Effects of interleukin-6 and granulocyte colony-stimulating factor on the proliferation of leukemic blast progenitors from acute myeloblastic leukemia patients.

The effects of recombinant human interleukin-6 (rh IL-6), which has homology with rh granulocyte colony-stimulating factor (rh G-CSF) at the amino acid sequence level, and rh G-CSF on normal human bone marrow cells, fresh leukemic blast progenitors from 16 acute myeloblastic leukemia (AML) patients, and G-CSF-dependent human AML cell line (OCI/AML 1a) were investigated. rh G-CSF stimulated the proliferation of leukemic blast progenitors from 13 out of 16 AML patients tested. rh IL-6 stimulated the proliferation of blasts from eight AML patients and enhanced the G-CSF-dependent proliferation of the fresh AML blasts from two out of eight patients tested. On the other hand, rh IL-6 suppressed the blast colony formation from two AML patients and OCI/AML 1a cells and also reduced the G-CSF-dependent proliferation of the blast progenitors from one of the two patients and the cell line, rh IL-6 had no effect on the colony formation of normal granulocyte-macrophage colony-forming units (CFU-GM) with or without rh G-CSF. Differentiation-induction by rh IL-6 was not observed in the fresh AML blasts but was observed in OCI/AML 1a. The effect of IL-6 on the blast colony formation and G-CSF-dependent blast cell growth was complicated and heterogenous among the AML cases; IL-6 stimulated blast colony formation in some cases and suppressed it in others. The heterogeneity of the response was supposed to be derived from the heterogeneity of the characteristics of AML cells. Although G-CSF simply stimulated the blast colony formation, IL-6 had a bimodulatory effect on the proliferation of leukemic blast progenitors from AML patients. IL-6 might be involved in the regulation of the proliferation of AML cells in vivo as well as in vitro.     

In vitro growth response to G-CSF and GM-CSF by bone marrow cells of patients with acute myeloid leukemia.

The in vitro growth response of bone marrow and blood cells to granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) was studied in 18 acute myeloid leukemia (AML) patients using semisolid and suspension cultures. In 80% of the cases growth of leukemic progenitor cells was stimulated by GM-CSF and/or G-CSF, as judged by colony or cluster formation. In acute promyelocytic leukemia [t(15;17)], G-CSF stimulated and maintained the leukemic progenitors only transiently but fully stimulated the residual normal granulocyte/macrophage colony-forming units (CFU-GM). In some cases of M2 and M4 leukemia, G-CSF enhanced markedly the production of mature but cytochemically abnormal neutrophils. In some cases of M1 leukemia, neither CSF stimulated leukemic progenitors but instead stimulated only residual normal granulopoiesis. Spontaneous colony formation was observed in 20% of cases and was correlated with high-grade leukemic growth in vivo and a poor response to chemotherapy. The differing effects of the CSFs upon leukemic cells and residual normal granulopoiesis may have some implications for the clinical use of GM-CSF and G-CSF to overcome infectious complications.     

Effect of Recombinant Human D-Factor on the Growth of Leukemic Blast Progenitors from Acute Myeloblastic Leukemia Patients

We studied the effects of D-factor on the growth of leukemic blast progenitors from 15 patients with acute myeloblastic leukemia and two leukemia cell lines in methylcellulose and suspension cultures. When stimulated by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor or interleukin-3, leukemic blast progenitors undergo terminal division with limited differentiation in methylcellulose culture, forming blast colonies. Leukemic blast progenitors can renew themselves. The self-renewal can be detected as secondary colony formation after replating primary blast colonies in fresh methylcellulose media and by the growth of clonogenic cells in suspension culture. D-Factor suppressed primary and secondary colony formation in methylcellulose culture. Furthermore, D-factor suppressed clonogenic cell recovery in suspension culture. The suppression by D-factor of the growth of leukemic blast progenitors was not significantly dependent upon the colony-stimulating factors used as growth-stimulating factors. High concentration of G-CSF did not overcome the suppressive effect of D-factor. The results indicate that D-factor is effective in suppressing not only terminal division but also self-renewal of leukemic blast progenitors.     

Effect of recombinant human D-factor on the growth of leukemic blast progenitors from acute myeloblastic leukemia patients.

