Search

Human vascular endothelial cells (HUVECs), which do not display the lipopolysaccharide (LPS) receptor CD14, were examined for protein tyrosine phosphorylation after LPS stimulation in the presence and absence of soluble CD14 (sCD14). By phosphotyrosine Western blotting and immunocomplex kinase assays we show that LPS was capable of inducing in these cells rapid protein tyrosine phosphorylation and kinase activation of two members of the mitogen-activated protein kinase (MAPK) family erk-1 and the newly discovered p38, requiring the presence of sCD14. LPS-induced tyrosine phosphorylation of MAPK was associated with increased transcript- and surface protein expression of intracellular adhesion molecule-1 by HUVECs. MAPK phosphorylation and activation was induced by LPS in concentrations as little as 30 ng/mL and as early as 15 minutes after stimulation. Furthermore, tyrosine kinase inhibitors such as Genistein partially inhibited this effect. These results show that LPS triggers similar signaling events in both CD14+ myelo-monocytic cells and cells lacking the putative LPS-receptor CD14, suggesting the presence of a common, yet unidentified element in LPS-signaling in both cell types.

The CD30 surface molecule is a recently identified member of the tumor necrosis factor/nerve growth factor receptor superfamily. Within the cytoplasmic signal transducing domain, CD30 shares no significant homology to other members of this family. Signaling events engaged via CD30 are still unknown. We here identify the NF-kappabeta transcription factor as a target of the CD30-induced signal pathway in Hodgkin's disease (HD) cells. Exposure of HD cells to CD30 ligand induces release of interleukin-6 (IL-6) that can be duplicated by cross-linking HD- cells to an agonistic anti-CD30 specific monoclonal antibody (alphaCD30), but not by cross-linking to an isotype-identical irrelevant monoclonal antibody. Cross-linking of HD cells to alphaCD30 leads to enhanced accumulation of IL-6 mRNA in a time-dependent fashion resulting from transcriptional activation of the IL-6 promoter. Transient transfection assays using a series of deleted IL-6 promoter constructs linked to the human growth hormone gene as a reporter gene furthermore indicate that transcriptional activation of the IL-6 promoter requires the presence of an intact NF-kappabeta binding site. In addition, introduction of an NF-kappabeta binding site appeared to be sufficient to confer inducibility of an heterologous promoter on activation of CD30 in HD cells. Cross-linking of CD30 promotes rapid and transient binding activity of nuclear proteins to the NF-kappabeta recognition site of the IL-6 promoter. Supershift experiments using a series of monoclonal antibodies recognizing distinct members of the NF- kappaBeta transcription factor family furthermore indicate that in CD30 cross-linked HD cells p50, p65/Rel-A, and Rel-B are present, whereas the c-rel protein is not.

Expression of the interleukin-2 receptor alpha-(IL-2Ralpha-), IL-2Rbeta- , and the recently identified IL-2Rgamma-chain was examined on a wide range of cells of myeloid origin including neutrophils, monocytes, normal bone marrow-derived myeloid progenitors enriched for CD34+ cells, bone marrow blasts obtained from acute myelogenous leukemia (AML) patients, and permanent myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction and surface membrane analysis using receptor chain-specific monoclonal antibodies and flow cytometry. Expression of the p75 IL-2Rbeta- and the p64 IL-2Rgamma-chain was a common finding in most of the myeloid cell samples investigated, whereas IL-2Ralpha-chain was less frequently expressed. Although the high-affinity IL-2R form (ie, the alpha+, beta+, gamma+ IL-2R form) was detectable in a small minority of primary AML samples as well as the KG- 1 cell line and IL-2 binding to these cells was sufficient to initiate signal transduction as evidenced by an increase in overall protein tyrosine phosphorylation and more specifically in tyrosine phosphorylation of the Janus kinase (JAK) 3, in none of these cell types did exposure to IL-2 affect cell growth kinetics. These results suggest that, in myeloid cells, the IL-2R may not stimulate mitogenic responses or that its components may be expressed in a combinational association with receptors for other cytokines and that IL-2Rgamma may play a regulatory role in normal and malignant myelopoiesis possibly independent from IL-2. Because recent studies by others have indicated that the IL-2Rgamma- chain may be shared by the IL-4R, the IL-7R, and most likely the IL-9R, expression of mRNA of these receptor types was also investigated in these cell samples. Surprisingly, in a substantial part of the myeloid lineage cells examined, an IL-2Rgamma+, IL-4R-, IL7R- configuration was noted that was, however, frequently associated with expression of IL-9R. Sharing of IL-9R/IL-2R components was furthermore suggested by inhibition of 125I-IL-2 binding to primary AML cells with excess of unlabeled IL-9.

