Distinct actions of interleukin-9 and interleukin-4 on a hematopoietic stem cell line, EMLC1

EMLC1 is a hematopoietic stem cell line that depends on stem cell factor (SCF) for growth and generates lymphoid, erythroid and myeloid progenitors in the presence of different cytokines. We have studied signaling events leading to cell proliferation and differentiation of EMLC1 mediated by interleukin (IL)-4 and IL-9. It was found that IL-9 enhances SCF-induced cell proliferation and promotes erythropoietin (EPO)-dependent erythroid differentiation of EMLC1 cells. However, IL-9 alone cannot support the growth of this cell line. In contrast, IL-4 by itself is sufficient to promote the growth of EMLC1 cells, even in the absence of SCF. Antiphosphotyrosine immunoblots of total cell lysates demonstrated that IL-4 and IL-9 induce tyrosine phosphorylation of different cellular substrates. Both IL-4 and IL-9 stimulated tyrosine phosphorylation of SHP-2, whereas the 90-kD tyrosine phosphorylated protein induced by IL-9 stimulation is Stat3. We have also shown that IL-4 is much more potent than IL-9 in inducing the expression of primary response gene c-myc. It was further determined that c-myc antisense oligodeoxynucleotide blocked IL-4 supported cell growth. Taken together, these results indicate that IL-4 may serve as a growth-promoting factor for hematopoietic stem cells, and IL-9 enhances both growth and erythroid differentiation of primitive hematopoietic progenitors. The results also suggest that differences in tyrosine phosphorylation induced by IL-4 and IL-9 may in part determine their distinct biological functions.     

Use of Mono Mac 6 human monocytic cell line and J774 murine macrophage cell line in parallel antimycobacterial drug studies

The Mono Mac 6 (MM6) human monocytic cell line was evaluated with the established J774 murine macrophage cell line to ascertain its effectiveness in determining the intracellular activities of antimycobacterial drugs. Cells were infected with Mycobacterium tuberculosis H37Ra and treated with drug concentrations corresponding to the MICs, as well as to threefold higher than and threefold less than the MICs. Changes in CFU were compared after 7 days to determine significant differences between treated and nontreated groups. The results suggest that MM6 will make a useful model for testing the intracellular activities of antituberculosis drugs.     

The NCI-H295 cell line: a pluripotent model for human adrenocortical studies

The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones has, in the past, required the isolation of cells from each of the adrenocortical zones. Indeed, the lack of a human adrenocortical cell line retaining the ability to produce any of the major adrenal steroid products has slowed studies on normal and abnormal adrenal function. This obstacle has now been largely overcome with the availability of H295 cells, which represents the first adrenocortical cell line to maintain the ability, under specified conditions, to produce all the adrenocortical steroids (i.e., mineralocorticoids, glucocorticoids, and adrenal androgens). Thus, H295 cells appear to act as pluripotent adrenocortical cells capable of being directed to produce each of the zone-specific steroids. The H295 cell line should prove to be of value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis.     

Regulation of interleukin-1ra, interleukin-1 alpha, and interleukin-1 beta production by human alveolar macrophages with phorbol myristate acetate, lipopolysaccharide, and interleukin-4

An attempt was made to estimate the risk of severe cutaneous adverse reactions (SCARs) to Fansidar (sulfadoxine plus pyrimethamine). Cases were identified through a spontaneous reporting system. Persons exposed were estimated using sales data of 27 countries reporting one SCAR case for either Fansidar or a related product, Bactrim (cotrimoxazole; sulfamethoxazole plus trimethoprim). Between 1974 and 1989, 126 cases were notified for Fansidar: 87 cases of erythema multiforme or Stevens-Johnson syndrome, and 39 cases of toxic epidermic necrolysis. 86% of cases were reported in Europe or North America. In 116 cases with use known, prophylaxis was the reason in 103, and treatment in 13. Toxic epidermolysis and erythema multiforme/Stevens-Johnson syndrome had case fatalities of 36 (95% confidence intervals 21 to 53%) and 9% (4 to 18%), respectively. Fansidar users were estimated at 117 million, and the overall SCAR risk to be 1.1 (0.9 to 1.3) per million. For developing countries with mainly single dose use, the risk was estimated to 0.1 (0.0 to 0.1) per million. For Europe and North America with mainly prophylactic use, the risk was 10 (8 to 12) and 36 (23 to 48) per million, respectively. Prophylactic use had a 40 times higher risk than single dose therapeutic use. The aggregated risk peaked in 1984-1985, with global and North American SCAR frequencies of 3.4 (2.4 to 4.3) and 72 (41 to 102) per million, respectively. After 1985, North America reported only one further case despite continued use by an estimated 0.3 million persons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppressive effect of hepatitis B virus on the induction of interleukin-1 beta and interleukin-6 gene expression in the THP-1 human monocytic cell line

