)%.转染24 h细胞凋亡率分别为(2.0±0.8)%、(5.3±1.0)%、(10.6±1.2)%、(12.4±1.0)%、(10.6±0.9)%;转染48 h的凋亡率分别为(3.0±1.0)%、(7.1±1.1)%、(17.5±0.9)%、(21.5±1.1)%、(15.7±1.0)%.结论 通过RNAi可以有效、特异地阻断SWl990细胞5-LOX的表达,并可以有效抑制肿瘤细胞的增殖,促进肿瘤细胞凋亡.     

IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression

Dendritic cell (DC) differentiation from human CD34(+) hematopoietic progenitor cells (HPCs) can be triggered in vitro by a combination of cytokines consisting of stem cell factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. The immune response regulatory cytokines, IL-4 and IL-13, promote DC maturation from HPCs, induce monocyte-DC transdifferentiation, and selectively up-regulate 15-lipoxygenase 1 (15-LO-1) in blood monocytes. To gain more insight into cytokine-regulated eicosanoid production in DCs we studied the effects of IL-4/IL-13 on LO expression during DC differentiation. In the absence of IL-4, DCs that had been generated from CD34(+) HPCs in response to stem cell factor/granulocyte-macrophage colonystimulating factor/tumor necrosis factor alpha expressed high levels of 5-LO and 5-LO activating protein. However, a small subpopulation of eosinophil peroxidase(+) (EOS-PX) cells significantly expressed 15-LO-1. Addition of IL-4 to differentiating DCs led to a marked and selective down-regulation of 5-LO but not of 5-LO activating protein in DCs and in EOS-PX(+) cells and, when added at the onset of DC differentiation, also prevented 5-LO up-regulation. Similar effects were observed during IL-4- or IL-13-dependent monocyte-DC transdifferentiation. Down-regulation of 5-LO was accompanied by up-regulation of 15-LO-1, yielding 15-LO-1(+) 5-LO-deficient DCs. However, transforming growth factor beta1 counteracted the IL-4-dependent inhibition of 5-LO but only minimally affected 15-LO-1 up-regulation. Thus, transforming growth factor beta1 plus IL-4 yielded large mature DCs that coexpress both LOs. Localization of 5-LO in the nucleus and of 15-LO-1 in the cytosol was maintained at all cytokine combinations in all DC phenotypes and in EOS-PX(+) cells. In the absence of IL-4, major eicosanoids of CD34(+)-derived DCs were 5S-hydroxyeicosatetraenoic acid (5S-HETE) and leukotriene B(4), whereas the major eicosanoids of IL-4-treated DCs were 15S-HETE and 5S-15S-diHETE. These actions of IL-4/IL-13 reveal a paradigm of eicosanoid formation consisting of the inhibition of one and the stimulation of another LO in a single leukocyte lineage.     

Adenovirus vector infection of chronic lymphocytic leukemia B cells

Adenovirus vectors have several features that make them attractive for potential use in gene therapy, including a broad tissue tropism and an ability to infect quiescent or postmitotic cells. In light of this, we examined whether recombinant adenovirus vectors could transfer genes into neoplastic cells of patients with chronic lymphocytic leukemia (CLL), a leukemia of "resting" B cells. Using high-titer recombinant adenovirus vectors, we found we could transfer genes encoding beta- galactosidase or murine CD80 (B7-1) into the CLL B cells of all patients tested (n = 10). The efficiency of gene transduction into CLL B cells was approximately 100 to 1,000-fold lower than into HeLa cells at any given multiplicity of infection (MOI). At a MOI of 500, 10% to 70% of the CLL B cells from different patients were made to express the transgene, as assessed by multiparameter flow cytometric analysis. Sustained levels of expression with little loss in the percentage of infected cells were maintained for up to 9 days, at which point the analysis was stopped. We found that CLL B cells have markedly lower expression levels of integrins that facilitate internalization of adenovirus particles into target cells, perhaps accounting, in part, for the reduced efficiency of adenovirus-mediated gene transfer compared with that in HeLa cells. Although HeLa cells express high levels of alpha(v)beta5, and detectable amounts of alpha(v)beta3, we find CLL cells from all patients tested express only low amounts of alpha(v)beta3, and no detectable alpha(v)beta5. Activation of CLL cells via CD40 cross-linking enhances expression of alpha(v)beta3, and induces expression of alpha(v)beta5. This phenotypic change is associated with a fivefold increase in the efficiency of adenovirus- mediated gene transfer into such activated CLL B cells. This study demonstrates that adenovirus vectors can transduce genes into CLL B cells and that the efficiency of gene transduction is enhanced by activation via CD40 cross-linking. This is the first demonstration that high proportions of CLL B cells can be made to express a selected transgene, suggesting that such gene transfer methods may become useful for the study of the pathogenesis and/or treatment of this disease.     

Mutagenicity of non-homologous end joining DNA repair in a resistant subset of human chronic lymphocytic leukaemia B cells

Non-homologous end joining (NHEJ) is an important determinant of genomic stability in mammalian cells. This DNA repair pathway is upregulated in a subset of B-cell chronic lymphocytic leukaemia (B-CLL) cells resistant to DNA damage-induced apoptosis. Using an in vitro assay for double-strand breaks (DSB) end ligation, we studied the fidelity of DSB repair in B-CLL cells which were resistant or sensitive to in vitro DSB-induced apoptosis with concomitant patients' resistance or sensitivity to chemotherapy, respectively. The fidelity of DNA repair was determined by DNA sequencing of polymerase chain reaction products cloned in pGEM-T vector. Sequence analysis of DNA end junctions showed that the frequency of accurate ligation was higher in sensitive B-CLL cells and control cell lines, than in resistant cells where end joining was associated with extended deletions. Upregulated and error-prone NHEJ in resistant cells could be a quite possible mechanism underlying both genomic instability and poor clinical outcome.     

