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BACKGROUND: Thrombocytopenia is frequently encountered in critically ill patients with the sepsis syndrome, but its mechanisms frequently remain undetermined. Hemophagocytosis has been reported as a cause of thrombocytopenia in various diseases. This prospective study was designed to assess: (1) the incidence of hemophagocytosis in patients suffering from both the sepsis syndrome and unexplained thrombocytopenia, and (2) the circulating level of the macrophage-colony-stimulating factor (M-CSF) according to the presence or absence of hemophagocytosis. METHODS: Fifty consecutive patients diagnosed with both the sepsis syndrome and thrombocytopenia of undetermined origin were studied. Hemophagocytosis was diagnosed based on microscopical examination of sternal bone marrow aspiration by two independent observers. Serum M-CSF concentrations were measured in each patient and compared with levels of a normal population (n = 59). Causes and severity of sepsis syndromes as well as serum M-CSF levels were compared beween patients with and without hemophagocytosis. RESULTS: Hemophagocytosis was diagnosed in 32 patients (64%). Mean serum M-CSF levels were increased in patients when compared with normal subjects (539 [+ or -] 141 versus 208 [+ or -] 82 IU/mL: P < 0.001), and higher in patients with than without hemophagocytosis (580 [+ or -] 145 versus 457 [+ or -] 89 IU/mL: P = 0.01). Multiorgan dysfunction and infection were independent risk factors of hemophagocytosis (odds ratio = 31.3 and 6.8, 95% confidence interval (CI) = 5.4 to 177.6 and 1.0 to 47.4, P < 0.0001 and P = 0.03, respectively). CONCLUSIONS: Hemophagocytosis is a frequent cause of unexplained thrombocytopenia in patients with severe sepsis syndrome. Our results suggest that M-CSF is overproduced in the sepsis syndrome, particularly when hemophagocytosis is present. The role of M-CSF in the initiation and development of hemophagocytosis remains to be determined. Am J Med. 1997;103:114-120. [C] 1997 by Excerpta Medica, Inc.

Proteasome inhibitors are a novel class of compounds that might increase sensitivity to chemotherapy for acute myeloid leukemia (AML). We quantified apoptosis in THP-1 cells incubated with idarubicin (IDA) alone or together with a low concentration of MG132 or bortezomib. The combination of both drugs yielded a percentage of apoptotic cells that was significantly higher than the additive effect of both drugs administered separately (p

The goal of new therapeutic strategies is to adapt the treatment of acute myeloid leukemia (AML) patients to the prognostic and/or to the hematological response.We analyzed in vivo apoptosis induction in blast cells and in lymphocytes of AML patients receiving remission induction treatment.We show, on 12 peripheral blood samples, that the increase of peripheral apoptotic blast cells cannot be considered as the earliest marker of the treatment efficiency, because the significant increase of apoptosis followed the white blood cell and the peripheral blast cell count reductions, probably due to an efficient clearance of circulating apoptotic cells. Furthermore, the study of 65 bone marrow samples at d15 showed that the treatment induced apoptosis of blast cells while sparing the lymphocytes. This apoptosis was evidenced both at the caspase and at the membrane levels using respectively fmk‐VAD‐FITC and Annexin V binding assays. We found that less than 50% of apoptosis, measured with the fmk‐VAD‐FITC, in the d15 residual bone marrow blast cells, correlated with lower disease‐free survival probability.More studies are needed in larger series and earlier during the remission induction treatment to confirm the possible prognostic significance of in vivo apoptosis induction. © 2006 International Society for Analytical Cytology

Objective:We describe a new human bladder carcinoma cell line (DAG–1) established from a resected bladder cancer fragment and maintained in culture for more than 5 years and over 300 passages. Methods and Results:Immunological, biochemical and molecular analysis showed that the DAG–1 cells (62 chromosomes) express the cytokeratines 8, 13, 18 and 20 that confirm their epithelial origin as well as numerous cytokine and cytokine receptor mRNAs. They secrete tissue–type plasminogen activator (t–PA), urokinase–type plasminogen activator (u–PA), plasminogen activator inhibitors (PAI–1 and PAI–2), and express u–PA receptors (u–PAR/CD87) at their surface. DAG–1 cells are resistant to TNFα– and IFNγ–induced apoptosis, two cytokines secreted in the urine of Calmette–Guérin bacillus–treated patients and involved in the tumor regression. Conclusion:The DAG–1 cell line is a useful tool, both in vitro and in vivo, to study the progression of bladder tumors and their mechanisms of resistance to immunotherapy in relation with PAI–2 and antioxidant enzymes.

Objectif : Cette e´tude avait pour but d'e´valuer le ro^le potentiel respectif du M-CSF et des re´activations virales dans la gene`se des he´mophagocytoses survenant lors des de´faillances multivisce´rales. Me´thodes : Vingt-cinq patients (a^ge moyen : 60 ± 16 ans ; Apache II : 23 ± 5) souffrant d'une de´faillance polyvisce´rale associe´e a` une thrombope´nie inexplique´e ont e´te´ e´tudie´s. Pour chaque patient, un mye´logramme, un dosage se´rique du M-CSF et une enque^te virologique (virus Herpe`s) ont e´te´ re´alise´s a` l'admission. Vingt autres patients (a^ge moyen : 57 ± 15 ans ; Apache II : 24 ± 7) avec au moins deux de´faillances d'organe mais sans thrombope´nie ont constitue´ le groupe contro^le. Le M-CSF circulant et la fre´quence de la re´activation virale ont e´te´ compare´s entre les groupes. Re´sultats : Une he´mophagocytose a e´te´ diagnostique´e chez 11/25 patients (44 %). Aucune re´activation de virus Herpe`s n'a e´te´ mise en e´vidence. Les concentrations se´riques de M-CSF e´taient plus e´leve´es en cas d'he´mophagocytose (699 ± 242 vs 438 ± 157 UI·mL ; p &lt; 0,05). La ferritine´mie e´tait aussi augmente´e en cas d'activation macrophagique (3 258 ± 2 807 vs 520 ± 280 mg·L ; p &lt; 0,0001). Au contraire, les concentrations se´riques de M-CSF et la ferritine´mie e´taient comparables chez les patients thrombope´niques sans he´mophagocytose et dans le groupe contro^le. Conclusions : Cette e´tude montre une incidence e´leve´e d'he´mophagocytose chez les patients de re´animation en de´faillance polyvisce´rale. Chez ces patients, les concentrations se´riques de M-CSF e´taient e´leve´es tandis qu'aucune re´activation de virus Herpe`s n'a e´te´ mise en e´vidence.

We have investigated the effect of growth factors, inflammatory and anti-inflammatory cytokines on the macrophage colony-stimulating factor (M-CSF) secretion by cultured human bone marrow stromal cells. Their production of M-CSF cultured in serum-free medium is enhanced in a time-dependent manner in response to tumour necrosis factor (TNF-)α and interleukin (IL-)4 but not to IL-1, IL-3, IL-6, IL-7, IL-10, SCF, granulocyte-macrophage colony-stumulating factor (GM-CSF), G-CSF, bFGF and transforming growth factor (TGF-)β. The co-addition of IL-4 and TNF-α has a greater than additive effect on the secretion of M-CSF suggesting that they act synergistically. The anti-inflammatory molecules IL-10 and TGF-β have no effect on the TNF-α-induced M-CSF synthesis by marrow stromal cells. In conclusion TNF-α and IL-4 are potent stimulators of the M-CSF synthesis by human bone marrow stromal cells, a result of importance regarding the role of M-CSF in the proliferation/differentiation of mononuclear-phagocytic cells and the role of marrow stromal cells as regulators of marrow haematopoiesis.