We studied the effects of D-factor on the growth of leukemic blast progenitors from 15 patients with acute myeloblastic leukemia and two leukemia cell lines in methylcellulose and suspension cultures. When stimulated by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor or interleukin-3, leukemic blast progenitors undergo terminal division with limited differentiation in methylcellulose culture, forming blast colonies. Leukemic blast progenitors can renew themselves. The self-renewal can be detected as secondary colony formation after replating primary blast colonies in fresh methylcellulose media and by the growth of clonogenic cells in suspension culture. D-Factor suppressed primary and secondary colony formation in methylcellulose culture. Furthermore, D-factor suppressed clonogenic cell recovery in suspension culture. The suppression by D-factor of the growth of leukemic blast progenitors was not significantly dependent upon the colony-stimulating factors used as growth-stimulating factors. High concentration of G-CSF did not overcome the suppressive effect of D-factor. The results indicate that D-factor is effective in suppressing not only terminal division but also self-renewal of leukemic blast progenitors.     

Human recombinant stem cell factor stimulates in vitro proliferation of acute myeloid leukemia cells and expands the clonogenic cell pool.

Stem cell factor (SCF) is a new growth factor acting on early hematopoietic progenitor and stem cells. In our experiments human recombinant SCF stimulated short-term proliferation of accessory cell-depleted acute myeloid leukemia (AML) cells in 13/14 cases, as determined by 3H-thymidine (3H-TdR) incorporation and cell counts. Stimulatory activity was significantly greater than in the presence of GM-CSF and was comparable to that of granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3), and 5637 cell line supernatant (SN). Conversely, the ability of SCF to induce primary colony formation by AML clonogenic cells (CFU-L) was lower than that of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 5637 SN in all but four cases. However, SCF potentiated the stimulatory effect of GM-CSF, G-CSF, and IL-3 on both 3H-TdR incorporation and colony formation. In a 7-day liquid culture SCF enhanced CFU-L recovery in all cases to a significantly greater extent than the other growth factors. A further increment was obtained by combinations of SCF with GM-CSF, G-CSF, or IL-3, and this was significantly more effective than 5637 SN. SCF did not induce leukemic cell differentiation. Human recombinant SCF is therefore highly efficient in stimulating AML cell proliferation and expanding the CFU-L pool. It was not, however, able to support long-term growth of AML cells (beyond 2-7 weeks) in five cases tested.     

Effects of Interleukin-6 and Granulocyte Colony-stimulating Factor on the Proliferation of Leukemic Blast Progenitors from Acute Myeloblastic Leukemia Patients

The effects of recombinant human interleukin-6 (rh IL-6), which has homology with rh granulocyte colony-stimulating factor (rh G-CSF) at the amino acid sequence level, and rh G-CSF on normal human bone marrow cells, fresh leukemic blast progenitors from 16 acute myeloblastic leukemia (AML) patients, and G-CSF-dependent human AML cell line (OCI/AML 1a) were investigated. rh G-CSF stimulated the proliferation of leukemic blast progenitors from 13 out of 16 AML patients tested. rh IL-6 stimulated the proliferation of blasts from eight AML patients and enhanced the G-CSF-dependent proliferation of the fresh AML blasts from two out of eight patients tested. On the other hand, rh IL-6 suppressed the blast colony formation from two AML patients and OCI/AML 1a cells and also reduced the G-CSF-dependent proliferation of the blast progenitors from one of the two patients and the cell line. rh IL-6 had no effect on the colony formation of normal granulocyte-macrophage colony-forming units (CFU-GM) with or without rh G-CSF. Differentiation-induction by rh IL-6 was not observed in the fresh AML blasts but was observed in OCI/AML 1a. The effect of IL-6 on the blast colony formation and G-CSF-dependent blast cell growth was complicated and heterogenous among the AML cases; IL-6 stimulated blast colony formation in some cases and suppressed it in others. The heterogeneity of the response was supposed to be derived from the heterogeneity of the characteristics of AML cells. Although G-CSF simply stimulated the blast colony formation, IL-6 had a bimodulatory effect on the proliferation of leukemic blast progenitors from AML patients. IL-6 might be involved in the regulation of the proliferation of AML cells in vivo as well as in vitro.     