Mononuclear cells (MNCs) containing peripheral blood stem cells (PBSCs) were obtained from solid-tumor patients undergoing mobilizing chemotherapy followed by granulocyte colony-stimulating factor for PBSC transplantation-supported dose-intensified anticancer chemotherapy and were transplanted into unconditioned “nonleaky” young severe combined immunodeficient mice. Multilineage engraftment was shown by flow cytometry and immunocytochemistry using monoclonal antibodies to various human cell surface antigens as well as identification of human immunoglobulin in murine sera. Within a dose range of MNCs suitable for transplantation (10 to 36 x 10(6) cells/graft) the number of CD34+ cells injected (optimal at > 0.7 x 10(6)/graft) determined the yield of human cells produced in recipient animals. Engraftment of hu PBSC preparations resulted in prolonged generation of physiologic levels of human cytokines including interleukin-3 (IL-3), IL-6, and granulocyte- macrophage colony-stimulating factor, which were detectable in the murine blood over a period of at least 4 months. In vivo survival of immature human progenitor cells was preserved even 9 months after transplantation. Because human IL-3 is known to stimulate early hematopoiesis, a rat fibroblast cell line was stably transfected with a retroviral vector carrying the human IL-3 gene and cotransplanted subcutaneously as additional source of growth factor. Cotransplants of this cell line producing sustained in vivo levels of circulating human IL-3 for at least 12 weeks significantly accelerated the process of engraftment of huPBSC and spurred the spread of mature human cells to the murine spleen, liver, thymus, and peripheral blood. Cotransplants of allogeneic human bone marrow stromal cells derived from long-term cultures resulted in a comparable--though less prominent--support of engraftment.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth factor-stimulated hematopoiesis in normal and leukemic conditions. Using the factor-dependent myelogenous leukemia cell lines GF-D8 and Mo7, we show that TGF-beta interferes with stem cell factor (SCF)-induced proliferation by downmodulating c-jun gene expression. The ability of SCF to induce accumulation of c-jun transcripts was abolished when TGF-beta was present in culture. Transcriptional nuclear run-on assays indicated that TGF-beta relieved the capacity of SCF to enhance the transcriptional rate of the c-jun gene. Deletion analysis of the c-jun promoter furthermore showed that SCF was activating the c- jun promoter via the NF-jun transcription factor. Gel mobility shift assays showed that SCF increased the binding activity of NF-jun to its recognition site within 5 to 15 minutes. Binding activity peaked at 1 hour after exposure to SCF and declined to starting levels within 4 hours. The ability of SCF to enhance NF-jun binding activity was also dose-dependent in the range of 5 to 100 ng/mL. Exposure of GF-D8 and Mo7 cells to TGF-beta before the addition of SCF antagonized SCF- induced NF-jun binding. Moreover, whereas SCF was capable of functionally activating a heterologous promoter containing the NF-jun binding site, pretreatment of GF-D8 cells with TGF-beta abolished transcriptional activation of this heterologous promoter. These findings indicate that SCF-mediated activation of c-jun via NF-jun is crucial for the SCF-inducible proliferative response and is inhibited by TGF-beta. In additional experiments, the antisense technique was used. Treatment of GF-D8 and Mo7 cells with an antisense oligodeoxyribonucleotide directed against the translation initiation site of c-jun abolished the capacity of SCF to induce a proliferative response, whereas sense and nonsense oligomers had no effect. Taken together, our data indicate that the counteracting modulation of the binding activity of NF-jun by SCF and TGF-beta regulates the expression of the c-jun gene and thereby the proliferative state of the GF-D8 and Mo7 target.