This study investigates the production of interleukin (IL-)1β by cultured human bone marrow stromal cells. RT-PCR experiments indicate that two-thirds of cultures constitutively express IL-1β mRNA transcripts. Their cell-associated IL-1β levels are elevated after stimulation with tumour necrosis factor (TNF-)α but not with cytokines such as IL-1α, IL-3, IL-4, IL-6, IL-7, IL-10, SCF, G-CSF, M-CSF and TGF-β or lipid mediators such as PGE2, LTB4, LXA4, LXB4, 12-HETE, 15-HETE and PAF. Addition of IL-4, but not IL-10 or TGF-β, reduces the TNF-α-induced cell-associated IL-1β. IL-1β is never detected in bone marrow stromal cell supernatants whatever the stimulant added. In conclusion the pro-inflammatory molecule TNF-α stimulates bone marrow stromal cell-associated IL-1β levels while the anti-inflammatory cytokine IL-4 reduces the TNF-α-induced effect. These results strengthen the key regulatory role of IL-4 on the production of haematopoietic cytokines by human bone marrow stromal cells.

The production of monocytic cytokines by isolated mononuclear cells after stimulation by phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) is generally increased in haemodialysed (HD) patients. We performed whole blood (WB) cultures to evaluate cytokine production by blood cells inside their complex cellular and humoral network.

Lipoxygenase inhibitors reduce the growth of K562 cells (chronic myelogenous human leukaemia blasts) suggesting a role for endogenous lipoxygenase products of arachidonic acid (AA) in their proliferation. The objectives of this work are to investigate the incorporation of AA into K562 cells and to assess the effects of the exogenous addition of AA and lipoxygenase products on their growth. The mechanism of acylation of [3H]-AA indicates that K562 cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]-AA is incorporated unmodified into K562 phospholipids and is linked by an ester bond. Prelabelling-chase experiments indicate a transfer of labelled AA from phosphatidylcholine to phosphatidylethanolamine. The addition of AA and lipoxygenase products of AA (leukotriene B4and C4, lipoxin B4, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE) has no effect on K562 cell proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while K562 cells readily incorporate AA into their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

In a patient with plasma cell leukemia and a kappa type Bence Jones protein in serum and urine, the immunofluorescence study of blood plasma cells showed intracellular gamma and kappa chain determinants. Biosynthesis experiments showed the production of abnormally short heavy chains (45,000 daltons) that assembled with normal sized light chains with a partial block. These abnormal heavy chains were secreted at a slow rate and were degraded after secretion.

Repeated analysis of peripheral blood lymphocytes (PBLs) from a patient with large granular lymphocytosis, neutropenia, and rheumatoid arthritis revealed that approximately 45% of PBLs displayed the following phenotype: CD3+, CD4-, CD8-, CD16+, HNK-1-, WT31-. This population was purified for further analysis by depletion with anti-CD4 and anti-CD8 monoclonal antibodies (MoAbs). Southern blot analysis showed preferential rearrangements of the V gamma 9 genes. Northern blot demonstrated the presence of V gamma 9 mRNA transcripts. With MoAbs directed against either the V gamma 9 peptide (Ti gamma A) or the delta chain of the gamma delta T-cell receptor (TCR delta 1), we further demonstrated that those cell surfaces expressed both V gamma 9 and a delta gene product. In addition, analysis of the gamma gene rearrangements on six clones derived from this population demonstrated a unique rearrangement on a single chromosome, strongly suggesting the monoclonality of this T-cell population. Significant cytotoxic activity against K562, U937 was observed only after an in vitro culture period with interleukin-2 (IL-2), whereas no specific inhibitory effect on autologous bone marrow (BM) CFU-G was noted.