Primary human myeloid leukemia cells: comparative responsiveness to proliferative stimulation by GM-CSF or G-CSF and membrane expression of CSF receptors

In vitro clonal culture of leukemic cells from patients with acute myeloid leukemia (AML) showed that cells from all subtypes tested could be stimulated to proliferate clonally either by purified recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) or by human cross-reactive, purified murine granulocyte CSF (G-CSF). The responsiveness of AML populations to CSF stimulation was quantitatively variable but was within the heterogeneous range exhibited by normal granulocyte-monocyte progenitor cells. A general concordance was noted between the proliferative effects of GM-CSF and G-CSF on the individual leukemic populations. All AML populations tested specifically bound 125I-labeled murine G-CSF; the level of labeling varied widely and correlated with AML subtype. Labeling levels on individual labeled leukemic cells were within the heterogeneous range exhibited by normal cells, but significant numbers of blast cells in M2, M4, and M5 AMLs appeared to lack membrane receptors for G-CSF. The level of labeling with G-CSF did not correlate with the frequency of clonogenic cells able to be stimulated by G-CSF. The data emphasized that GM-CSF and G-CSF are equivalent proliferative stimuli for human myeloid leukemia cells. Further, despite the potential ability of G-CSF to suppress murine leukemic cells, many AML blast cells lack significant numbers of G-CSF receptors. These considerations warrant caution in future attempts to use G-CSF in the therapy of acute myeloid leukemia.     

Interleukin-4 as a growth regulator of clonogenic cells in acute myelogenous leukemia in suspension culture.

Using two complementary culture systems, suspension and clonal cultures, and with a method of graphic display (star diagram), we studied the effects of recombinant human interleukin-4 (IL-4) on leukemic stem cell renewal and differentiation in acute myelogenous leukemia (AML). The interactions between IL-4 and other recombinant human cytokines, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF) and interleukins-1 alpha, -2, -3, -5, and -6 were also studied. IL-4 alone had significant effects on both self-renewal and differentiation of blast progenitors in some cases; in clonogenic assay, IL-4 stimulated blast colony formation and in one case IL-4 was the most powerful stimulator among the nine growth factors tested. Star diagrams, constructed using the data from both suspension and clonal cultures, showed that IL-4 could influence the balance between self-renewal and differentiation of clonogenic cells. Negative and positive interactions were detected between IL-4 and other cytokines in suspension culture. These results indicate that IL-4 is a cytokine with a potential role in regulating the growth of myeloid leukemic stem cells, and that IL-4 may be useful in treating selected AML patients.     

Human bladder carcinoma cell line HTB9, which secretes a factor to stimulate clonogenic leukemic blast growth, expresses the granulocyte-macrophage colony-stimulating factor gene

Media conditioned by the human bladder carcinoma cell line HTB9 contained high leukemic blast growth factor activity and also showed granulocyte-macrophage colony-stimulating factor (GM-CSF) activity. Northern blotting analysis of total RNA from HTB9 cells using GM-CSF cDNA as a probe demonstrated abundant expression of the GM-CSF gene. Thus, this cell line secretes GM-CSF, and this factor contributes to the proliferation of clonogenic leukemic blast cells.     

Aclarubicin induces differentiation of leukemic progenitors in myelodysplastic syndrome cooperating with granulocyte colony-stimulating factor

We have reported that low-dose aclarubicin (ACR) therapy is effective in some patients with myelodysplastic syndrome (MDS). Here, we demonstrate that a low concentration of ACR induces the in vitro differentiation of leukemic progenitor cells from patients with MDS. ACR (0.1 ng/ml) significantly increased the number of granulocyte colony-stimulating factor (G-CSF)-dependent colonies from circulating blast cells in vitro in six out of seven MDS patients with refractory anemia with excess of blast in transformation or chronic myelomonocytic leukemia, but not in all four patients with primary acute myelogenous leukemia. In these MDS patients, the effect of ACR gradually disappeared along with the progression of MDS. Interestingly, the majority of G-CSF/ACR-dependent colonies consisted of rather differentiated myeloid cells such as myelocytes and metamyelocytes, whereas colonies formed by G-CSF alone were composed mainly of immature blastic cells. The number of G-CSF-responding progenitors significantly increased during a 24-48 h incubation with ACR alone. The circulating blasts in MDS patients expressed G-CSF receptors at unchanged levels before and after the incubation with ACR. It is suggested that ACR might increase clonogenic progenitor responsiveness to G-CSF in MDS, probably through modulating downstream signaling cascades associated with G-CSF receptors, and induce these progenitors to differentiate in response to G-CSF.     

Receptor binding of human granulocyte colony-stimulating factor to the blast cells of myeloid leukemia.