The major target organ for Hepatitis B Virus (HBV) is the liver, but extrahepatic sites including monocytes express receptors for HBV and may become infected. Therefore, we investigated the effect of HBV on the in vitro expression of interleukin-beta (IL-1 beta) and interleukin-6 (IL-6) by the monocytoid cell line THP-1, exposed to various stimuli (LPS, PMA or both). Nonstimulated THP-1 cells did not synthesize IL-1 beta and IL-6, even after in vitro exposure to HBV. LPS stimulation alone induced moderate secretion of both IL-1 beta and IL-6 (300 pg/ml). After induction of macrophage differentiation by PMA, THP-1 cells acquired adherence and expressed a higher level of IL-1 beta (up to 2 ng/ml) but did not synthesize IL-6. Treatment of THP-1 cells with PMA and LPS caused the highest production of both IL-1 beta and IL-6 (> 5ng/ml). In vitro exposure of PMA + LPS-stimulated THP-1 cells to HBV resulted in secretion of both HBsAg and preS2Ag which was maintained over 10 days of culture. Southern blot technique was used to study the state of HBV DNA in the cells. Hybridization of non-digested cellular DNA showed only high molecular weight HBV DNA forms. The HindIII restriction pattern revealed bands corresponding to large DNA fragments and the presence of bands at the 3.2 kb position. Under these conditions (PMA + LPS), HBV inhibited the production of IL-1 beta and IL-6 proteins and completely suppressed the IL-1 beta and IL-6 mRNA. Thus, our findings (i) strongly support a relationship between the state of cell differentiation and susceptibility of cells to HBV infection, and (ii) demonstrate that HBV exerts an inhibitory effect on the induction of IL-1 beta and IL-6 genes expression in monocytic THP-1 cells. These results suggest that HBV leads to a fall of pro-inflammatory cytokine production by monocytes/macrophages, which may contribute to impaired host immune response during infection.     

MaMi, a human endometrial stromal sarcoma cell line that constitutively produces interleukin-6, interleukin-8, and monocyte chemoattractant protein-1

BACKGROUND: Uterine endometrial stromal sarcoma is a rare neoplasm with a morphology that closely resembles that of the proliferative endometrial stroma. To understand its pathologic characteristics, we established a novel cell line, MaMi, from a primary culture of an endometrial stromal sarcoma obtained from a 65-year-old Japanese woman. METHODS: We observed the morphology of MaMi cells and performed immunohistochemical analysis on the primary tumor and transplants in nude mice. Prolactin, insulin-like growth factor-binding protein-1, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1, and fibronectin production in the culture medium of MaMi cells were also examined. RESULTS: MaMi cells were shown to exhibit a fibroblast-like morphology in vitro, and they adopted a more elongated appearance in response to 12-O-tetradecanoyl phorbol-13-acetate (TPA). On injection into nude mice, the cells gave rise to subcutaneous tumors. Immunohistologically, both the primary tumor and MaMi cell-induced tumors stained positively with antibodies to neuron-specific enolase or vimentin. MaMi cells constitutively produced IL-6, IL-8, and monocyte chemoattractant protein-1 in vitro. Interleukin-1beta, (100 pmol/L), tumor necrosis factor-alpha (1 nmol/L), and lipopolysaccharide (1 microg/mL) each increased the release of IL-6, IL-8, and monocyte chemoattractant protein-1 by MaMi cells. TPA (10 nmol/L) also stimulated the production of IL-6 and IL-8 by these cells, but inhibited that of monocyte chemoattractant protein-1. CONCLUSIONS: We demonstrated that MaMi cells closely resemble proliferative endometrial stromal cells not only morphologically, but also functionally. This cell line may prove valuable in understanding the role of cytokines produced by tumor cells in the pathogenesis of endometrial stromal sarcoma and may also be useful as an in vitro model of functioning endometrial stromal cells.     