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have previously been reported to induce rapid phosphorylation of the mitogen-activated protein (MAP) kinase. However, little is known about signaling events initiated by both hematopoietins that occur downstream of the MAP kinase. MAP kinase has been shown to phosphorylate the AP-1 transcription factor and also to activate two kinases designated insulin-stimulated protein kinase-1 and MAP kinase- activated protein (MAP-KAP) kinase 2. We show here that IL-3 and GM-CSF induce MAPKAP kinase 2 activity in the human megakaryoblastic leukemia cell line MO7 and phosphorylate the human small heat shock protein Hsp 27 on serine residues in vitro. GM-CSF also induced Hsp 27 phosphorylation in neutrophils in a range similar to that observed in MO7 cells, suggesting that MAPKAP kinase 2-mediated Hsp 27 activation occurs independently of proliferation. Hsp 27 phosphorylation was dose- dependent, occurred as early as 5 minutes after factor exposure, and was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A. Furthermore, the protein phosphatase A2 abolished IL-3- and GM-CSF-induced serine phosphorylation of Hsp 27. Taken together, our findings indicate that tyrosine phosphorylation of MAP kinase is a prerequisite for serine phosphorylation of Hsp 27, which is mediated by MAPKAP kinase 2. Hsp 27 has shown activation-dependent translocation from the cytosolic to the nuclear region and has been linked to the cellular stress response. However, its precise function is largely unknown. Our data identify Hsp 27 as a target of the IL-3/GM-CSF stimulation pathway that involves MAP kinase and MAPKAP kinase 2. In addition, our results indicate that Hsp 27 may be target of phosphorylation events not only in the stress response but also in unstressed cells responding to cytokine stimulation.

In the absence of appropriate stimuli, polymorphonuclear neutrophils (PMN) undergo programmed cell death (PCD), also termed apoptosis. We show that granulocyte-macrophage colony-stimulating factor (GM-CSF), but not the chemotactic factors formyl-methionyl-leucyl-phenylalanine (FMLP), recombinant human (rh) C5a, transforming growth factor (TGF)- beta, and interleukin-8 (IL-8), or other cytokines including IL-3, IL-4, IL-6, and G-CSF, maintains viability of PMN in culture by preventing these cells from undergoing PCD. Prevention from PCD by GM-CSF was associated with induction of RNA and protein synthesis in PMN. Inhibition of RNA and protein synthesis by actinomycin-D and cycloheximide impeded the protection of apoptosis by GM-CSF. Similarly, neutralization of GM-CSF biologic activity by a specific antiserum abrogated GM-CSF-mediated inhibition of PCD.

Recent studies have indicated that the leukemia inhibitory factor (LIF) induces secretion of interleukin-6 (IL-6) in myeloid cells. We here show that synthesis of IL-6 by human mononuclear phagocytes exposed to recombinant human (rh) LIF is preceded by an increase of IL-6 transcript levels as a result of transcriptional activation of the IL-6 gene. Analysis of deleted fragments of the IL-6 promoter indicated that transcriptional activation of the IL-6 promoter was associated with enhanced binding activity of the transcription factor nuclear factor (NF)-kappa B. Binding of activation protein (AP)-1 and NF-IL-6, also known to transcriptionally activate the IL-6 promoter, was not inducible by LIF. Furthermore, introduction of the NF-kappa B sequence into a heterologous promoter construct, but not of AP-1- and NF-IL-6- binding sequences, conferred inducibility by LIF to this promoter. Deletion of the NF-kappa B binding site in the IL-6 promoter was associated with loss of inducibility by LIF, lending further support for the notion that the NF-kappa B binding site is crucial for LIF- mediated induction of the IL-6 promoter. Taken together, our results show that rhLIF induces IL-6 gene expression in mononuclear phagocytes through transcriptional gene activation involving NF-kappa B.