Human granulocyte colony-stimulating factor (G-CSF) rapidly loses the biological activity and the receptor binding capacity following radioiodination. We have made a mutein of human G-CSF, KW-2228, in which Thr-1, Leu-3, Gly-4, Pro-5, and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg, and Ser; showed more potent G-CSF activity; and retained full biological activity and receptor binding capacity at least 2 weeks of radioiodination. G-CSF is an effective growth factor for the blasts of myeloid leukemia. Radioiodinated KW-2228 was prepared using solid-phase glucose oxidase-lactoperoxidase. Human leukemia cell lines and the blast cells from leukemia patients were examined for binding. High affinity binding sites were identified on myeloid cell lines and on the blasts obtained from acute myeloid leukemia patients. Scatchard analysis showed that a single binding site for G-CSF was observed (361-1688 receptors/cell; Kd 128-1400 pM). In contrast, specific binding of 125I-KW-2228 was not demonstrated on lymphoblastic cell lines or the blast cells of acute lymphoid leukemia or lymphoma. This difference was reflected in the effectiveness of G-CSF to stimulate colony formation in acute myeloid leukemia blasts, while G-CSF did not stimulate colony formation of the blast cells from acute lymphoid leukemia.     

Growth of CD34+ acute myeloblastic leukemia colony-forming cells in response to recombinant hematopoietic growth factors.

In order to minimize the interactions of clonogenic cells with accessory cells and characterize the direct effect of recombinant hematopoietic growth factors (HGF) on acute myelogenous leukemia colony-forming cells (AML-CFU), the response of CD34+ AML-CFU to individual or combined recombinant HGF, i.e., interleukin-1 (IL-1), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF), was studied in 10 patients and compared with the growth response obtained from unfractionated marrow cells. IL-3 and GM-CSF had a similar stimulating activity on AML-CFU growth. G-CSF resulted the most efficient stimulus for colony formation and was additive or synergistic with IL-3 and GM-CSF, M-CSF, used alone, had a negligible stimulating activity. When CD34+ cells were used, IL-1 by itself had a low stimulating activity and displayed little or no synergy with IL-3, GM-CSF, and G-CSF. On the contrary, when unfractionated cells were used, IL-1 was very effective in inducing AML-CFU formation and was markedly synergistic with IL-3 and GM-CSF. These results show that IL-1-induced leukemic colony formation is prevalently mediated by accessory cells. IL-6 supported AML-CFU growth in seven of 10 cases, thus showing a direct effect on CD34+ leukemic cells, and enhanced the growth of IL-3-(+47 to +167%) and GM-CSF-dependent (+60 to +110%) AML-CFU. Recloning studies of single colonies demonstrated that primary CD34+ AML-CFU, stimulated by IL-3 and GM-CSF, generated secondary and tertiary colonies, whereas primary AML-CFU stimulated by G-CSF and IL-6 failed to give rise to secondary colonies, thus indicating a complete suppression of self-renewal. Sequential recloning of colonies grown in the presence of IL-3 + IL-6 demonstrated that addition of IL-6 and IL-3-containing plates resulted in a nearly complete suppression of self-renewal. In conclusion, these results demonstrate the heterogeneity of the CD34+ leukemic cell fraction and indicate the existence of complex regulatory events at the level of CD34+ leukemic cells. Data obtained from recloning experiments are of therapeutic interest in view of the clinical application of HGFs in the treatment of myeloid leukemias.     

Effect of protein kinase inhibitors on the proliferation of leukemic cells stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor or interleukin-3.

The effects of the inhibitor for protein kinase A or C, or tyrosine kinase (H-8, staurosporine, or genistein, respectively) on the proliferation of leukemic and normal bone marrow cells stimulated by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3) were studied using the MTT assay. These inhibitors suppressed the proliferation of leukemic and normal bone marrow cells in a dose-dependent manner. Although the suppressive effect of each inhibitor on cell proliferation was varied in each instance, the effects were almost similar whichever CSF was added. A significant difference was not recognized between leukemic and normal bone marrow cells in terms of sensitivity to these inhibitors. The data indicate that protein kinase inhibitors have an inhibitory effect on leukemic and normal hematopoietic cell proliferation and that further studies are required to determine if this effect is due to the inhibition of protein kinases acting as the second messenger of CSFs.     

Positive and negative effects of tumor necrosis factor on colony growth from highly purified normal marrow progenitors.

