Expression of human tissue kallikrein in differentiated HL-60 cells

The kallikrein-kinin system is a mediator of inflammation in humans. In order to elucidate the range of expression of human tissue kallikrein and its substrates, high and low molecular weight kininogen, in inflammatory cells in vitro, we examined their biosynthesis in the HL-60 cell line by RT-PCR and Southern blot analyses. Prominent expression of tissue kallikrein mRNA occurred in untreated promyelocytic cultures as well as in HL-60 cells that were induced to differentiate toward neutrophilic, monocytic, and macrophagic cells. Under the same inducing conditions, kininogen biosynthesis was undetectable at each differentiation state of HL-60 cultures. These results indicate that the myelomonocytic lineage of human leukocytes is a source of tissue kallikrein, which may be secreted as part of the inflammatory process.     

Diverse effects of sphingosine on calcium mobilization and influx in differentiated HL-60 cells

Sphingosine induces a biphasic increase in cytosolic-free Ca(2+)([Ca(2+)](i)) with an initial peak followed by a sustained increase in HL-60 cells differentiated into neutrophil-like cells. The initial peak is not affected by the presence of ethylene glycol bis (beta-aminoethyl ether) N, N, N', N-tetraacetic acid (EGTA) in the buffer and appears to be dependent on conversion of sphingosine to sphingosine -1-phosphate (S1P) by sphingosine kinase, since it is blocked by the presence of N, N-dimethylsphingosine (DMS), which, like sphingosine, causes a sustained increase itself. The sustained increase that is elicited by sphingosine or DMS is abolished by the presence of EGTA in the buffer. The sustained sphingosine-induced Ca(2+)influx does not appear due to Ca(2+)influx through store-operated Ca(2+)(SOC) channels, since the influx is not inhibited by SKF 96365, nor is it augmented by loperamide. In addition, sphingosine and DMS attenuate the Ca(2+)influx through SOC channels that occurs after depletion of intracellular stores by ATP or thapsigargin. Both the initial peak and the sustained increase in [Ca(2+)](i)elicited by sphingosine can be blocked by phorbol 12-myristate 13-acetate (PMA)-elicited activation of protein kinase C. Thus, in HL-60 cells sphingosine causes a mobilization of Ca(2+)from intracellular Ca(2+)stores, which requires conversion to S1P, while both sphingosine and DMS elicit a Ca(2+)influx through an undefined Ca(2+)channel and cause a blockade of SOC channels.     

Autoantibodies recognizing proteins copurified with PCNA in patients with connective tissue diseases

Objective. Proliferating cell nuclear antigen (PCNA), one of the target antigen recognized by lupus sera, has been reported to be present as a subnuclear multi-peptide complex. But autoantibodies reacting with components of PCNA complex are poorly understood. To study the specificity of those autoantibodies, immunoreactivities of autoimmune sera against purified PCNA antigen were studied. Methods. PCNA antigens were purified from rabbit thymus extract by affinity column using murine monoclonal antibodies (mAbs) to PCNA, TOB7, TO17 and TO30. Immunoreactivities of autoimmune sera against purified PCNA were analyzed by WB. Results. PCNA antigen purified by serum AK predominantly showed a 34 kD band specific for PCNA in SDS-PAGE. When antigens were purified by anti-PCNA mAb TOB7 and TO30 which are known to be targeting different epitopes on PCNA antigen, SDS-PAGE analysis showed various mol. wt of proteins in addition to the 34 kD PCNA while both AK and mAbs reacted only with 34 kD PCNA in WB. In WB using PCNA purified by TOB7, various immunoreactivities were observed at 150, 66, 58, 48, 45, 37, 32 and 16 kDa in sera from patients with connective tissue diseases. Conclusions. These results suggested that many of the proteins copurified with PCNA were also targets of autoimmune responses and these autoantibody experssion may be induced through antigen-driven mechanisms.Abbreviations mAb monoclonal antibody - PCNA proliferating cell nuclear antigen - PCNA/AK PCNA affinity purified by antibodies from patient serum AK - PCNA/TO30 PCNA purfied by mAb TO30 - PCNA/TOB7 PCNA purified by mAb TOB7 - SLE systemic lupus erythematosus     

Appearance of specific leukotriene B4 binding sites in myeloid differentiated HL-60 cells