Human bone marrow stromal cells regulate haematopoiesis by releasing cytokines such as leukaemia inhibitory factor (LIF) and interleukin 6 (IL-6). We have investigated the effects of 5 lipid mediators (i.e. 12-HETE, 15-HETE, platelet-activating factor (PAF), LTB4and prostaglandin E2(PGE2)) on their LIF and IL-6 synthesis. 12-HETE, 15-HETE and LTB4(both at 1 μM) stimulate the LIF production by human bone marrow stromal cells grown in 10% fetal calf serum (FCS). In contrast, PAF and PGE2have no effect. 12-HETE (1 μM) enhances LIF synthesis in serum free medium 7.7-fold and stimulates IL-1 induced LIF production. 12-HETE, 15-HETE, PAF and LTB4have no effect on the spontaneous, serum- and cytokine-induced IL-6 synthesis by bone marrow stromal cells. In contrast PGE2significantly stimulates serum-induced IL-6 synthesis. This study reports for the first time that lipid mediators may act on human haematopoiesis by modulating LIF and IL-6 synthesis by bone marrow stromal cells. Particularly, 12-HETE enhances LIF but not IL-6 synthesis. The different regulation of IL-6 and LIF synthesis in response to lipid mediators highlights the complexity of the cytokine regulation into the human bone marrow.

Leukaemia inhibitory factor (LIF) acts on the growth and differentiation of haematopoietic cells. By using a specific enzyme-linked immunosorbent assay for human LIF, we demonstrate that human bone marrow stromal cells produce LIF. LIF synthesis is enhanced in a dose-dependent manner after stimulation with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMAS). LIF production in response to PMA is PKC-dependent since the two PKC inhibitors sphingosine and staurosporine markedly diminished it. Interleukin 1α (IL-1α), IL-1β, IL-3, IL-6, IL-8, tumour necrosis factor (TNF-α) and SCF (both at 10 ng/ml) stimulate LIF production. By contrast macrophage colony-stimulating factor (M-CSF), granulocyte (G)-CSF, GM-CSF, basic fibroblast growth factor (bFGF), platelet-activating factor (PAF), protaglandin E2(PGE2), leukotriene B4(LTB4), and leukotriene C4(LTC4) did not. These results suggest that bone marrow stromal cells might represent a major source for the cytokine-regulated local production of LIF inside human bone marrow.

Myelofibrosis with myeloid metaplasia (MMM) is a myeloproliferative disorder characterized by clonal expansion of hematopoiesis and marrow fibrosis. Previous results from our group have shown an increased production of two potent fibrogenic factors also involved in the regulation of primitive hematopoietic cells, namely transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF), in patients with MMM. It is likely to assume that the myeloproliferation characteristic of this disease may result from an abnormal proliferation of CD34+ hematopoietic progenitors. Thus, we were particularly concerned in studying the gene and protein expression of these cytokines and their receptors in CD34+ progenitors purified from the peripheral blood of MMM patients by using semiquantitative reverse transcriptase-polymerase chain reaction and immunolabeling methods. Our data showed that the expression of TGF-beta1 is not altered in patients CD34+ cells; in contrast, the expression of TGF-beta type II receptor is significantly decreased in such cells, as compared with CD34+ cells from healthy subjects. Regarding bFGF, the very low expression of the cytokine and its type I and II receptors detected in normal CD34+ cells contrasts with that observed in patients' CD34+ cells, which is significantly higher. Our results might be a clue for a better understanding of the mechanism(s) involved in the dysregulation of hematopoiesis in MMM. Actually, the increased expression of bFGF and its receptors associated with the reduction of the TGF-beta binding receptor in CD34+ progenitors from MMM patients might facilitate the expansion of hematopoietic progenitors, not only by stimulating their growth and/or survival, but also by overcoming negative regulatory signals.