The effects of recombinant human tumor necrosis factor alpha (TNF alpha) on colony growth were studied using highly enriched progenitor cells from normal human bone marrow. Supplementation of TNF to culture resulted in a dose-dependent suppression of granulocyte colony-stimulating factor (G-CSF) induced granulocytic colony formation and also erythropoietin (Epo) induced erythroid burst formation. However, the number of erythroid bursts, stimulated by interleukin-3 (IL-3) plus Epo, increased when TNF was added at comparable concentrations. Further, TNF enhanced eosinophilic colony growth induced by IL-3 or granulocytic-macrophage colony-stimulating factor (GM-CSF). In GM-CSF cultures TNF (100-1000 U/ml) also induced granulocytic and macrophage colonies. The addition of neutralizing antibodies against G-CSF, GM-CSF, or interleukin-6 (IL-6) to culture did not abrogate the observed effects of TNF, so that stimulation of myeloid colony growth was unlikely to result from the secondary induction of G-CSF or GM-CSF. TNF therefore exerts favourable effects on hematopoietic progenitors responsive to the more primitive colony-stimulating factors (IL-3, GM-CSF) and potent negative effects on precursors reactive to the single lineage G-CSF and Epo. These contrasting effects of TNF suggest that TNF, when available to marrow progenitors at similar tissue concentrations, may drive hematopoiesis within the progenitor cell compartment into selected directions.     

Proliferative effect of human granulocyte colony-stimulating factor on blast cells of acute promyelocytic leukemia

The effect of human granulocyte colony-stimulating factor (G-CSF) on leukemic cells of acute promyelocytic leukemia (APL) was examined. Mononuclear cells obtained from bone marrow cells containing more than 90% blasts from seven APL patients were incubated in the presence of G-CSF using semisolid and liquid culture systems. On day 7, the cells from all the patients produced many clusters consisting of 8-40 cells. These cells appeared to be promyelocyte-like blast cells in four patients and had differentiated to more mature neutrophils in three patients. On day 14, the number of clusters decreased except for two patients. Blast cells from the two patients showing the increase of blast clusters could proliferate in a liquid culture containing G-CSF. Blast cells cultured for 14 days formed many secondary cultures after replating on a methylcellulose medium. Moreover, chromosomal analyses of blasts cultivated in the presence of G-CSF for 7 days showed t(15;17) in all metaphases in one patient. It appears that the leukemic cells from APL patients could proliferate in the presence of G-CSF.     

Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on lung cancer: roles of cyclooxygenase-2

We examined the effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the lung cancer cell lines PC-9, LA-1 and A549. In addition, we examined if the effects of the cytokines on the cell lines are mediated by activation of cyclooxygenase (COX)-2. The three cell lines did not constitutively produce either G-CSF or GM-CSF. G-CSF did not influence cell growth in the three cell lines, while GM-CSF increased cell growth in the A549 and LA-1 lines. G-CSF and GM-CSF dose-dependently decreased cell death in the three cell lines. RT-PCR demonstrated GM-CSF receptor expression in the three lung cancer cell lines, whereas the G-CSF receptor exists only in the PC-9 line. We suggest that G-CSF might rescue the tumor cells from cytotoxicity due to serum deprivation through cellular pathways independent of the G-CSF receptor. G-CSF and GM-CSF increased cyclooxygenase-2 (COX-2) expression in PC-9 and LA-1 cells whereas they decreased COX-2 expression in A549 cells. The COX-2 inhibitor NS-398 increased cell death in PC-9 and LA-1 cells, whereas it decreased cell death in A549 cells. PC-9 and LA-1 clones transfected with sense G-CSF- or GM-CSF showed an increase in COX-2 expression, while COX-2 expression was decreased in transfected A549 clones. COX-2 expression was increased in anti-sense G-CSF- and GM-CSF-transfected A549 clones. Thus, although COX-2 activation seems to induce different biological behavior depending on the cell type, we propose that G-CSF and GM-CSF might accelerate tumor progression by directly regulating COX-2 expression, independently of an autocrine mechanism.     

Cooperative autocrine and paracrine functions of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the progression of skin carcinoma cells