Exposure of HL-60 cells for 6 days to a combination of 1.25% (v/v) dimethyl sulfoxide and 10 microM dexamethasone induces myeloid differentiation which results in a cell with many of the characteristics of a mature granulocyte. At 4 degrees C myeloid differentiated, but not undifferentiated, monocytic differentiated or eosinophilic differentiated HL-60 cells display marked specific leukotriene B4 binding. Leukotriene B4 binding at 4 degrees C reaches a maximum within 10 min, is readily reversed by unlabeled leukotriene B4, and is stereospecific. Only molecules with structural and biological similarity to leukotriene B4 can competitively inhibit leukotriene B4 binding. Scatchard analysis at 4 degrees C in differentiated cells shows two classes of binding sites. The high affinity sites have a Kd of 0.27 nM and a Bmax of 14.8 fmol/10(7) cells; the low affinity sites have a Kd of 0.58 microM and a Bmax of 2453 fmol/10(7) cells. The appearance of specific leukotriene B4 binding sites in the myeloid differentiated cells correlates with their ability to chemotax in response to leukotriene B4. Undifferentiated cells do not chemotax to leukotriene B4. At 37 degrees C leukotriene B4 is incorporated into phospholipid and triglyceride species in both undifferentiated and myeloid differentiated HL-60 cells making binding studies at 37 degrees C in intact cells impossible. No evidence of omega-hydroxylase activity was found in HL-60 cells. These data suggest that the HL-60 cell may be an excellent model system for the study of leukotriene B4 receptor binding, processing, and gene expression.     

Interaction of guanine-nucleotide-binding regulatory proteins with chemotactic peptide receptors in differentiated human leukemic HL-60 cells.

Human leukemic HL-60 cells were differentiated into neutrophil-like cells by treatment with dimethylsulfoxide (Me2SO) or N6,O2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP), and membrane fractions were prepared from the differentiated cells. Receptors for fMLF (fM,N-formylmethionine) and guanine-nucleotide-binding regulatory proteins (G proteins) serving as the substrate for pertussis toxin (islet-activating protein; IAP) were extracted from cell membranes then reconstituted into phospholipid vesicles. The binding of fMLF to the reconstituted vesicles (or the membranes) was determined with 10 nM [3H] fMLF. In both cases, high-affinity binding to vesicle preparations from the Me2SO- and Bt2cAMP-induced cells was abolished following treatment with IAP, suggesting that fMLF receptors were functionally coupled to IAP-sensitive G proteins in each of the two vesicle types. However, the high-affinity fMLF binding was much higher in vesicle preparations originating from Bt2cAMP-induced cells than in those from Me2SO-induced cells, although the amount of IAP-substrate G protein reconstituted into the each phospholipid vesicles preparation was not significantly different from the other. The G proteins of the two differentiated cells were both identified as inhibitory forms (Gi-2) based on their electrophoretic mobilities and immunoblot analyses. When purified Gi-2 from rat brain was reconstituted into the two IAP-treated vesicles, high-affinity fMLF binding was restored in a similar manner in both. IAP-substrate G proteins partially purified from the two differentiated HL-60 cells were also effective in restoring high-affinity fMLF binding to the IAP-treated vesicles. However, a significant difference was observed that the reconstituted binding was higher with the G-protein-rich fraction from Bt2cAMP-induced cells than with that from Me2SO-induced cells, with each of the two IAP-treated vesicle types. These results suggest that the different high-affinity binding of fMLF observed in the two differentiated HL-60 cells are due to a difference in the property of endogenous G proteins rather than fMLF receptors, though the two G proteins are indistinguishable from each other in terms of the subtype of G protein, Gi-2.     

Managanese stimulates the oxidative brust of differentiated HL-60 cells

Incubation with manganese results in a twofold increase in the oxidative burst of differentiated HL-60 cells. This stimulation was characterized by examining the dose response, length of incubation time, and specifity of manganese. Managese only stimulated the burst in cells induced to differentiated with retinoic acid and not in undifferentiated HL-60 cells. Incubation with manganese did not result in a greater number of differentiated cells. The maximum stimulation occurred at 0.2 μmol/L manganese. Stimulation of the oxidative burst required 96 h of incubation with manganese, since cells incubated with the same levels of manganese for the last 24 h of culture did not result in any stimulation. Magnesium, present in the incubation medium at physiological serum levels (820 μmol/L) also stimulated the oxidative burst, whereas iron (0.3 μmol/L), zinc (18 μmol/L), and copper (12 μmol/L) had no effect. To determine whether manganese and magnesium stimulated the burst differently, the initial rates of superoxide anion production was determined. The initial rate of the reaction proceeded rapidly in cells incubated with managnese, whereas there appeared to be a lag before magnesiumtreated cells produced superoxide anion. Thus, manganese seems to stimulate the oxidative burst differently than magnesium.     