The c-kit proto-oncogene encodes a transmembrane glycoprotein identical to the receptor for the recently cloned stem cell factor (SCF). The present study examines constitutive synthesis of transcripts in primary acute myelogenous leukemia (AML) blasts and the effects of recombinant human tumor necrosis factor (TNF)-alpha on c-kit mRNA expression in these cells. The c-kit transcripts were detectable at low levels in 10 of 10 different AML samples investigated. TNF treatment of AML cells was associated with enhanced c-kit mRNA expression in all specimens. Nuclear run-on transcription assays indicated that the c-kit gene was transcriptionally active in all leukemias examined and the rate of transcription was unaffected by exposure to TNF, suggesting posttranscriptional control mechanisms of c-kit mRNA accumulation. In the absence of TNF, the half-life of c-kit transcripts was 2 to 3 hours, while in TNF-treated AML cells, c-kit half-life was found to be 5 to 9 hours. Inhibition of protein synthesis reduced TNF-induced c-kit mRNA expression by Northern blot analysis, but did not affect the rate of c-kit gene transcription. In the presence of inhibition of protein synthesis, the half-life of c-kit transcripts in TNF-induced leukemia cells decreased to 2 to 4 hours. These findings indicate that levels of c-kit mRNA are controlled by a labile protein that is involved in TNF- mediated stabilization of c-kit transcripts. The effects of TNF-alpha also extended to the protein level in that TNF-alpha treatment of primary AMLs was associated with enhanced surface expression of the SCF receptor by some of these cells. While exogenous SCF induced clonogenic growth of all primary AML samples investigated, TNF-alpha failed to stimulate leukemic cells to proliferate. However, the combination of SCF and TNF-alpha resulted in synergistic growth stimulation in seven of nine different AML specimens investigated. The finding of transmodulation of the SCF receptor through posttranscriptional modifications might further contribute to our understanding of the synergistic interplay of TNF-alpha and SCF.

In a phase I study, the sequentially administered combination of recombinant human interleukin-3 (rhIL-3) and rhGM-CSF was compared with treatment with rhIL-3 alone in 15 patients with advanced tumors but normal hematopoiesis. Patients were initially treated with rhIL-3 for 15 days. After a treatment-free interval, the patients received a second 5-day cycle of rhIL-3 at an identical dosage, immediately followed by a 10-day course of rhGM-CSF, to assess the toxicity and biologic effects of this sequential rhIL-3/rhGM-CSF combination. rhIL-3 doses tested were 125, and 250 micrograms/m2, whereas rhGM-CSF was administered at a daily dosage of 250 micrograms/m2. Both cytokines were administered by subcutaneous (SC) bolus injection. rhIL-3/rhGM-CSF treatment was more effective than rhIL-3 but equally effective to each other in increasing peripheral leukocyte counts, especially neutrophilic and eosinophilic granulocyte counts. In contrast, both modes of cytokine therapy raised the platelet counts to the same degree. rhIL-3/GM-CSF treatment was more effective than rhIL-3 in increasing the number of circulating hematopoietic progenitor cells BFU- E and CFU-GM. High-dose rhIL-3, but not low-dose rhIL-3, was as effective as the rhIL-3/rhGM-CSF combinations in increasing the number of circulating CFU-GEMM. The increase in absolute neutrophil counts correlated with the increase in the number of circulating CFU-GM. Side effects, mainly fever, headache, flushing, and sweating, were generally mild, but in two patients the occurrence of chills, rigor, and dyspnea after initiation of GM-CSF treatment necessitated dose reduction and discontinuation, respectively. These results indicate that sequential treatment with rhIL-3 and rhGM-CSF is as effective as single-factor treatment with rhIL-3 in stimulating platelet counts, whereas the effect of combination therapy on neutrophil counts and circulating progenitor cells is superior.