Distinct regulation of glucose transport by interleukin-3 and oncogenes in a murine bone marrow-derived cell line

Growth factors and oncogenes promote glucose uptake, but the extent to which increased uptake is regulated at the level of glucose transporter function has not been clearly established. In this paper, we show that interleukin-3 (IL-3), a cytokine growth factor, and the transforming oncogenes ras and abl alter the activation state of glucose transporters by distinct mechanisms. Using bone marrow-derived IL-3-dependent 32Dc13 (32D clone 3) cells and 32D cells transformed with ras and abl oncogenes, we demonstrated that IL-3 enhanced [3H]-2-deoxyglucose (2-DOG) uptake in parental 32Dc13 cells by 40-50% at 0.2 mM 2-DOG, and this was associated with a 2.5-fold increase in transporter affinity for glucose (reduced Km). In comparison, ras and abl oncogenes enhanced 2-DOG uptake by 72-112%, associated with a 2-fold greater transporter affinity for glucose. The tyrosine kinase inhibitor genistein reversed the effects of both IL-3 and oncogenes on glucose uptake and reduced transporter affinity for glucose. Likewise, with exponentially growing 32D cells in the presence of IL-3, a protein kinase C inhibitor, staurosporine, and a phosphatidylinositol 3-kinase (PI-3) kinase inhibitor, wortmannin, inhibited 2-DOG uptake and decreased transporter affinity for glucose. In contrast, in oncogene-transformed cells, staurosporine inhibited 2-DOG uptake but failed to decrease transporter affinity for glucose, whereas wortmannin did not affect 2-DOG uptake. Inhibition of protein tyrosine phosphatases with vanadate enhanced 2-DOG uptake and transporter affinity for glucose in parental cells and in ras-transformed cells but had little effect on abl-transformed cells. Consistently, the serine/threonine phosphatase type 2A inhibitor okadaic acid enhanced 2-DOG uptake and transporter affinity for glucose in parental cells but had little effect on ras- or abl-transformed cells. These results demonstrate differences in the regulation of glucose transport in parental and oncogene-transformed 32D cells. Thus, IL-3 responses are dependent upon tyrosine, serine/threonine, and PI-3 kinases, whereas ras and abl effects on glucose transport depend upon tyrosine phosphorylation but are compromised in their dependence upon serine/threonine and PI-3 kinases.     

PGG-Glucan activates NF-kappaB-like and NF-IL-6-like transcription factor complexes in a murine monocytic cell line

Basic fibroblast growth factor (bFGF) and FGF receptors have been localized to photoreceptors and retinal pigmented epithelium (RPE), but the function of bFGF in adult retina and RPE is unknown. Exogenous bFGF has a neuroprotective effect in retina and brain and its expression is increased in some neurons in response to cytokines or stress. In this study, we investigated the effect of light, other types of stress, neurotrophic factors, and cytokines on bFGF levels in cultured human RPE. Some agents that protect photoreceptors from the damaging effects of constant light, including brainderived neurotrophic factor (BDNF), ciliary neurotrophic factor, and interleukin-1 beta, increase bFGF mRNA levels in RPE cells. Intense light and exposure to oxidizing agents also increase bFGF mRNA levels in RPE cells and cycloheximide blocks the increase. An increase in bFGF protein levels was demonstrated by ELISA in RPE cell supernatants after incubation with BDNF or exposure to intense light or oxidizing agents. These data indicate that bFGF is modulated in RPE cells by stress and by agents that provide protection from stress and support the hypothesis that bFGF functions as a survival factor in the outer retina.     