Dystroglycan depletion inhibits the functions of differentiated HL-60 cells

Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.     

Action of extremely low frequency electric fields on the cytosolic calcium concentration of differentiated HL-60 cells: Nonactivated cells

The effect of sinusoidal electric fields on the cytosolic free [Ca²⁺]_i concentration in differentiated HL-60 cells was measured. The calcium concentration was measured in a fluorescence spectrometer using the fluorescence sample fluo-3. In the fluorescence spectrometer two samples can be measured simultaneously, one as the sham-exposed control and the other as the field-exposed sample. The effects of an external field, applied using two capacitor plates outside the cuvettes, and a field applied directly to the medium, using two platinum electrodes inside the cuvettes, were measured at selected frequencies between 0 and 100 Hz and field strengths from 1 to 2000 V_pp/m (external field) and from 0.1 to 1000 V_pp/m (in medium). No significant effects of the fields on the cytosolic free [Ca²⁺]_i concentration in HL-60 cells have been observed at the measured frequencies and field strengths. Bioelectromagnetics 19:32–40, 1998. © 1998 Wiley-Liss, Inc.     

结缔组织病伴发疾病分析

目的：对结缔组织病与其常见伴发疾病进行研究分析,探讨肿瘤等疾病与与结缔组织病的相关性。方法：收集确诊有结缔组织病患者共333例,对其临床资料并进行回顾性统计分析。结果：本文中333例结缔组织病中发生肺间质病变79例（23.7%）,发生肿瘤8例（2.4%）,出现肾损害有124例（37.2%）。结论：间质性肺病、肿瘤、肾损害在结缔组织病患者中占一定的比例,其机制可能与结缔组织病本身的病理生理改变、外源性因素等共同作用所致。     

结缔组织病伴发疾病分析

目的:对结缔组织病与其常见伴发疾病进行研究分析,探讨肿瘤等疾病与与结缔组织病的相关性.方法:收集确诊有结缔组织病患者共333例,对其临床资料并进行回顾性统计分析.结果:本文中333例结缔组织病中发生肺间质病变79例(23.7%),发生肿瘤8例(2.4%),出现肾损害有124例(37.2%).结论:间质性肺病、肿瘤、肾损害在结缔组织病患者中占一定的比例,其机制可能与结缔组织病本身的病理生理改变、外源性因素等共同作用所致.     

IL-5 up-regulates cysteinyl leukotriene 1 receptor expression in HL-60 cells differentiated into eosinophils

The cysteinyl leukotrienes, leukotriene (LT) C(4), LTD(4), and LTE(4), are lipid mediators that have been implicated in the pathogenesis of several inflammatory processes, including asthma. The human LTD(4) receptor, CysLT(1)R, was recently cloned and characterized. We had previously shown that HL-60 cells differentiated toward the eosinophilic lineage (HL-60/eos) developed specific functional LTD(4) receptors. The present work was undertaken to study the potential modulation of CysLT(1)R expression in HL-60/eos by IL-5, an important regulator of eosinophil function. Here, we report that IL-5 rapidly up-regulates CysLT(1)R mRNA expression, with consequently enhanced CysLT(1)R protein expression and function in HL-60/eos. CysLT(1)R mRNA expression was augmented 2- to 15-fold following treatment with IL-5 (1-20 ng/ml). The effect was seen after 2 h, was maximal by 4 h, and maintained at 8 h. Although CysLT(1)R mRNA was constitutively expressed in undifferentiated HL-60 cells, its expression was not modulated by IL-5 in the absence of differentiation. Differentiated HL-60/eos cells pretreated with IL-5 (10 ng/ml) for 24 h showed enhanced CysLT(1)R expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-CysLT(1)R Ab. They also showed enhanced responsiveness to LTD(4), but not to LTB(4) or platelet-activating factor, in terms of Ca(2+) mobilization, and augmented the chemotactic response to LTD(4). Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil functions and particularly their responsiveness to LTD(4), and thus contribute to the pathogenesis of asthma and allergic diseases.     