In this report we show that recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and rh macrophage (M)-CSF induce accumulation of interleukin-1 beta (IL-1 beta) mRNA in blood-derived mononuclear phagocytes (MNP). GM-CSF and M-CSF treatment of MNP is also associated with IL-1 beta secretion. Regulation of GM- and M-CSF- induced IL-1 beta mRNA expression involves transcriptional and posttranscriptional mechanisms. However, the action of IL-3 on synthesis of IL-1 beta mRNA differs from that of other CSFs: While GM- CSF and M-CSF induce binding activity of the nuclear factor (NF) kappa B, IL-3 treatment of MNP has no profound effect on NF kappa B binding to DNA. Moreover, IL-3 decreases the transcription rate of the IL-1 beta gene and has only little effect on stability of IL-1 beta mRNA, which is increased by GM- and M-CSF. However, IL-3 enhances M-CSF- induced accumulation of IL-1 beta mRNA by unknown posttranscriptional means that may relate to an increased expression of M-CSF receptor (ie, c-fms) mRNA, detectable in mononuclear phagocytes on exposure to IL-3.

The cytogenetic hallmark of chronic myelogenous leukemia (CML) is the Philadelphia chromosome (Ph1), which reflects a chromosomal translocation t(9;22) and a rearrangement of the ABL and bcr genes. This marker is found in all cells arising from the same malignant precursor cell and can be detected in CML cells of the myeloid, monocytic, erythroid, and B-lymphocyte lineage. It is, however, controversial as to whether T lymphocytes of CML patients carry this gene rearrangement. An answer to this question would clarify whether the translocation in CML occurs in a pluripotent hematopoietic stem cell or in a precursor cell already committed to certain lineages, but not the T-cell lineage. To address this question, we established T-cell clones from peripheral venous blood cells of four patients with CML and screened these clones for bcr-abl fusion transcripts by means of polymerase chain reaction and Southern blot analysis. In four T-cell clones of three of these patients, the bcr-abl transcript could be detected. None of 12 T-cell clones of the fourth patient disclosed detectable bcr-abl amplification product. Both CD4+ as well as CD8+ clones displayed fused bcr-abl sequences. These data imply that in CML some but not all T lymphocytes may originate from the Ph1-positive stem cell.

Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara- C) induces transcription of the c-jun immediate early response gene in human myeloid leukemia cells. The present work has examined the mechanisms responsible for this effect. Deleted forms of the c-jun promoter were linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into KG-1 cells. The results demonstrate that ara- C-induced c-jun transcription is mediated by an element between positions -74 and -20 upstream to the start site. Electrophoretic mobility shift assays with the fragment f(-74/-20) showed an increase in binding with nuclear proteins from ara-C-treated cells as compared with untreated cells. Competition with an oligonucleotide containing the AP-1 consensus sequence indicated that ara-C stimulates binding of nuclear proteins at the AP-1 site in the c-jun promoter. These findings were confirmed in other gel shift studies with the collagenase enhancer AP-1 consensus sequence and with a DNA fragment containing an altered AP-1 site. The binding of JUN/AP-1 was maximal at 1 hour of ara-C treatment and decreased to baseline levels at 12 hours. The finding that ara-C induces AP-1 binding in the absence of protein synthesis indicated that this agent activates already synthesized JUN/AP-1. To confirm these findings, the AP-1 consensus sequence was introduced 5′ to the heterologous SV40 promoter. The results show that AP-1 enhances SV40 promoter activity in ara-C-treated cells. Taken together, these findings indicate that: (1) enhancement of JUN/AP-1 activity in ara-C- treated cells involves a posttranslational modification of JUN/AP-1; and (2) binding of activated JUN/AP-1 to the AP-1 site in the c-jun promoter confers ara-C inducibility of this gene.

