Observations of endotoxin-like activity associated with the Legionnaires' disease bacterium

Suspensions of Legionnaires' disease bacterium stored in sterilized tap water 279–287 days produced gelation ofLimulus amebocyte lysate. A 1-ml suspension of washed cells containing 10⁹ viable organisms had aLimulus amebocyte lysate activity equivalent to 4 mg of endotoxin. This activity remained stable in samples that had been autoclaved at 121°C for 15 min. Both the autoclaved cells and filtrate of autoclaved cells were pyrogenic in inoculated rabbits. The Legionnaires' disease bacterium produces a substance or substances that have biological properties associated with endotoxin of more typical Gram-negative bacteria.     

Classification of the Legionnaires' disease bacterium: An interim report

Deoxyribonucleic acid (DNA) from strains of the Legionnaires' disease bacterium (LDB) was characterized in order to aid in the proper classification of this organism. The genome size of LDB DNA was estimated at 2.5×10⁹ daltons by reassociation kinetics; a guanine-plus-cytosine content of LDB of 39% was established by optical thermal denaturation and buoyant density ultracentrifugation measurements. DNA relatedness studies on 12 strains of the LDB indicated that they were all members of the same species. DNA relatedness studies have thus far failed to show that the LDB is significantly related to any other organism, including all members of Enterobacteriaceae,Pasteurella multocida, Francisella tularensis, Rochalimaea quintana, Vibrio species,Staphylococcus epidermidis, andFlavobacterium meningosepticum.     

Detection of complement-fixing antibodies to the Legionnaires' disease bacterium andChlamydia in animal and human sera

Because the sera of almost half of the Legionnaires' disease (LD) survivors we tested had antibodies toChlamydia psittaci as well as to the LD bacterium, we evaluated the possibility of LD/Chlamydia serological cross-reactivity by testing sera from Philadelphia LD survivors, other persons and animals exposed to or infected withC. psittaci, and unexposed humans and animals against identically prepared antigens of both agents. Using a complement fixation method, high-titered chlamydial antisera from cattle, sheep, horses, and guinea pigs in general had no antibodies to the LD antigen. Sera from a few chlamydia-infected animals had low LD antibody titers, but no substantial cross-reaction was observed. Of 21 human LD survivors, 100% had antibodies to the LD agent and 9 (43%) had chlamydial antibodies. Of 24 controls, 7 (29%) had LD antibodies, and 5 (21%) had chlamydial antibodies. Of 58 other persons who had no clinical history of pneumonia, 5 (9%) had LD antibodies and 15 (26%) had chlamydial antibodies. The serological data on animal antisera suggest there is no significant antigen sharing between the LD and chlamydial agents and therefore the increased incidence of chlamydial antibody in LD survivors is not due to exposure to LD antigen.     

β-Lactamase of the Legionnaires' bacterium

Nine strains of the Legionnaires' bacterium produced a β-lactamase that was basically a cephalosporinase, but which also had some effect on all the penicillins tested. Cefoxitin, cefuroxime, and cephalexin were resistant to the enzyme. Minimal inhibitory and bactericidal concentrations were very similar.     

Effect of supplementall-tyrosine on pigment production in cultures of the Legionnaires' disease bacterium

The etiologic agent of Legionnaires' disease grows on certain agar media. Cultures of this organism on supplemented Mueller-Hinton agar are characterized by the appearance of brown pigment in and around areas of bacterial growth. The major peptone source in Mueller-Hinton agar is an acid hydrolysate of casein. Legionnaires' disease bacterium also grows on a medium in which the peptone source is 0.25% yeast extract, but no pigment is produced. If the yeast extract agar is enriched withl-tyrosine, pigment formation can occur. Pigmentation of cultures of Legionnaires' disease bacterium may be mediated by a phenolo-monooxygenase, or tyrosinase.     

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.     

Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis

We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of inhibitors of signalling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter assay for chemotaxis. We also assessed expression of signalling and cytoskeletal proteins using Western blotting.Chemotactic peptide induced a front-tail polarity in HL-60 cells comparable to that of neutrophils. Chemokinetic and chemotactic responses to chemotactic peptide were also very similar for both cell types, concerning mean speed of migration, the fraction of migrated cells and the concentration of stimulus optimal for activation. The cytokine interleukin-8 was in contrast clearly less effective in activating motile responses of differentiated HL-60 cells as compared to neutrophils.An important functional role of Rho-activated kinases and phosphatidylinositol 3-kinase in motile responses of HL-60 cells, consistent with their upregulation during differentiation, could be confirmed using inhibitors with specificity for the corresponding enzymes. The only difference observed here between HL-60 cells and neutrophils concerned the differential effects of a protein kinase C inhibitor.In summary, the results presented here show that differentiated HL-60 cells, stimulated with chemotactic peptide, are a valid model system to study molecular mechanisms of neutrophil emigration.     

The expression of receptors for IgA on human monocytes and calcitriol-treated HL-60 cells

In this study, we investigated the expression of IgA Fc receptors (FcR alpha) by human myeloid cells. By using a sensitive cytofluorometric IgA binding assay, we found that approximately 85% of peripheral blood monocytes bound IgA in an isotype-specific fashion. The HL-60 human promyelocytic leukemia cell line failed to express FcR alpha; however, treatment of HL-60 cells with the differentiating agent calcitriol induced the expression of FcR alpha on greater than 90% of these cells. Monocytes and calcitriol-treated HL-60 cells were capable of ingesting IgA-coated erythrocyte targets, suggesting that phagocytosis could be mediated through FcR alpha. The induced HL-60 cell system represents a useful model for further studies on FcR alpha expression and function.     

mRNA species regulated during the differentiation of HL-60 cells to macrophages and neutrophils

Using cDNA clone banks from differentiated and undifferentiated HL-60 promyelocytic leukemia cells, we have selected clones for genes which are regulated during this differentiation. Regulation of the corresponding mRNAs in HL-60 cells during both monocytic and neutrophilic differentiation was measured for 21 of these clones. The levels of mRNA hybridizing to some of these clones changed by more than 100-fold during differentiation. Unlike erythropoiesis or myogenesis, in which the synthesis of a few new proteins is synchronously regulated, mRNAs in differentiating HL-60 cells are asynchronously regulated, suggesting a complex series of regulatory events. About half of these regulation-selected clones contained repeat sequences, including both Alu and novel repeat families. Most of the regulated genes are members of extensive gene families.     

Effect of DNA methylation on protein-DNA interaction of HL-60 cells

HL-60 cells have been induced with differentiation index 16 % by S-adenosyl-L-rnethionine (SAM) as inducer in the presence of optimum conceptration of 10 μmol/L. The methylation level of genorne DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma I, Sal I, XhoI and Hind Ⅲ which are sensitive to 5-methylcytosine were used to cleave the genorne DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.     

Effect of DNA methylation on protein-DNA interaction of HL-60 cells

HL-60 cells have been induced with differentiation index 16% by S-adenosyl-L-methionine (SAM) as inducer in the presence of optimum concentration of 10 pmol/L. The methylation level of genome DNA determined by HPLC is increased during cell differentiation. When restriction endonucleaseHae III,Sma I,Sal I,XhoI andHind III which are sensitive to 5-methyicytoside were used to cleave the genome DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.     

Gene expression spectra in human leukemia HL-60 cells treated with EGCG

To decipher the molecular mechanism of EGCG induced HL-60 cell apoptosis, alterations of gene expression spectra in HL-60 cell line cells after treatment with EGCG were screened by cDNA chip, and analyzed with the GenePix 3.0 twice; and the cDNA chip results further identified by RT-PCR. Ninety-seven genes among the total 8398 (1.15%) showed consistent significant differential expression in the duplicated cDNA chip assessments. Thirty-nine genes (40.2%) were up-regulated and 58 genes (59.8%) were down-regulated; and the randomly selected four performed RT-PCR results agreed with the cDNA chip data. The results suggest that the apoptosis of HL-60 cells induced by EGCG is a progressive transformation process regulated by a variety of genes.     