Circulating erythroid progenitors from 14 patients with acute lymphoblastic leukemia (ALL) and from 8 healthy subjects were studied in culture to determine the frequence and size of CFU-E- and BFU-E-derived colonies. Cells were cultured in a plasma clot system, and hemoglobinized colonies identified by diaminobenzidine reaction. The numbers of CFU-E and BFU-E per milliliter of peripheral blood were greatly increased in 10 patients when compared to controls. In 13 patients, the size distribution of BFU-E-derived colonies, analyzed by counting the number of subunits in each colony, was also found to differ significantly from controls, with a large excess of small colonies and a low percentage or a total lack of large colonies. This abnormal BFU-E size distribution was partially corrected, in the 5 patients tested, by the addition to the culture medium of 10% phytohemaglutinin-leukocyte-conditioned medium (PHA-LCM). Bone marrow crowding out of the normal progenitors, as well as disturbances in the cellular interactions involved in their normal development, most likely explain these results and these factors could be implicated in the frequent pancytopenia of ALL.

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation present in the human bone marrow. Freshly isolated human mononuclear bone marrow cells and marrow stromal cell cultures produced PAF under calcium ionophore (2µM) and LPS (10 µg/ml) stimulation. By contrast M-CSF (1000 U/ml), GM-CSF (100 ng/ml), IL1. IL3, IL6 and stem cell factor (10 ng/ml) did not stimulate PAF production. Marrow stromal cells produced 50-fold more PAF than freshly isolated mononuclear marrow cells, suggesting that stromal cells might be the major source of the human marrow-derived PAF. Mononuclear marrow cells and stromal cell cultures metabolized PAF with 1-alkyl-2-acyl-glycerophosphocholine as the major metabolic product. PMSF and p-BPB decreased the catabolism of PAF by freshly isolated marrow cells, but not by stromal cell cultures. While stromal cells rather than haematopoietic progenitors might be a major source of the human bone-marrow-derived PAF, both cell types metabolize it, suggesting their putative role in the regulation of PAF concentration in the human bone marrow.

Macrophage colony stimulating factor (CSF-1) is one of several cytokines that control the differentiation, survival, and proliferation of monocytes and macrophages. A set of 11 human T-cell clones, chosen for their phenotypic diversity, were tested for their ability to express CSF-1 mRNA. After 5 hours of stimulation with phorbol myristate acetate (PMA) + calcium ionophore (Cal), all T-cell clones expressed a major 4-kb transcript, a less abundant 2-kb transcript, and several other minor species. This pattern of expression is typical for CSF-1 mRNAs. Furthermore, of the two alloreactive T-cell clones analyzed, only one showed a definitive message for CSF-1 on specific antigenic stimulation, but with delayed kinetics and less efficiency. Both conditions of stimulation induced the release of CSF-1 protein by T cells in the culture medium. Together, these findings demonstrate for the first time that normal T cells are able to produce CSF-1, previous reports being limited to two cases of tumoral cells of the T-cell lineage.

The murine myeloproliferative syndrome induced by the myeloproliferative sarcoma virus (MPSV) has numerous similarities to human primary myelofibrosis. We have shown that medium conditioned by spleen cells of MPSV-infected mice has the capacity to support the growth of primitive blast cell colonies. The detection of this activity associated with MPSV infection stimulated us to characterize the hematopoietins responsible for this activity. Northern blot analysis showed a large increase, or induction, of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage- CSF (CSF-1), and granulocyte-CSF (G-CSF) transcripts in the hematopoietic organs of MPSV-infected mice; however, no IL-3 transcript could be detected in either MPSV-infected or normal mice. Significant levels of IL-1 alpha, IL-6, G-CSF, and CSF-1 bioactivities were found in the serum of MPSV-infected mice, but not in controls. Additionally, analysis of medium conditioned by spleen cells of MPSV-infected mice showed the presence of tumor necrosis factor alpha bioactivity. The increased production of cytokines that are able to stimulate pluripotent hematopoietic stem cells corroborates the hypothesis of a possible involvement of hematopoietic growth factors in the development of some myeloproliferative disorders.