Effects of tunicamycin on the expression and function of formyl peptide chemotactic receptors of differentiated HL-60 cells

We previously showed that formyl peptide chemotactic receptors (FPCR) of human phagocytic cells contain at least two asparagine-linked oligosaccharide chains located at the distal end of the receptor. The requirement of these N-linked oligosaccharide chains for expression and function of FPCR was investigated in HL-60 cells induced to differentiate by N6,O2-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of 5 micrograms/ml tunicamycin. Tunicamycin did not prevent the changes in morphology associated with Bt2cAMP-induced differentiation of HL-60 cells. Autoradiographic analysis after SDS-PAGE of FPCR affinity labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (formyl 125I-hexapeptide) and ethylene glycol bis(succinimidyl succinate) demonstrated that greater than 95% of FPCR expressed by tunicamycin-treated cells completely lacked N-linked oligosaccharide (Mr 32,000), and no fully glycosylated FPCR (Mr 62,000 to 85,000) was detectable. Scatchard analysis of formyl 125I-hexapeptide binding indicated the presence of two classes of binding sites for both control and tunicamycin-treated cells (control cells, 82,000 +/- 32,000 sites/cell with Kd 10.0 +/- 4.3 nM and 520,000 +/- 40,000 sites/cell with Kd 250 +/- 80 nM; tunicamycin-treated cells, 11,000 +/- 5000 sites/cell with Kd 3.0 +/- 1.9 nM and 470,000 +/- 70,000 sites/cell with Kd of 500 +/- 140 nM). Both control and tunicamycin-treated cells augmented superoxide anion release, exhibited a migratory response, and showed a transient rise in intracellular free Ca2+ upon stimulation with N-formyl-Nle-Leu-Phe. However, the responses of the tunicamycin-treated cells were less than that of the control cells. The present studies demonstrate that N-glycosylation of FPCR is not essential for cell surface expression or for several FPCR-mediated cell responses.     

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL－60 cells

INTRODUCTIIONThe nuclear matrix is an essential component ofthe nucleus which is important for the nuclear structural integrity and specific genomic functions[1, 2].Several articles have reported that the nuclear matrix, as a higher order framework structures, mightbe disassembled du-ring the apoptotic process[3-5].Accordingly3 nuclear lamins A/C or B have beenfound to decrease in apoptotic thymocytes[6], Tcells[7], and carcinoma cell line[8, 9]. The nucleolar protein B23, an obscure ma…     

Modulation of cytokine production by interferential current in differentiated HL-60 cells

The influence of interferential current (IFC) on the release of four cytokines was investigated. IFC is an amplitude-modulated 4 kHz current used in therapeutic applications. Human promyelocytes (HL-60) were differentiated to monocytes/macrophages by treatment with calcitriol. Release of tumor necrosis factor alpha (TNFalpha) and interleukines 1beta, 6, and 8 (IL-1beta, IL-6, and IL-8) into the supernatant was measured after exposure to IFC at different modulation frequencies. TNFalpha release was stimulated about twofold by 4 kHz sine waves alone. The influences of exposure time (5-30 min) and current density (2.5-2500 microA/c m(2)) were tested. A maximum field effect was found at an exposure time of 15 min and a current density of 250 microA/cm(2). With these exposure conditions (15 min and 250 microA/cm(2) ), cells were treated at different modulation frequencies and reacted for TNFalpha, IL-1beta, and IL-8 release in a complex manner. Within the frequencies studied (0-125 Hz), we found stimulation as well as depression of the release. In a second run the cells were activated by pretreatment with 10 microg/ml lipopolysaccharide (LPS) and exposed in the same way as the nonactivated cells. Again the modulation frequency influenced, in a complex way, the induction of TNFalpha, IL-1beta, and IL-8, resulting in a pattern of stimulation and depression of release different from that found in nonactivated cells. For IL-6 production no significant changes were detected in activated or non-activated cells.     