The interaction of carbamylated low-density lipoprotein with cultured cells. Studies with human fibroblasts, rat peritoneal macrophages and human monocyte-derived macrophages

We determined the effects of various degrees of chemical modification of low-density lipoprotein (LDL) on its interaction with receptors present on human fibroblasts, human monocyte-derived macrophages and rat peritoneal macrophages. We isolated LDL (d = 1.019-1.063 g/ml) and carbamylated different numbers of lysine residues and tested its cell-interactive properties, including binding, degradation, and stimulation of [3H]oleate incorporation into cholesteryl oleate. Small carbamylation of LDL (approximately 1-2% of lysine residues) resulted in a reduced ability (70-80% of control) to displace 125I-labeled LDL from fibroblast receptors. Modification of 12.5-25% of lysine residues resulted in a marked increase in the ability of LDL to interact with scavenger receptors and an almost total loss in the ability to interact with apolipoprotein B-E receptors. Acetylated LDL and malondialdehyde-modified LDL inhibited competitively the degradation of 125I-carbamylated LDL by human macrophages. Thus, the extent of modification plays an important role in recognition of modified LDL by scavenger receptors. There also seems to be a range of modification over which LDL is not yet recognized by the scavenger receptor, but its interaction with the apolipoprotein B-E receptor is markedly reduced. This perhaps explains how a small in vivo modification of LDL can result in an increase in residence time of LDL in the subendothelial tissue which can lead to further local interactions, ultimately increasing the atherogenicity of the LDL particle.     

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.     

Cytotoxic activity of acridin-3,6-diyl dithiourea hydrochlorides in human leukemia line HL-60 and resistant subline HL-60/ADR

A series of acridin-3,6-diyl dithiourea hydrochloride derivatives (alkyl-AcrDTU) was prepared and tested against sensitive and drug resistant leukemia cell lines for their cytotoxic/cytostatic activity. The products (ethyl-, n-propyl-, n-butyl-, n-pentyl-AcrDTU) showed high DNA binding affinity via intercalation (K = 7.6 ? 2.9 × 10⁵ M^?1). All derivatives inhibited proliferation of HL-60 cells and its resistant subline HL-60/ADR, unexpectedly the resistant subline was more sensitive than the parental one (IC₅₀ = 3.5 μM, 48-treatment of HL-60/ADR with pentyl-AcrDTU). Cytotoxicity of tested compounds was associated with their DNA-binding properties and the level of intracellular thiols has been changed in the presence of AcrDTU.     

Prostaglandin A2 activates intrinsic apoptotic pathway by direct interaction with mitochondria in HL-60 cells

HL-60 cells treated by prostaglandin (PG) A₂ showed characteristics of apoptosis such as accumulation of hypodiploid and annexin V positive cells, condensed and fragmented nuclei, cytochrome c (Cyt C) release from mitochondria and activation of caspase-1, -2, -3, -7 and -9. PGA₂-induced cell death was rescued by inhibitors of caspase-9 and -3, but PGA₂-induced Cyt C release was not prevented by caspase inhibitors. During Cyt C release by PGA₂, mitochondrial transmembrane potential was maintained and mitochondrial permeability transition pore was not formed. In addition, anti-apoptotic BCL-2 family proteins like BCL-2 and BCL-XL, and ROS scavengers including ascorbic acid and 2,2,6,6-tetramethyl-1-piperidinyloxy were not able to inhibit Cyt C release as well as apoptosis by PGA₂. Finally, it was shown that PGA₂-induced Cyt C release in vitro from purified mitochondria in the absence of cytosolic components. Furthermore, thiol-containing compounds such as N-acetylcysteine, l-cysteine and monothioglycerol prevented Cyt C release, and hence induction of apoptosis. Taken together, these results suggest that PGA₂ activates intrinsic apoptotic pathway by directly stimulating mitochondrial outer membrane permeabilization to release Cyt C, in which thiol-reactivity of PGA₂ plays a pivotal role.     

Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model

We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca²⁺ levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca²⁺ release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo.