Neutrophil serine proteinases and defensins in chronic obstructive pulmonary disease: effects on pulmonary epithelium

Neutrophils have the capacity to accumulate in high numbers in the lung during infection and inflammation. Because they play an important role in host defence against infection, but may also cause tissue injury, these cells are thought to be involved in the pathogenesis of various inflammatory lung disorders, including chronic bronchitis and chronic obstructive pulmonary disease. Neutrophil products that may mediate tissue injury at sites of neutrophil-dominated inflammation include the neutrophil serine proteinases elastase, cathepsin G and proteinase 3, and the nonenzymatic defensins. One of the targets of the neutrophil is the lung epithelium, and in vitro studies have revealed that both the serine proteinases and neutrophil defensins markedly affect the integrity of the epithelial layer, decrease the frequency of ciliary beat, increase the secretion of mucus, and induce the synthesis of epithelium-derived mediators that may influence the amplification and resolution of neutrophil-dominated inflammation. Both neutrophil elastase and defensins induce the release of the neutrophil chemoattractant chemokine interleukin-8 from respiratory epithelial cells. The alpha1-proteinase inhibitor (alpha1-PI) is a well-characterized inhibitor of neutrophil elastase, that also blocks the cytotoxic and stimulatory activity of defensins towards epithelial cells. The elastase inhibitory activity of alpha1-PI is also abrogated by the binding of defensins to this inhibitor. Incubation of epithelial cells with neutrophil defensins in combination with either elastase or cathepsin G resulted in decreased effects on the epithelial cells compared with those observed when the cells were incubated with defensins, elastase or cathepsin G separately. These results suggest that neutrophil defensins and serine proteinases cause injury and stimulate epithelial cells to produce chemokines that attract more neutrophils to the site of inflammation. The effects of neutrophil defensins and serine proteinases on epithelial cells appear to be restricted by proteinase inhibitors and by inhibitory interactions between these sets of neutrophil granule proteins.     

Inhibitory activity in human marrow plasma directed against mouse marrow cell proliferation: reduced in subjects with decreased granulopoiesis

Human venous plasma is known to contain a lipoprotein-inhibitor of mouse marrow cell growth which we have found does not have cell type-specificity, in that it inhibits mouse lymphoma cell as well as marrow cell colony formation in vitro. Following its removal by CHCl3, we have identified residual inhibitory activity which reduces the growth of mouse marrow cells but not lymphoma cells. This inhibitory activity was found to be present in marrow and to much lesser extent in peripheral venous plasma obtained from subjects without disturbances in granulopoiesis. It was markedly reduced in subjects with disorders in which normal granulopoiesis was reduced, such as acute leukemia. The deficiency of this inhibitory activity in the marrow plasma of subjects with leukemia and related disorders may be due to a reduction in the cells from which it is derived (e.g. normal neutrophils or stromal cells), although further studies will be required to verify its presence and to determine its source and physiological role in granulopoiesis in man.     

At issue: are traditional societies schizophrenogenic?

Bone marrow is a major granulopoietic organ whose hematopoietic microenvironment is comprised of stromal cells. In the present work, we examined the regulation of in vitro granulopoiesis with an established line of bone marrow stromal cells. In coculture of the progenitor cells on the particular stromal cell lines from bone marrow, large granulocyte (G) colonies consisting of over 200 cells were formed without G-CSF for 5 days. Stromal cells supported development of Gr-1 (granulocyte specific surface marker)-negative progenitors into Gr-1 and myeloperoxidase positive granulocytes. Seventy percent of the large G-colonies were formed on the stromal layers even in the presence of anti-G-CSF antibody, which indicates the G-CSF independent pathway of granulopoiesis. Inhibition of the large G-colony formation by the addition of anti-adhesion molecules, such as very late activation antigen-4 (VLA-4) and CD31 (PECAM-1), suggested the role of cell-to-cell adhesion in stroma-supported granulopoiesis.     