We have examined the in vitro effects of recombinant human (rh) interleukin-1 (IL-1) on the growth of purified megakaryoblasts obtained from patients with acute megakaryoblastic leukemia. We demonstrate that both IL-1 alpha and IL-1 beta treatment of these cells led to stimulation of DNA synthesis (as shown by increase of 3H-thymidine incorporation up to 35-fold) and also resulted in colony formation of leukemic megakaryoblasts. However, the stimulatory effect of IL-1 was dependent on endogenous production of IL-6, because addition of neutralizing monoclonal antibody (MoAb) to IL-6 abrogated the stimulatory activity of IL-1. In contrast, neutralizing MoAbs to granulocyte (G)-colony stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF failed to counteract the growth- enhancing effects of IL-1. Leukemic megakaryoblasts accumulated IL-6 mRNA and released IL-6 protein into their culture supernatant when exposed to rh IL-1 but failed to disclose transcripts for G-, GM-, and M-CSF under these conditions. Analysis of IL-6 receptor (IL-6R) transcript levels demonstrated that megakaryoblasts constitutively expressed IL-6R mRNA and that these transcripts are down-regulated to undetectable levels upon exposure to IL-1 and IL-6. Increase of 3H- thymidine incorporation by megakaryoblasts could be duplicated by exogenous IL-6 that could be blocked by neutralizing MoAb to IL-6. In conclusion, our results suggest that leukemic megakaryoblasts could produce and secrete IL-6, and express IL-6R, and that the growth- enhancing effect of IL-1 on these cells is indirect, via production of IL-6 by leukemic cells.

In a phase I/II study, 19 patients with advanced tumors but normal hematopoiesis and nine patients with bone marrow failure and prolonged severe cytopenias were treated with recombinant human interleukin-3 (rhIL-3) to assess the toxicity and biological effects of this multipotential hematopoietic growth factor. Doses ranging from 30 micrograms/m2 to 500 micrograms/m2 were administered as subcutaneous bolus injection daily for 15 days. A dose-dependent increase in platelet counts ranging from 1.3-fold at 60 micrograms/m2 to 1.9-fold at 250 micrograms/m2 was induced by rhIL-3 in 15 of 18 evaluable patients with normal hematopoiesis. An increase in reticulocyte counts was observed in 14 patients. The blood leukocyte counts dose dependently increased 1.4- to 3.0-fold. In patients with bone marrow failure, platelet counts increased by a mean of sixfold (range, 1.3- fold to 14.3-fold) in five of eight evaluable patients. Reticulocyte counts increased 4.4-fold in six patients, and neutrophil counts increased by a mean of 3.1-fold in all eight patients. Platelet transfusions could be discontinued after treatment with rhIL-3 in two of three transfusion-dependent patients. Only mild side effects, mainly fever, headache, and flushing, were observed. These results indicate that rhIL-3 functions as a multilineage hematopoietin in vivo in patients with normal bone marrow function and in patients with secondary bone marrow failure.

In a phase I-II study, nine patients with myelodysplastic syndromes and concomitant severe transfusion-dependent cytopenias were treated with recombinant human interleukin-3 (rhIL-3) to improve hematopoietic function. Doses of rhIL-3 ranged from 250 micrograms/m2 to 500 micrograms/m2 and were given as daily subcutaneous bolus injections for 15 days. Blood leucocyte counts increased 1.3- to 3.6-fold in all nine patients, including neutrophils, eosinophils, lymphocytes, basophils, and monocytes. The mean absolute neutrophil counts increased from 1,350/microL (range, 150 to 2,420) to 2,660/microL (range, 300 to 9,380) (P less than .05) immediately after the end of rhIL-3 therapy and to a maximum count of 4,096/microL (range, 350 to 10,820) (P less than .01). Platelet responses were seen in two of four profoundly thrombocytopenic patients, resulting in discontinuation of platelet transfusion. The requirements for red blood cell transfusion temporarily improved in one patient. Stimulation of plasma cells was evident by a significant increase in serum IgM and IgA levels. Mild side effects (fever, headache, local erythema, and bone pain) were observed in some patients, while transient thrombocytopenia developed in two patients. Disease progression with an increase in blast cells was seen in one patient. These results suggest that rhIL-3 is effective in stimulating hematopoiesis of all lineages in patients with myelodysplastic syndromes and may produce at least short-term hematologic improvement.