Cooperative effects of interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor: a new myeloid cell line inducible to eosinophils

The cytokines interleukin-3 (IL-3); IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to contribute to the proliferation and differentiation of eosinophil progenitors. Recently, it was determined that the cellular receptors for these three cytokines share a common beta-chain while having unique alpha-chains. Thus, there is considerable interest in how these cytokines and their receptors interact in promoting production of eosinophils. We have established a cell line (AML14) from a patient with acute myelogenous leukemia that will consistently exhibit eosinophilic differentiation in suspension in response to IL-3, IL-5, and GM-CSF. Proliferation with only modest differentiative effects was observed in response to a single cytokine. Combinations of two cytokines gave variable results, with GM-CSF + IL-3 and IL-3 + IL-5 causing more proliferation than a single cytokine but little more differentiation. The combination of GM-CSF + IL-5 caused marked enhancement of eosinophilic differentiation with only modest augmentation of proliferation. The combination of all three cytokines was most effective in stimulating both proliferation and eosinophilic differentiation (up to 70% of cells) of AML14 cells. Specific binding of GM-CSF and IL-5 to AML14 cells can be conveniently studied by flow cytometric methods, and cross-competition of these two cytokines for their respective receptors was demonstrated. IL-3 was shown to partially compete for IL-5 binding on AML14 cells. Although specific IL-3 binding could not be demonstrated by flow cytometry, mRNA for the alpha-chains of the IL-3, IL-5, and GM-CSF receptors and the beta-chain common to all three receptors was detected in AML14 cells. The AML14 cell line may be a useful model for the study of cooperative interactions of IL-3, IL-5, GM-CSF, and their respective receptors in the promotion of eosinophil progenitor growth and differentiation.     

Basophilic differentiation of the human leukemia cell line KU812 upon treatment with interleukin-4

To assess the effect of right ventricular pacing on rate regularity during exercise and daily life activities, 16 patients with sinoatrial disease and chronic atrial fibrillation (AF) were studied. Incremental ventricular pacing was commenced at 40 beats/min until > 95% of ventricular pacing were achieved during supine, sitting, and standing. Thirteen patients also underwent randomized paired submaximal exercise tests in either a fixed rate mode. (VVI) or a ventricular rate stabilization (VRS) mode in which the pacing rate was set manually at 10 beats/min above the average AF rate during the last minute of each exercise stage. The pacing interval for rate regularization was shortest during standing (692 +/- 26 ms) compared with either supine or sitting (757 +/- 30 and 705 +/- 26 ms, respectively, P < 0.05). During exercise VRS pacing significantly increased the maximum rate (119 +/- 5.2 vs 106 +/- 4.2 ms, P < 0.05), percent of ventricular pacing (85% +/- 5% vs 23% +/- 7%, P < 0.05), rate regularity index (5.8% +/- 1.6% vs 13.4% +/- 1.9%, P < 0.05), and maximum level of oxygen consumption (12.4 +/- 0.5 vs 11.3 +/- 0.5 mL/kg, P < 0.05) compared with VVI pacing. There was no change in oxygen pulse or difference in symptom scores in this acute study between the two pacing modes. It is concluded that right ventricular pacing may significantly improve rate regularity and cardiopulmonary performance in patients with chronic AF. This may be incorporated in a pacing device for rate regularization of AF using an algorithm that is rate adaptive to postural and exercise stresses.     

Establishment of an osteocyte-like cell line, MLO-Y4

Although osteocytes are the most abundant cells in bone, their functional role remains unclear. In part, this is due to lack of availability of osteocyte cell lines which can be studied in vitro. Since others have shown that cell lines can be readily developed from transgenic mice in which the SV40 large T-antigen oncogene is expressed under the control of a promoter which targets the cells of interest, we used this approach to develop an osteocyte cell line. We chose as a promoter osteocalcin, whose expression is essentially limited to bone cells and which is expressed more abundantly in osteocytes than in osteoblasts. From these transgenic mice, we isolated cells from the long bones using sequential collagenase digestion and maintained these cells on collagen-coated surfaces which are optimal for osteocyte maintenance and growth. We describe here the properties of a cell line cloned from these cultures, called MLO-Y4 (for murine long bone osteocyte Y4). The properties of MLO-Y4 cells are very similar to primary osteocytes. Like primary osteocytes and unlike primary osteoblasts, the cell line produces large amounts of osteocalcin but low amounts of alkaline phosphatase. The cells produce extensive, complex dendritic processes and are positive for T-antigen, for osteopontin, for the neural antigen CD44, and for connexin 43, a protein found in gap junctions. This cell line also produces very small amounts of type I collagen mRNA compared with primary osteoblasts. MLO-Y4 cells lack detectable mRNA for osteoblast-specific factor 2, which appears to be a positive marker for osteoblasts but may be a negative marker for osteocytes. This newly established cell line should prove useful for studying the effects of mechanical stress on osteocyte function and for determining the means whereby osteocytes communicate with other bone cells such as osteoblasts and osteoclasts.     

Cloning and characterization of four types of cDNA encoding myeloperoxidase from human monocytic leukemia cell line, SKM-1

Four cDNA clones encoding myeloperoxidase were isolated from a cDNA library of monocytic leukemia SKM-1 cells. The sequences of two of them were identical to those of cDNA clones previously isolated from a HL-60 cell cDNA library. The sequences of the other two cDNA clones, MP-S34 and MP-S29, differed from those previously described. There was a deletion of 57 bp in the MP-S34 sequence, which was generated by partially skipping exon 9. MP-S29 had a 171 bp deletion, which was generated by completely skipping exon 10. Thus MP-S34 and MP-S29 encoded polypeptides lacking 19 and 57 amino acids, respectively. Both deletions were located on the sequence encoding the heavy subunit. These results indicate that the heterogeneity of the heavy subunit of MPO observed in leukocytes or leukemia could be in part produced by partial or complete skipping of an exon.     

4-Oxoretinol, a metabolite of retinol in the human promyelocytic leukemia cell line NB4, induces cell growth arrest and granulocytic differentiation

All-trans-retinoic acid (RA) is used as a differentiation therapy for acute promyelocytic leukemia. Patients can become resistant to RA, and this resistance is thought to be mediated in part by an increase in the rate of RA metabolism. We have characterized the metabolism of all-trans-retinol (ROL; vitamin A) in NB4 cells, which are human promyelocytic leukemia cells. NB4 cells metabolize ROL into a variety of compounds, including all-trans-4-hydroxyretinol, all-trans-4-oxoretinol (4-oxoROL), 14-hydroxy-4,14-retro-retinol, anhydroretinol, and several ROL esters. No metabolism of ROL to RA or to RA derivatives in NB4 cells was detected. The rate of ROL metabolism increased after cell differentiation; in a 24-h period, differentiated cells metabolized 2-fold more ROL than did undifferentiated cells. The major difference in the ROL metabolism pattern between undifferentiated and differentiated cells was an approximately 10-fold increase in the production of all-trans-4-hydroxyretinol and 4-oxoROL in differentiated cells. Furthermore, exogenously added 4-oxoROL was capable of eliciting NB4 cell differentiation, as measured by growth inhibition, nitroblue tetrazolium reduction, nuclear body relocalization of PML, and surface expression of CD11b. In addition, 4-oxoROL synergized with IFN-gamma in the promotion of NB4 cell growth arrest. Following treatment of NB4 cells with 4-oxoROL to induce differentiation, the production of 4-oxoROL from ROL was observed; this indicated that 4-oxoROL induces its own synthesis in NB4 cells. In addition, 48 h after the administration of 1 microM 4-oxoROL, NB4 cells maintained a high intracellular concentration (17 microM) of 4-oxoROL. These unique properties of 4-oxoROL may provide advantages over RA in the treatment of promyelocytic leukemia cells because it may be possible to maintain cytodifferentiating concentrations of 4-oxoROL in the cells for extended periods of time.     

Bacterial lipopolysaccharide confers resistance to G418, doxorubicin, and taxol in the murine macrophage cell line, RAW264

Many bacterial pathogens including Salmonella and Listeria replicate within macrophages. The susceptibility of these organisms to various antibiotics is dependent on the ability of macrophages to take up, retain, and deliver the antibiotic to the correct intracellular compartment. In this context, macrophages are known to express proteins that are involved in efflux of antibiotics and cytotoxic drugs, thereby reducing intracellular accumulation of such compounds. In our studies on the action of bacterial lipopolysaccharide (LPS) on the macrophage-like cell line, RAW264 we found that LPS treatment of these cells conferred resistance to the neomycin-related aminoglycoside G418 (geneticin). This phenotype was stable and was specific to LPS since colony-stimulating factor 1 and phorbol myristate acetate had no effect on G418 resistance. We have extended this observation to show that LPS induces transient resistance to the cytotoxic drugs taxol and doxorubicin. Macrophage resistance to cytotoxic drugs and antibiotics may have a number of important clinical consequences.