Immune complexes inhibit apoptosis of chronic lymphocytic leukaemia B cells

In the present study we examined the effect of immune complexes (IC) on the survival of chronic lymphocytic leukaemia (B-CLL) B cells. Our results showed that either precipitating IC (pIC), Ab-coated erythrocytes (E-IgG) or heat-aggregated IgG (aIgG) significantly inhibited spontaneous apoptosis of B-CLL cells, as well as that induced by fludarabine, chlorambucil or dexamethasone. After depletion of T lymphocytes, monocytes and NK cells, incubation with IC was no longer able to delay B-CLL cells apoptosis, suggesting that prevention of apoptosis depends on IC interaction with accessory leucocytes. The release of IFNgamma by non-malignant cells upon activation with IC was responsible, to some extent, for IC effects as shown by the fact that neutralizing anti-IFNgamma MoAb partially prevented their ability to inhibit B-CLL cells apoptosis. The observation that treatment with IC resulted in increased expression of HLA-DR on B-CLL cells suggests that inhibition of apoptosis is associated with cellular activation.     

Activation of 5-lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis ofproinflammatory leukotrienes. This study showed that various forms ofcell stress, such as chemical stress (sodium arsenite), osmotic stress,or heat shock lead to substantial formation of 5-LO products in freshlyisolated human polymorphonuclear leukocytes (PMNLs), when exogenousarachidonic acid (10 µM) was present. In parallel, cellstress led to activation of p38 MAPK (mitogen-activated protein kinase)and mitogen-activated protein kinase-activated proteinkinases (MAPKAPKs) kinases, which can phosphorylate 5-LO invitro. Interestingly, arsenite also caused redistribution of 5-LOfrom the cytosol to the nuclear membrane. Only minor activation ofextracellular signal-regulated kinases and c-junNH₂-terminal kinases was observed, implying that theseMAPKs are less important for 5-LO product formation instress-stimulated PMNLs. Stimulation of 5-LO product formation byCa⁺⁺-ionophore A23187 or thapsigargin depended onCa⁺⁺; almost no 5-LO product formation was observed infreshly isolated PMNLs when Ca⁺⁺ was depleted bychelating agents. Also the response toN-formylmethionyl-leucyl-phenylalanine (fMLP) was clearly diminished, but some 5-LO product formation remained. In contrast, stress-induced product formation andtranslocation of 5-LO, as well as activation of p38 MAPK, occurred alsoafter Ca⁺⁺ depletion. Moreover, the p38 MAPKinhibitor SB203580 blocked stress-induced 5-LO product formationefficiently, whereas ionophore- or thapsigargin-induced formation of5-LO products was less sensitive. These data show that cell stress canactivate 5-LO in isolated PMNLs by a mechanism that does not involveCa⁺⁺ mobilization. This mechanism could functionindependently of Ca⁺⁺-mediated 5-LO activation forstimulation of leukotriene biosynthesis under physiologic conditions aswell as in inflammatory diseases.     

Nuclear localization of 5-lipoxygenase as a determinant of leukotriene B4 synthetic capacity

The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of leukotrienes from arachidonic acid. In resting cells, 5-LO can accumulate in either the cytoplasm or the nucleoplasm and, upon cell stimulation, translocates to membranes to initiate leukotriene synthesis. Here, we used mutants of 5-LO with altered subcellular localization to assess the role that nuclear positioning plays in determining leukotriene B4 (LTB4) synthesis. Mutation of either a nuclear localization sequence or a phosphorylation site reduced LTB4 synthesis by 60%, in parallel with reduced nuclear localization of 5-LO. Mutation of both sites together or mutation of all three nuclear localization sequences on 5-LO inhibited LTB4 synthesis by 90% and abolished nuclear localization. Reduced LTB4 generation in mutants could not be attributed to differences in 5-LO amount, enzymatic activity, or membrane association. Instead, 5-LO within the nucleus acts at a different site, the nuclear envelope, than does cytosolic 5-LO, which acts at cytoplasmic and perinuclear membranes. The significance of this difference was suggested by evidence that exogenously derived arachidonic acid colocalized with activated nuclear 5-LO. These results unequivocally demonstrate that the positioning of 5-LO within the nucleus of resting cells is a powerful determinant of the capacity to generate LTB4 upon subsequent activation.     

Inhibition of 5-lipoxygenase pathway of arachidonic acid metabolism in human neutrophils by sulfasalazine and 5-aminosalicylic acid

The possible effect of sulfasalazine, 5-aminosalicylic acid, and acetyl-5-aminosalicylic acid on endogenous arachidonic acid release and metabolism was studied in human polymorphonuclear leukocytes (PMNs). A new in vitro assay was used by which [1-14C]arachidonic acid is incorporated by purified peripheral PMNs until steady state was obtained (5 hr). After preincubation with the test drugs prior to activation with calcium ionophore A23187, the released eicosanoids were isolated by extraction and thin-layer chromatography (TLC) and quantitated by autoradiography and laser densitometry. Median drug concentrations needed for 50% inhibition of leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) release was 4-5 mM (range 1-9 mM) for both sulfasalazine and 5-aminosalicylic acid. The acetylated derivative of 5-aminosalicylic acid was ineffective. The present data suggest that inhibition of arachidonic acid lipoxygenation may be an essential action of sulfasalazine and its active metabolite, 5-aminosalicylic acid. Interference with lipoxygenase enzymes, rather than a steroid-like inhibition of arachidonic acid release from intracellular phospholipids, seems to be the mode of action.