Myeloproliferative disease (MPD) is heterogeneous in phenotypic expression and may display features consistent with expansion and activation of the monocyte/macrophage population during its course. The role of colony-stimulating factor-1 (CSF-1) in the pathophysiology of MPD was investigated by measuring circulating CSF-1 levels and examining their relationship to disease phenotype. Serum CSF-1 concentrations, measured by radioimmunoassay, were elevated in all MPD phenotypes. CSF-1 levels differed significantly between groups of patients with essential thrombocythemia, polycythemia vera, and postpolycythemic or agnogenic myeloid metaplasia (in ascending order). CSF-1 serum levels were positively correlated with spleen size and the degree of peripheral bone marrow extension, determined by scintigraphy using a macrophage-seeking isotope. There was no correlation between CSF-1 concentration and circulating levels of erythrocytes, neutrophils or platelets, or the presence of bone marrow fibrosis. Elevated serum CSF-1 levels appear to be associated with an expanded monocyte/macrophage population in MPD. In view of the known cooperativity between CSF-1 and other growth factors in regulating hematopoiesis, the finding of increased serum CSF-1 concentrations and its association with myeloid metaplasia and bone marrow extension may indicate a pathophysiologic role for CSF-1 in determining the phenotypic expression of MPD.

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder characterized by clonal expansion and marrow fibrosis. Although marrow fibrosis appears to be a reactive process, it substantially contributes to impaired haemopoiesis. During the last few years the implication of megakaryocyte-derived growth factors in its pathogenesis has been documented.
We previously reported increased expression of TGF-beta in patients with idiopathic myelofibrosis. In the present study we show that circulating megakaryocytic cells from such patients expressed high levels of basic fibroblast growth factor (bFGF). An increased expression of bFGF was also detected in patients' platelets. Under culture conditions, bFGF present in megakaryocytic cells was not exported into the medium, consistent with the fact that bFGF is devoid of a secretion peptide signal. Interestingly, this lack of bFGF secretion was observed in all patients but one, who was in an accelerated phase of the disease and presented an important percentage of circulating megakaryoblasts.

In a patient with plasma cell leukemia and a ktype Bence Jones protein in serum and urine, the immunofluorescence study of blood plasma cells showed intracellular yand kchain determinants. Biosynthesis experiments showed the production of abnormally short heavy chains (45,000 daltons) that assembled with normal sized light chains with a partial block. These abnormal heavy chains were secreted at a slow rate and were degraded after secretion.

Platelet-activating factor (PAF) is a phospholipid compound with major immunoregulatory activities. The present study shows that human bone marrow contains 576 ± 39 pg PAF/ml (n = 35). Bone marrow-derived PAF exhibits the same biophysical and biological properties that synthetic PAF. PAF concentrations in bone marrow are correlated with the granulocyte (r = 0.4, P = 0.02) but not with the lymphocyte (r = 0.24, P = 0.17) and the monocyte (r = 0.12, P = 0.48) counts. In bone marrow PAF is inactivated by a plasma PAF acetylhydrolase activity (48.0 ± 2.3 nmol/min per ml, n = 34). Experiments with [3H]PAF indicate that human bone marrow cells actively metabolize this potent molecule by the deacetylation-transacylation pathway. Results of this investigation indicate the permanent presence of significant amounts of PAF in bone marrow suggesting its putative involvement in the processes of bone marrow cell proliferation and maturation.

The expression of the low-affinity NGF receptor (p75) and the trkA proto-oncogene product was analyzed in a series of human hematopoietic cell lines at protein and RNA levels. We did not detect any form of NGF receptor in cell lines displaying a myelomonocytic phenotype (HL60 and U937). In contrast, cells displaying a more immature erythroleukemic phenotype (TF1 and K562) expressed TrkA in the absence of detectable p75. Scatchard analysis showed a single high-affinity site for NGF (kd = 10(-10) mol/L), with a copy number ranging from 300 to 3,000 sites per cell depending on the studied cell line. In addition, NGF induced autophosphorylation of TrkA and could substitute for granulocyte- monocyte colony-stimulating factor to trigger the proliferation of the TF1 cell line, with a half-maximal signal observed at 50 pmol/L, indicating that p75 is not required for DNA synthesis in this cell line. The physiologic relevance of NGF in early hematopoiesis was confirmed by showing that 12% to 15% of progenitor blood cells from mice treated with 5-fluorouracil expressed TrkA and that these cells could be induced to proliferate and differentiate in response to NGF in association with macrophage colony-stimulating factor. Our study demonstrates for the first time that trkA proto-oncogene expression and activation is not restricted to the nervous system, but is also an important element in early hematopoiesis.