The effects of histamine H2 receptor agonist, antagonist and antineoplastic agent on the in vitro growth of PB CFU-GM from normal individuals and HL-60 leukemia cells

Colony forming unit of granulocytes and macrophages from peripheral blood (PB CFU-GM) represents stem and/or progenitor cells from human blood. In this paper, the effects of histamine H2 receptor agonist 4-methylhistamine (4-MH) and its antagonist ranitidine (Ranit) on the growth of PB CFU-GM cultured in methylcellulose system were studied and their differential effects on normal PB CFU-GM and leukemic HL-60 cells were compared with the effect of the antineoplastic agent cytosine arabinoside (Ara-C). It was found that the histamine H2 receptor agonist 4-MH stimulated the growth of PB CFU-GM when 4-MH was added at the concentrations from 10(-9) mol.L-1 to 10(-6) mol.L-1 among which the dose 10(-8) mol.L-1 exerted most potent stimulating effect (the PB CFU-GM colony numbers was 137.68% +/- 8.20% vs the control, P < 0.01). In contrast, the antagonist Ranit showed inhibitory effect on the growth of PB CFU-GM when at the concentrations 10(-9)-10(-5) mol.L-1 cultured for 14 d in the same methylcellulose system. The inhibition rate was 23.73% +/- 1.16% (10(-9) mol.L-1) and 41.42% +/- 6.75% (10(-6) mol.L-1), respectively. Although both Ranit and Ara-C could inhibit the growth of PB CFU-GM in vitro, Ranit exerted much greater inhibition on HL-60 leukemic cells than on normal PB CFU-GM at the dose of 10(-6) mol.L-1 (100% inhibition for HL-60 and < 50% inhibition for PB CFU-GM). However, the inhibition rate of Ara-C for both HL-60 and PB CFU-GM was 100% at the intensive chemotherapeutic dose of 10(-5) mol.L-1. It would appear that the histamine H2 receptor agonist 4-MH possesses stimulating effect on the growth of PB CFU-GM similar to its effect on CFU-GM from bone marrow as documented before. It is suggested that the histamine H2 receptor antagonist Ranit has, to some extent, potential in the treatment of myeloid leukemia, especially when combined with antineoplastic agent Ara-C at suboptimal doses.     

Late results after resection of benign hepatic tumors: clinical and radiological findings

Our previous work showed the existence of low molecular weight tumor suppressors in human fetal tissues. In this paper, two tumor cell suppressors were isolated and purified from methanol extract of human fetal liver by C18 reversed-phase medium pressure chromatography, gel filtration on Sephadex LH-20, and high-performance liquid chromatography, directed by suppression of growth of HL-60 cells. The structures of the suppressors were identified to be 7-ketocholesterol and 7-beta-hydroxycholesterol. Under the condition of in vitro agar plate culture, 7-ketocholesterol and 7-beta-hydroxycholesterol showed preferentially inhibitory effects on the growth of both human and murine leukemic cell lines including human HL-60 and murine S-180 cells, but less effective on the growth of normal human and murine bone marrow granulocyte-macrophage progenitors (CFU-GM).     

Mechanism-based inhibitors of serine proteinases based on the Gabriel-Colman rearrangement

Neutrophil-derived mediators such as, for example, the serine proteinase elastase, cathepsin G and proteinase 3, play a critical role in inflammatory lung disease. This report describes the design, synthesis and in vitro inhibitory activity of some novel mechanism-based inhibitors of human leukocyte elastase and cathepsin G. The design of the inhibitors is based on the Gabriel-Colman rearrangement. The behavior of the synthesized compounds toward elastase and cathepsin G with respect to inhibitory prowess, mode of interaction, specificity, etc., has been found to be dependent on the recognition and reactivity elements present in each inhibitor.     

Proteinase 3, the major autoantigen of Wegener's granulomatosis, enhances IL-8 production by endothelial cells in vitro

Proteinase 3 is the major target antigen of antineutrophil cytoplasmic autoantibodies (ANCA) in Wegener's granulomatosis and is contained in the azurophilic granules of polymorphonuclear neutrophils, the dominant cell type in vascular lesions during the early stages of systemic vasculitis. This study questioned whether neutrophil lysosomal enzymes, once released at the site of inflammation, are able to potentiate the influx of additional neutrophils by enhancing the production of the chemotactic cytokine interleukin-8 (IL-8) by endothelial cells. Therefore, human umbilical vein endothelial cells in culture were incubated with varying concentrations of highly purified proteinase 3, human neutrophil elastase, and cathepsin G for different time periods. The supernatants were subsequently assessed for IL-8 antigen by using a sandwich ELISA. The presence of both proteinase 3 and elastase resulted in an increased production of IL-8, up to 15.6- and 4.2-fold, respectively, in a dose- and time-dependent fashion. Cathepsin G did not influence IL-8 production. Although the addition of an alpha 1-proteinase inhibitor completely abrogated elastase-mediated IL-8 production, it did not significantly influence the effect of proteinase 3. Both proteinase 3-and elastase-mediated production of IL-8 was inhibited by cycloheximide, indicating de novo synthesis. This was supported by the finding of increased IL-8 mRNA levels in proteinase 3-treated human umbilical vein endothelial cells by using Northern blot analysis. Taken together, the neutrophil lysosomal enzymes proteinase 3 and human neutrophil elastase may contribute to a self-perpetuating process of neutrophil recruitment in acute inflammation by increasing de novo synthesis of IL-8 by endothelial cells. The studies presented here also show that proteinase 3 mediates its effect independently of its enzymatic activity, indicating a hitherto unknown mode of action on endothelial cells.     

Temporal relationship of CDK1 activation and mitotic arrest to cytosolic accumulation of cytochrome C and caspase-3 activity during Taxol-induced apoptosis of human AML HL-60 cells

The antimicrotubule anticancer drug, Taxol, suppresses microtubule dynamics, causes mitotic arrest, and induces caspase-3 cleavage and activity resulting in apoptosis of human AML HL-60 cells. Caspase-3 cleavage is triggered by the mitochondrial release and cytosolic accumulation of the electron transfer protein, cytochrome c (cyt c). Taxol-induced G2/M transition is mediated by p34(cdc-2) (CDK1) which, if prematurely activated, may also trigger apoptosis. In the present studies following S-phase synchronization and release, HL-60 cells with enforced expression of the bcl-xL (HL-60/Bcl-xL) and/or neomycin resistance gene (HL-60/neo) were exposed to Taxol to examine CDK1-related cell-cycle events and the cyt c-triggered molecular cascade of apoptosis. At various time-intervals after Taxol treatment, immunoblot analyses of cyclin B1 and CDK1 levels were performed. In addition, the in vitro histone H1 kinase activity of immunoprecipitated CDK1 and its tyrosine phosphorylation status (by anti-phosphotyrosine immunoblot analysis) were determined. Data presented here show that, while Taxol-induced peak CDK1 kinase activity occurs earlier in HL-60/neo cells, there are no significant differences in cyclin B1 accumulation, tyrosine dephosphorylation of CDK1, and mitotic arrest of Taxol-treated HL-60/neo vs HL-60/Bcl-xL cells. Taxol-induced CDK1 activation and mitosis preceded the cytosolic accumulation (approximately six-fold) of cyt c. The latter event was blocked by Bcl-xL overexpression but not by inhibitors of caspase-3. Although the caspase inhibitors and high Bcl-xL levels inhibited caspase-3 cleavage and activity, they did not significantly affect Taxol-induced CDK1 activation or mitotic arrest. These findings indicate that Bcl-xL overexpression does not affect Taxol-induced CDK1 activity leading to G2/M transition, which temporally precedes the cytosolic cyt c-mediated cleavage and activity of caspase-3 and apoptosis.     

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.     

Spinal tuberculosis in an unusual location

OBJECTIVE: To study the effect of transforming growth factor beta 1 on tumor cells. METHOD: HL-60 cells were transfected with transforming growth factor beta 1 expression vector by electroporation. Stable cell clones resistant to G 418 were obtained after 2-3 week growth with the drug-containing medium. A series of malignant phenotype were compared between transfected and untransfected HL 60 cells. RESULTS: The growth speed of HL-60 cells which overexpressed transforming growth factor beta 1 mRNA decreased significantly. Clonigenecity in soft agarase and tumor formation in athymic mice were lower than those untransfected HL-60 cells. CONCLUSION: Suppression effect of transforming growth factor beta 1 on malignant phenotype in HL-60 cells were demonstrated.     

Interaction of lactoferrin, monocytes, and T lymphocyte subsets in the regulation of steady-state granulopoiesis in vitro

Colony-stimulating activities (CSA) are potent granulopoietic stimulators in vitro. Using clonogenic assay techniques, we analyzed the degree to which mononuclear phagocytes and T lymphocytes cooperate in the positive (production/release of CSA) and feedback (inhibition of CSA production/release) regulation of granulopoiesis. We measured the effect of lactoferrin (a putative feedback regulator of CSA production) on CSA provision in three separate assay systems wherein granulocyte colony growth of marrow cells from 22 normal volunteers was stimulated by (a) endogenous CSA-producing cells in the marrow cells suspension, (b) autologous peripheral blood leukocytes in feeder layers, and (c) medium conditioned by peripheral blood leukocytes. The CSA-producing cell populations in each assay were varied by using cell separation techniques and exposure of isolated T lymphocytes to methylprednisolone or to monoclonal antibodies to surface antigens and complement. We noted that net CSA production increased more than twofold when a small number of unstimulated T lymphocytes were added to monocyte cultures. Lactoferrin's inhibitory effect was also T lymphocyte dependent. The T lymphocytes that interact with monocytes and lactoferrin to inhibit CSA production are similar to those that augment CSA production because their activities are neither genetically restricted not glucocorticoid sensitive, and both populations express HLA-DR (Ia-like) and T3 antigens but not T4 or T8 antigens. These findings are consistent with results of our studies on the mechanism of lactoferrin's inhibitory effect with indicate that mononuclear phagocytes produce both CSA and soluble factors that stimulate T lymphocytes to produce CSA, and that lactoferrin does not suppress monocyte CSA production, but does completely suppress production or release by monocytes of those factors that stimulate T lymphocytes to produce CSA. We conclude that mononuclear phagocytes and a subset of T lymphocytes exhibit important complex interactions in the regulation of granulopoiesis.     

Apoptosis of endothelial cells induced by the neutrophil serine proteases proteinase 3 and elastase

The pathogenesis of vasculitis associated with anti-neutrophil cytoplasmic antibodies is not established. The anti-neutrophil cytoplasmic antibody autoanigens proteinase 3 (PR3) and elastase induce detachment and cytolysis of endothelial cells in vitro. We investigated whether PR3 and elastase trigger endothelial cell apoptosis. Primary bovine pulmonary artery endothelial cells were treated with either PR3, elastase, or myeloperoxidase (MPO) and apoptosis assessed by four different methods. By the cell death detection enzyme-linked immunosorbent assay, DNA fragmentation increased to 208 +/- 84% or 153 +/- 27% of control with 1 micrograms/ml PR3 or elastase at 24 hours. By ultraviolet light microscopy, the percentage of apoptotic cells significantly increased (P < 0.05) with 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase at 6, 12, or 24 hours. Values at the 24-hour time point are 15.3 +/- 6.4% or 25.8 +/- 6.6% for 5 or 10 micrograms/ml PR3 and 13.9 +/- 3.6% or 20.7 +/- 1.8% for 25 or 50 micrograms/ml elastase compared with 2.2 +/- 1.2% for control. Similarly, with flow cytometry, 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase for 6, 12, or 24 hours demonstrated increasing apoptosis in a dose- and time-dependent manner with the highest values achieved at 24 hours (23.4 +/- 4.0% and 35.6% for 5 and 10 micrograms/ml PR3 and 31.8 +/- 4.0% and 47.8% for 25 and 50 micrograms/ml elastase compared with 7.9 +/- 2.2% in control). Typical DNA laddering was apparent from 6 to 24 hours at 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase. Myeloperoxidase did not induce cell apoptosis. Release of PR3 and elastase by activated neutrophils during acute inflammation, including anti-neutrophil cytoplasmic antibody-associated vasculitis, may result in vascular damage by endothelial cell apoptosis.     

Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides

Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.     

Stromal factors support the expansion of the whole hemopoietic spectrum from bone marrow CD34+DR- cells and of some hemopoietic subsets from CD34+ and CD34+DR+ cells

Ex vivo expanded bone marrow CD34+DR- cells could offer a graft devoid of malignant cells able to promptly reconstitute hemopoiesis after transplant. We investigated the specific expansion requirements of this subpopulation compared to the more mature CD34+ and CD34+DR+ populations. The role of stromal factors was assessed by comparing the expansion obtained when the cells were cultured in (1) long-term bone marrow culture (LTBMC) medium conditioned by an irradiated human BM stroma (CM), (2) medium supplemented with 15% FBS (FBSM) and (3) non-conditioned LTBMC medium (LTM) for 21 days. The effect of the addition of G-CSF (G) and/or of MIP-1alpha (M) to a combination of IL-3, SCF, IL-6 and IL-11 (3, S, 6, 11) was analyzed. Compared to CD34+DR- cells, CD34+ and CD34+DR+ cells gave rise to a similar number of viable cells and to a lower progenitor expansion. The expansion potential of CD34+ and CD34+DR+ cells was equivalent in CM and in FBSM except for both the emergence of CD61 + megakaryocytic cells and LTC-IC maintenance which were improved by culture in CM. In contrast, expansion from CD34+DR- cells was enhanced by CM for all the parameters tested. Compared to FBSM, CM induced a higher level of CFU-GM and BFU-E expansion and allowed the emergence of CD61+ cells. HPP-CFC were maintained or expanded in CM but decreased in FBSM. Compared to input, the number of LTC-IC remaining after 21 days of CD34+DR- expansion culture was strongly decreased in FBSM and variably maintained or expanded in CM. Comparison with LTM indicated that stroma conditioning is responsible for this effect. G-CSF significantly improved CFU-GM and HPP-CFC expansion from CD34+DR- cells without being detrimental to the LTC-IC pool. The growth of CD61+ cells was significantly enhanced by G-CSF in CM. Addition of MIP-1alpha had no significant effect either on progenitor expansion or on LTC-IC, regardless of culture medium. We conclude that factors present in stroma- conditioned medium are necessary to support the expansion of the whole spectrum of hematopoietic cells from CD34+DR- cells and to support the expansion of cell subsets from CD34+ and CD34+DR+.     

Purification and identification of a novel and four known serine proteinase inhibitors secreted by human glioblastoma cells

Our previous studies have shown that some human cancer cell lines produce pancreatic trypsinogen, plasminogen, and tissue-type kallikrein. To understand the regulatory mechanism of these proteinases, serine proteinase inhibitors secreted by human glioblastoma cell line T98G were analyzed by gelatin reverse zymography with trypsin. The serum-free conditioned medium of T98G cells showed more than ten trypsin inhibitor bands ranging from 16 to 150 kDa in the reverse zymography. Major trypsin inhibitors were purified by trypsin-affinity chromatography. Analysis of their N-terminal amino acid sequences demonstrated that the purified inhibitors were identical to the secreted forms of amyloid protein precursors (APPs), tissue factor pathway inhibitor (TFPI), placental protein 5 (PP5)/TFPI-2, and secretory leukocyte proteinase inhibitor (SLPI). In addition, a novel 25-kDa trypsin-binding protein, tentatively named p25TI, was identified. p25TI showed weak inhibitory activity against trypsin in reverse zymography as compared with the other inhibitors. The secretion of multiple forms of serine proteinase inhibitors by human cancer cells raises the possibility that they might be involved in the abnormal growth of cancer cells.     

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK1, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30-300 micromol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK1 cells (100 micromol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 micromol/L. Flow cytometric analysis showed that DBCP (1-10 micromol/L) exposure for 24 h caused an accumulation of LLCPK1 cells in the G2/M-phase. In HL-60 cells the accumulation in G2/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK1 cells exposed to 100-500 micromol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G1 cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK1 cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G2/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.     

Cell-water transfer and stability of biological structures (resonance of biological structures)

We demonstrated that adrenomedullin (AM) is produced and secreted from human leukemia cell lines (THP-1 and HL-60) as well as peripheral blood granulocytes, lymphocytes, monocytes and monocyte-derived macrophages. Immunoreactive AM accumulated in the culture media of THP-1 and HL-60 cells increased according to their differentiation into macrophage-like cells. Retinoic acid exerted synergistic effects on AM secretion from THP-1 and HL-60 cells when administered with tumor necrosis factor-alpha, lipopolysaccharide or 12-O-tetradecanoyl phorbol-13-acetate. AM was shown to increase the scavenger receptor activity on THP-1 cells. Thus, monocytes/macrophages should be recognized as sources of AM, and the secreted AM may modulate the function of macrophages.     

Thrombopoietin stimulates myelodysplastic syndrome granulocyte-macrophage and erythroid progenitor proliferation

Thrombopoietin (TPO) has been successfully used to stimulate megakaryocyte progenitor proliferation and platelet production both in vitro and in vivo. We and other investigators have found that TPO also stimulates normal marrow colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) growth. In contrast to its effect on normal marrow precursors, TPO stimulates acute myelogenous leukemia (AML) progenitor proliferation in only 25% of the cases. Because the hematopoietic cells in Myelodysplastic syndrome (MDS) originate from both the normal and leukemic clones, we hypothesized that TPO may be a useful therapeutic agent for MDS. To test this hypothesis, we used fresh marrow samples taken from 14 MDS patients. We found that in the presence of fetal calf serum (FCS) and erythropoietin (EPO) TPO (5 to 40 ng/ml) MDS CFU-GM and BFU-E colony-forming cell proliferation were stimulated in a dose-dependent fashion by up to 103% and 93% respectively. This effect was similar to the stimulation obtained with optimal concentrations of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or interleukin-3 (IL-3). Furthermore, TPO increased the colony-stimulatory effects of G-CSF, GM-CSF, IL-3, and stem cell factor (SCF) on MDS marrow cells. However, depletion of either T lymphocytes or adherent cells abrogated the effect of TPO, suggesting that the effect is not a direct one but is mediated through interaction with cytokines produced by accessory cells. Taken together, our data suggest that the therapeutic role of TPO in the management of MDS warrants further investigation.