In a phase I/II study, nine patients with aplastic anemia were treated with recombinant human interleukin-3 (rhIL-3) to assess the toxicity and biologic effects of this multipotential hematopoietic growth factor. Doses ranging from 250 micrograms/m2 to 500 micrograms/m2 were administered as subcutaneous bolus injections daily for 15 days. An increase in platelet counts from 1,000/microL to 31,000/microL was induced by rhIL-3 in one patient, and an increase in reticulocyte counts by more than 10,000/microL in four patients. The blood leukocyte counts temporarily increased in eight patients 1.5- to 3.3-fold (median, 1.8-fold), mainly due to an increase in the number of neutrophils, eosinophils, lymphocytes, and monocytes. In two patients, bone marrow cellularity increased from 7% to 33% and from 10% to 80%, respectively, but without resulting in a substantial improvement of peripheral blood counts. Mild side effects (headache and flushing) were observed in some patients, while low-grade fever occurred in all patients. Transient thrombocytopenia necessitating discontinuation of rhIL-3 treatment occurred in one patient. In conclusion, rhIL-3 can stimulate hematopoiesis in patients with aplastic anemia; however, no lasting effects were obtained.

We studied the effects of a series of well-characterized clones of human natural killer (NK) cells on the proliferation of highly purified normal marrow hematopoietic progenitor cells. Individual NK clones suppressed granulocyte, monocyte, erythroid, or mixed colony formation in a heterogeneous but clonally stable manner. Inhibition of colony growth required a period of close cell contact between NK cell and progenitor cell with maximum inhibition occurring after 8 to 18 hours of preincubation time. The mechanism of killing was at least partially humoral, however, as cell-free supernatants generated by NK clones “activated” by contact with a target cell also inhibited progenitor cell growth. One of the possible humoral mediators was identified as gamma-interferon by studies with specific neutralizing monoclonal antibodies. These results show that clonal NK lines can be further activated by coming in contact with hematopoietic progenitor cells, resulting in substantial inhibition of colony formation in vitro.

Proliferation of acute myeloblastic leukemia (AML) cells in vitro is limited in most cases to a small subset of blasts that have several properties of stem cells. These leukemic colony-forming cells (AML-CFU) generally require addition of exogenous growth factors for proliferation in agar or methylcellulose. These factors can be supplied by media conditioned by phytohemagglutinin-stimulated normal leukocytes or by CSF-secreting tumor cell lines. However, the exact factor or factors required for stimulation of AML-CFU growth have not been defined. We compared the AML-CFU stimulatory activity of a human recombinant GM-CSF with that of GCT-CM, Mo-CM, and the PHA-leukocyte feeder system in 15 cases of AML. In each of the 12 cases that required exogenous growth factors for maximum AML-CFU growth, recombinant GM-CSF could replace either GM-CSF or Mo-CM, and could partially replace the PHA-leukocyte feeder system. These results indicate that this GM-CSF is a growth promoter of AML-CFU in these culture systems.

We assessed the capacity of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and multilineage (Multi)-CSF to induce release of granulocyte-CSF (G-CSF) by highly purified peripheral blood monocyte (Mo) preparations. Our results reveal that GM-CSF and Multi-CSF, either alone or in a synergistic concert, activate Mo to transcribe G-CSF messenger (m) RNA and release biologically active G- CSF protein into their culture supernatants. G-CSF had no regulatory effect on Mo expression of cytoplasmic G-CSF mRNA levels and G-CSF protein secretion by itself. These differential actions of CSFs provide further insight into self-regulatory mechanisms within the growth factor hierarchy system.

Tumor necrosis factor (TNF)-alpha has been identified as a major inducer of colony stimulating factor (CSF)-secretion by human vascular endothelial cells and fibroblasts. In the present study we assessed the capacity of TNFs to induce release of CSF-1 from highly purified peripheral blood monocyte preparations. Whereas monocytes do not accumulate CSF-1 messenger (m)RNA constitutively and consequently do not produce CSF-1 protein, CSF-1 mRNA and protein secretion became detectable, when monocytes were cultured in the presence of TNF-alpha, that was synergistically enhanced by interferon-gamma (IFN-gamma). However, under identical experimental conditions TNF-beta failed to induce monocyte CSF-1 synthesis. Cultures of monocytes in the presence of TNF-beta before addition of TNF-alpha abolished the CSF-1 inducing capacity of TNF-alpha, suggesting that TNF-beta may act as antagonist to TNF-alpha for CSF-1 production. These data point out a previously unrecognized function of TNF-alpha to modulate CSF-1 release by monocytes and demonstrate disparate biological properties of different TNF species in hematopoiesis.