Ciliary neurotrophic factor (CNTF) associates with an alpha subunit (CNTFRalpha) of the receptor complex to initiate signal transduction by facilitating heterodimerization of the gp130 transducing protein and the leukemia inhibitory factor receptor (LIFR) beta. CNTFRalpha is anchored to the membrane by a glycosylphosphatidylinositol linkage; however, a soluble form of the alpha subunit can still bind CNTF to recruit the signal transducing components of the receptor complex. In the present study we show that alanine substitution for residues Thr268 and Asp269 of the CNTFRalpha subunit results in a mutated receptor subunit (R3), which can bind CNTF with an affinity similar to that of the wild type CNTFRalpha but, when expressed as a soluble receptor subunit, lowers the binding of CNTF to its tripartite receptor. In addition, CNTFR3alpha inhibits the proliferation of the TF1 hematopoietic cell line triggered by CNTF plus soluble wild type CNTFRalpha but not by IL-6 or oncostatin M. Similarly, CNTFR3alpha specifically antagonizes the induction of gp130 and LIFRbeta tyrosine phosphorylation observed in response to CNTF and wild type soluble CNTFRalpha in the HepG2 hepatoma cell line, as well as the subsequent events leading to haptoglobin synthesis. Positions 268 and 269 of CNTFRalpha appear to be critical for its interaction with gp130 and LIFRbeta, whereby alanine substitution of the residues at these positions results in antagonism of the CNTF-induced response.

By using a specific enzyme-linked immunosorbent assay, the authors demonstrated that human bone marrow stromal cells produce IL-6 and IL-8. Their synthesis is enhanced in a dose-dependent manner after stimulation with lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). Interleukin 6 (IL-6) and IL-8 production in response to PMA were markedly diminished by the PKC inhibitor staurosporine. IL-6 (10 ng/ml) stimulated IL-8 production with 0% and 10% fetal calf serum (FCS) in the culture medium. In similar conditions, IL-8 (10 ng/ml) enhanced IL-6 production. IL-1α, IL-1β, IL-3, tumour necrosis factor α (TNF-α), Stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (at 10 ng.ml) stimulated IL-6 and IL-8 production in 0% and 10% FCS. G-CSF stimulated and IL-4 inhibited IL-8 production in 10% FCS. IL-2, IL-4 and bFGF stimulated IL-6 production in 0% FCS. These results suggest that bone marrow stromal cells might represent a major source for the cytokine-regulated local production of IL-6 and IL-8 inside human bone marrow.

Macrophage colony-stimulating factor (M-CSF), initially described as a growth factor of the mononuclear phagocytic lineage, also participates in immunological and inflammatory reactions, bone metabolism and pregnancy. All its biological activities are mediated by a tyrosine kinase receptor (M-CSF-R) that is encoded by the c-fmsprotooncogene. After a brief overview on the synthesis, structure, metabolism and signalling of M-CSF and its receptor, we present with more details the major in vitro and/or in vivo biological activities of this cytokine. A particular attention has been devoted to the results suggesting that the various M-CSD isoforms (i.e. soluble, cell-associated and matrix anchored forms) play different specific roles on target cells bearing M-CSF-R at their surface. Infectious, inflammatory and neoplastic disease in which M-CSF is involved and could participate to their physiopathology are mentioned. Finally, the role that the various isoforms of M-CSF could play in the regulation of “physiological and pathological cytokine networks” during inflammation and immune responses is discussed.