Expression of selected genes in differentiated HL-60 cells and primary cells from human leukemias

Expression of three clones (6-1E, 7-3G and 9-5C) selected from a chronic lymphocytic leukemia cDNA library was studied by nucleic acid hybridization in human promyelocytic leukemia cells (HL-60) treated with chemical inducers of cell differentiation and in primary cells derived from 27 patients with leukemia or myelodysplastic syndrome. The differentiation of HL-60 cells into macrophage-like cells upon induction by 12-0-tetradecanoyl phorbol-13-acetate (TPA) was accompanied by rapid induction of the expression of 6-1E and 7-3G genes. The levels of expression of the 9-5C gene were not altered during macrophage-monocytic or granulocytic differentiation of HL-60 cells. The expression of the 6-1E and/or 7-3G gene was induced by TPA in four of 6 samples derived from patients who achieved complete remission, but not in any of the acute nonlymphocytic leukemia samples from patients who failed to achieve complete remission. These findings suggest that expression of the 6-1E and 7-3G genes is related to macrophage-monocytic differentiation and that alterations of these gene expressions in fresh leukemia cells after one hour of TPA treatment are of prognostic significance in predicting the response to therapy.     

Characteristics of U-937 and HL-60 leukemia cells differentiated by spinach extract

Some characteristics of U-937 and HL-60 leukemia cell lines treated with a fraction of non-dialyzable extract of spinach are reported. The absorbed fraction separated by a DEAE-Tyopearl 650 column chromatography of the non-dialyzable extract induced NBT reducing activity of U-937 and HL-60 cells. This fraction also induced substrate adhesion of U-937 cells, and the non-specific esterase activity of HL-60 cells. The expression of CD11b, CD11c and CD36 antigens on the U-937 cell surface was enhanced by the treatment with the fraction, whereas CD24 antigen was not. The treatment of HL-60 cells with the fraction also induced the expression of CD11b and CD11c antigens, but CD24 and CD36 were not expressed. These results indicated that the non-dialyzable extract of spinach induced immature differentiation of U-937 and HL-60 cells into monocyte/macrophages.Abbreviations NBT nitroblue tetrazolium - TPA 12-O-tetradecanoyl-phorbol-13-acerate - PBS phosphate buffered saline - FITC fluorescein isothiocyanate     

Retinoic acid-induced expression of tissue transglutaminase in human promyelocytic leukemia (HL-60) cells

Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.     

Membrane-bound heparin binding proteins from HL-60 cells purified in a two-step affinity chromatography differentially eluted with divalent cations

Solubilized membrane proteins from HL-60 cells were separated by two-step affinity chromatography. Proteins eluted with MgCl2 in the first heparin-gel were applied to the second heparin-gel and eluted with CaCl2. The eluted proteins were analysed and purified by electrophoresis. N-terminal amino acid sequences of eight proteins on the characteristic bands were determined. Homology search for the sequences indicated that three microsomal proteins, two nuclear proteins and a glycolytic enzyme were eluted with divalent cations, whereas a nuclear ribonucleoprotein and a membrane-cytoskelton linker protein were not dissociated with divalent cations, but with 2 M NaCl. Heparin affinity chromatography combined with differential elution with divalent cations can be a useful method for separation of membrane proteins.     

Autoantibodies to angiotensin-converting enzyme 2 in patients with connective tissue diseases

Introduction  

Angiotensin-converting enzyme (ACE) 2, a homolog of ACE, converts angiotensin (Ang) II into Ang(1-7), and the vasoprotective effects of Ang(1-7) have been documented. We explored the hypothesis that serum autoantibodies to ACE2 predispose patients with connective tissue diseases to constrictive vasculopathy, pulmonary arterial hypertension (PAH), or persistent digital ischemia.     

Postvaccinal antidiphtheria and antitetanus immunity in patients with systemic connective tissue diseases

The retrospective evaluation of the intensity of postvaccinal immunity at different periods after the primary course of immunization against diphtheria and tetanus was made. In the sera taken from 130 children with systemic connective tissue diseases the content of antibodies to diphtheria and tetanus was determined. As revealed in this study, the protective levels of antibodies to diphtheria and tetanus toxoids were retained by both sick and healthy children for 5 years and longer. Significantly lower titers of antibodies to diphtheria and tetanus toxoids were registered in children undergoing therapy with glucocorticosteroids and cytostatics at the time of the study.