Expression of HLA-DR surface antigens by granulocyte/monocyte colony- forming cells (CFU-GM) may be important in the regulation of proliferation of these cells. Using immunological techniques to enrich for progenitor cells, we investigated the expression of HLA-DR in subsets of CFU-GM. “Early” (day 14) CFU-GM express higher levels of HLA- DR than do “late” (day 7) CFU-GM. Among late CFU-GM, cells destined to form monocyte (alpha-naphthyl acetate esterase-positive) colonies express higher levels of HLA-DR than do CFU-GM destined to form granulocyte (chloroacetate esterase-positive) colonies. Because high- level expression of DR antigen was a marker for monocyte differentiation, we examined several lymphokines for their effects on both DR expression and in vitro commitment to monocyte differentiation by myeloid precursor cells. DR antigen density could be increased by more than twofold over 48 hours upon exposure to gamma-interferon (gamma-IFN), whereas colony-stimulating factors had no effect. This was associated with a dose-dependent inhibition of total CFU-GM number, and a relative, but not absolute, increase in the ratio of monocyte colonies to granulocyte colonies. Similarly, in day 7 suspension cultures of purified myeloid precursor cells, gamma-IFN inhibited cell proliferation and increased the ratio of monocytes to granulocytes. Thus, despite the induction of high levels of HLA-DR antigen on precursor cells (a marker of monocyte commitment), the dominant in vitro effect of gamma-IFN was inhibition of granulocyte differentiation.

Hairy cell leukemia (HCL) is frequently associated with severe pancytopenia. The authors detected high levels of tumor necrosis factor (TNF)-alpha in the bone marrow serum of patients with HCL and found anti-TNF-alpha neutralizing monoclonal antibodies (MoAbs) to be able to enhance hematopoiesis of HCL patients in in vitro colony assays. As potent producers of TNF-alpha, hairy cells could be identified, thus implicating the malignant population in the pathogenesis of hematopoietic failure due to inappropriate secretion of this cytokine.

Hairy cell leukemia (HCL) is frequently associated with severe pancytopenia. The authors detected high levels of tumor necrosis factor (TNF)-alpha in the bone marrow serum of patients with HCL and found anti-TNF-alpha neutralizing monoclonal antibodies (MoAbs) to be able to enhance hematopoiesis of HCL patients in in vitro colony assays. As potent producers of TNF-alpha, hairy cells could be identified, thus implicating the malignant population in the pathogenesis of hematopoietic failure due to inappropriate secretion of this cytokine.

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on hematologic parameters was evaluated in a phase I clinical study in 18 patients with advanced malignancy. G-CSF was administered once daily as a 30-minute infusion for 14 days; three patients each were treated at increasing dose levels of 1, 3, 10, 30, and 60 micrograms kg-1 day-1. A transient decrease in neutrophil and monocyte counts was observed immediately after the G-CSF infusion, followed by a dose-dependent increase of up to 15-fold. G-CSF-induced neutrophils exhibited an increased O2- radical production, and serum levels of enzymes related to granulocyte turnover, including lysozyme and elastase, were markedly elevated during therapy. A dose-dependent depression of platelet counts occurred in the second third of the treatment course, followed by a spontaneous recovery despite continuing therapy. G-CSF was well-tolerated; minor to moderate bone pain was the most common side effect. The primary course of the malignant diseases studied was not significantly altered. G-CSF appears to be an appropriate means to selectively increase the number of functionally competent polymorphonuclear phagocytes.

T cells are known to interact cooperatively with monocytes to produce Colony-Stimulating Factors (CSF), although T cell-mediated signals leading to CSF secretion by monocytes are not completely understood. We have made use of Northern blot hybridization and specific bioassays to study the effects of the T cell product interleukin-4 (IL-4) on monocyte CSF expression. The results suggest a previously unrecognized role of IL-4 as a CSF inducer since exposure of monocytes to IL-4 resulted in accumulation of transcripts for granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF). Consequently, IL-4-activated monocytes released factors in their culture supernatants biologically and antigenically indistinguishable from G- and M-CSF.