Six human T lymphomas and an NK-like cell line were tested for their ability to produce HILDA, one of the two human growth promoting activities for the DA1.a cells. Among them, the HSB2 cell line turned out to be the only one secreting significant HILDA activity (200–400 units/ml) after activation with 50 nMphorbol myristate acetate. Subclones of the HSB2 cell line were obtained by limiting dilution experiments. One of them (2B3) was found to secrete 1,000-5,000 units/ml of HILDA after phorbol myristate acetate activation in the presence of 10% fetal calf serum and 200–500 units/ml in serum-free conditions. 2B3-HILDA was purified from serum-free conditioned medium by a four-step procedure including fast flow cationic exchange at pH 6, concanavalin A chromatography, reverse-phase high performance liquid chromatography and gel-filtration high performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified radiolabeled cytokine revealed a single band of Mr43,000, which co-electrophoresed with the biological activity. The NH2-terminal amino acid sequence of the first residues of the protein were determined and found to be similar to the equivalent residues deduced from the molecularly cloned cytokine. Isoelectric point determination revealed some charge heterogeneity of HILDA, which focused to pH 8.5–9 after neuraminidase treatment. Carbohydrate content of the cytokine was studied by deglycosylation experiments which showed that the O-linked oligosaccharides represented 2,000–3,000 and that the N-linked sugars account for half of the apparent molecular weight of HILDA.

Haematopoietic organs of rats were examined for the presence of platelet-activating factor (PAF) and acetylhydrolase before and after treatment with 5-fluoro-uracil (5-FU) (200 mg/kg) a chemotherapeutic compound with apoptotic effects. PAF was reported in thymus, spleen and femoral bone marrow of rats with or without 5-FU. Although acetylhydrolase activity in organs was not affected by 5-FU treatment, elevated levels of PAF were observed in thymus and spleen. For the first time PAF is reported in haematopoietic organs of rats, strengthening in vitro data suggesting its role in the apoptotic processes in thymus, in the modulation of the immune response, and in the regulation of haematopoiesis.

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) is a physiological inhibitor of the proliferation of haematopoietic stem cells. In 12 healthy volunteers treated with the angiotensin-converting enzyme (ACE) inhibitor enalapril (20 mg day-1 for 15 days), we studied plasma and urinary AcSDKP levels, the in vitro degradation of AcSDKP by plasma ACE and the numbers of circulating haematopoietic progenitors (granulocyte-monocytic colony forming unit: CFU-GM; burst forming unit-erythroid: BFU-E; and mixed colony forming unit: CFU-mixed). During treatment, plasma and urinary AcSDKP concentrations increased 2- to 5-fold, degradation of AcSDKP was reduced, and CFU-mixed significantly increased by 100% while BFU-E and CFU-GM significantly decreased by 16% and 26%, respectively. These results indicate that ACE inhibitors may be of value during chemotherapy or radiotherapy, warranting further study.

PAF is a potent inflammatory compound known to stimulate the release of various cytokines involved in rheumatic diseases. Elevated blood PAF levels are reported in these patients. We report that serum PAF acetylhydrolase activity (AHA) levels are decreased in patients with rheumatoid arthritis or osteoarthritis as compared to healthy controls. Serum and synovial fluid AHA levels were correlated in these patients. The present study suggests the potential role of AHA in controling systemic and/or local PAF levels in patients with rheumatic diseases.

This study reports that TNF-α is a potent mitogen for human bone marrow sternal cells in vitro (assessed by [3H]-thymidine incorporation into DNA and cell counts). In contrast, cytokines such as IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, LIF, SCF, M-CSF, G-CSF and GM-CSF had no effect. The effect of TNF-α on the growth of human bone marrow stromal cells could be of importance during inflammatory processes which take place in the marrow, for example marrow fibrosis.