Tumor growth and progression are critically controlled by alterations in the microenvironment often caused by an aberrant expression of growth factors and receptors. We demonstrated previously that tumor progression in patients and in the experimental HaCaT tumor model for skin squamous cell carcinomas is associated with a constitutive neoexpression of the hematopoietic growth factors granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), causing an autocrine stimulation of tumor cell proliferation and migration in vitro. To analyze the critical contribution of both factors to tumor progression, G-CSF or GM-CSF was stably transfected in factor-negative benign tumor cells. Forced expression of GM-CSF resulted in invasive growth and enhanced tumor cell proliferation in a three-dimensional culture model in vitro, yet tumor growth in vivo remained only transient. Constitutive expression of G-CSF, however, caused a shift from benign to malignant and strongly angiogenic tumors. Moreover, cells recultured from G-CSF-transfected tumors exhibited enhanced tumor aggressiveness upon reinjection, i.e., earlier onset and faster tumor expansion. Remarkably, this further step in tumor progression was again associated with the constitutive expression of GM-CSF strongly indicating a synergistic action of both factors. Additionally, expression of GM-CSF in the transfected tumors mediated an earlier recruitment of granulocytes and macrophages to the tumor site, and expression of G-CSF induced an enhanced and persistent angiogenesis and increased the number of granulocytes and macrophages in the tumor vicinity. Thus both factors directly stimulate tumor cell growth and, by modulating the tumor stroma, induce a microenvironment that promotes tumor progression.     

Effects of recombinant human granulocyte colony stimulating factor (rG-CSF) on murine myeloid leukemia: stimulation of proliferation of leukemic cells in vitro and inhibition of development of leukemia in vivo

We have established an experimental murine myeloid leukemia model and investigated the effects of recombinant granulocyte colony stimulating factor (rG-CSF) on myeloid leukemia in vitro and in vivo. rG-CSF stimulated colony formation by the leukemic cells in semisolid agar medium, and exponential growth of the clonogenic cells in suspension medium. Thus, rG-CSF was able to stimulate both the differentiation and self-renewal processes of the leukemic stem cells in vitro. However, 14 consecutive daily injections of rG-CSF prolonged the mean survival time of the mice implanted with the leukemic cells. This effect of rG-CSF was accompanied by a delay in the emergence of the blast cells in peripheral blood and by a decreased blast population in the spleen, suggesting that development of leukemia was suppressed in the rG-CSF-treated-mice. The prolongation of the survival time by rG-CSF was more evident when rG-CSF was administered in therapeutic combination with cyclophosphamide. These results indicate that the effect of rG-CSF on the development of leukemia is not exactly predicted from in vitro experiments.     

Granulocyte colony-stimulating factor receptors on human acute leukemia: biphenotypic leukemic cells possess granulocyte colony-stimulating factor receptors.

Granulocyte colony-stimulating factor (G-CSF) receptors on the gated leukemic blast cells from newly diagnosed patients with acute leukemia or crisis of chronic myelogenous leukemia were investigated using flow cytometric detection. Surface marker analysis and cytochemical studies were conducted simultaneously to characterize the blast cells. Among 24 leukemia cases examined, G-CSF receptor-positive blast cells were detected in all 11 cases of acute myeloblastic leukemia even though the percentage range of positive cells was widely variable. On the other hand, they were not detected on the blast cells from patients with peroxidase-negative acute lymphoblastic leukemia with no myeloid surface antigens. However, G-CSF receptors were demonstrated in significant amounts on blast cells from 5 of 8 cases of peroxidase-negative acute leukemia expressing both myeloid and lymphoid surface antigens (biphenotypic leukemia). The percentage of blast cells positive for G-CSF receptors was significantly smaller in biphenotypic cases [33 +/- 14% (SD)] than in acute myeloblastic leukemia cases [65 +/- 22%] (P less than 0.01). The percentage expression of CD13 antigen by blast cells was significantly related to their percentage positivity for G-CSF receptors (rs = 0.50, P less than 0.05). These findings indicate that the distribution of flow cytometrically detectable G-CSF receptors on leukemic cells possessing myeloid characteristics may be related to the maturation process.     

Recombinant human TNF-alpha stimulates the secretion of granulocyte colony-stimulating factor in vivo.

Tumor necrosis factor (TNF) is a macrophage-derived cytokine that causes hemorrhagic necrosis of several human tumors in vitro. It has a wide range of biologic effects including stimulation of secretion of both granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) by normal adult lung fibroblasts in culture. No in vivo data are available on the effect of exogenously administered TNF on cytokine production. In the studies reported here, we show that G-CSF accumulates in the serum in vivo in response to recombinant TNF (rTNF) administration. At the peak of the response circulating levels of 2-6 ng/ml of biologically active G-CSF are detectable. Surprisingly, circulating levels of GM-CSF, interleukin-3 as well as a number of other cytokines were not detectable within the limits of the assays. The results indicate that the levels of GM-CSF or interleukin-3 are minimally 100-fold lower than the peak levels of G-CSF. These data illustrate the complex interplay that cytokines have in vivo. Understanding these interactions in humans is crucial to the correct use of this new class of agents in the clinic.