Modulatory in vitro effects of interleukin-1 receptor antagonist (IL-1Ra) or antisense oligonucleotide to interleukin-1 beta converting enzyme (ICE) on acute myeloid leukaemia (AML) cell growth.

We investigated the effects of interleukin-1 receptor antagonist (IL-1Ra) on the spontaneous proliferation and AML colony forming unit (CFU-AML) formation of bone marrow and peripheral blood cells in 50 acute myeloid leukaemia (AML) patients. Exposure to IL-1Ra (10 micrograms/ml) caused either decreased, unaltered or increased AML cell proliferation, as well as of CFU-AML colony formation, depending on the individual patient, but the inhibitory effects were dominant. To evaluate the involvement of IL-1 beta converting enzyme (ICE) in the autonomous AML cell growth, the effects of an antisense oligonucleotide on ICE were examined in 19 of these patients. In a majority of patients, antisense ICE suppressed both AML cell proliferation and CFU-AML although a stimulatory effect was sometimes evident. The proportion of AML patients with suppression obtained by antisense ICE was higher than with IL-1Ra, suggesting the involvement of additional ICE-dependent cytokine(s) in AML cell growth besides IL-1. The presence of IL-1Ra or antisense ICE also suppressed the endogenous IL-1 beta production of AML cells, at both the level of pro-IL-1 beta and mature IL-1 beta. Although inhibition by IL-1Ra or antisense ICE on growth parameters of AML cells in vitro prevailed, indicating the importance of IL-1 activity in autonomous AML cell growth, stimulatory effects on the cells of some patients suggest that AML is a heterogenous disorder regarding IL-1 beta regulation.     

Effect of interleukin-1 beta converting enzyme inhibitor on acute myelogenous leukemia progenitor proliferation

Interleukin-1 beta (IL-1 beta) converting enzyme (ICE) is a cysteine protease that specifically cleaves precursor IL-1 beta to its biologically active form. Recent studies have also implicated ICE in the induction of apoptosis in vertebrate cells. Because IL-1 plays a major role in acute myelogenous leukemia (AML) blast proliferation, we sought to investigate the effect of ICE inhibition on AML progenitors. To do this, we used bocaspartyl (benzyl) chloromethylketone (BACMK) an inhibitor designed to penetrate cells and bind covalently to the active site of ICE. Our preliminary experiments showed that incubation of activated peripheral blood cells with 2.5 mumol/L of BAMCK downregulated production of mature IL-1 beta but had no effect on tumor necrosis factor-alpha. To test the effects of the inhibitor on AML cells, we first used the OCI/AML3 cell line. We found that these cells produce IL-1 beta and bind the biotinylated cytokine and that IL-1 inhibitors, such as IL-1 neutralizing antibodies, IL-1 receptor antagonist, and soluble IL-1 receptors, specifically inhibit OCI/AML3 proliferation, indicating that IL-1 beta is an autocrine growth factor for OCI/AML3 cells. The ICE inhibitor suppressed OCI/AML3 growth in a dose-dependent manner (at 0.4 to 4 mumol/L) and downregulated mature IL- 1 beta production, as assessed by Western immunoblotting. Similar results were obtained with marrow aspirates from 16 AML patients. The ICE inhibitor suppressed proliferation of AML precursors (by up to 78%; mean, 44%) in a dose-dependent fashion at concentrations ranging from 0.4 to 5 mumol/L but not proliferation of normal marrow progenitors; the suppressive effect was reversed by IL-1 beta. Furthermore, incubation of AML cells with 4 mumol/L BAMCK downregulated the production of mature IL-1 beta, suggesting that the growth-inhibitory effect is mediated through suppression of the biologically active cytokine. Our data indicate that inhibition of ICE suppresses AML blast proliferation and suggest that ICE inhibitors may have a role in future therapies for AML.     

Effect of interleukin-1, tumor necrosis factor-alpha, and interferon-alpha on the blast cells of acute myeloblastic leukemia.

In this study, we further established the role of interleukin-1 alpha (IL-1 alpha), interleukin-1 beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-alpha (IFN-alpha) as regulators of proliferation of acute myeloid leukemia (AML) cells. AML cells from 8 of 15 patients incorporated high levels of 3H-thymidine (3H-TdR) in the absence of exogenous growth factors. The spontaneous DNA synthesis could be abrogated with monospecific antibodies directed toward IL-1 alpha, IL-1 beta, or TNF-alpha, as well as with antigranulocyte-macrophage colony-stimulating factor (GM-CSF). Human recombinant GM-CSF reversed the inhibitory action of each of these antibodies and reinduced DNA synthesis in AML cells. Thus, in these cases, constitutively produced IL-1 or TNF-alpha had stimulated the synthesis of GM-CSF, which resulted in GM-CSF-dependent proliferation of AML blasts. Exogenous IL-1 up-regulated the endogenous production of GM-CSF, suggesting a positive regulation of autocrine growth factor production. We also present evidence that TNF-alpha may exert both stimulative as well as inhibitory effects on DNA synthesis in AML cells. The enhancing effect of TNF-alpha was mediated through the induction of GM-CSF production, as stimulation of DNA synthesis in AML blasts could be abrogated with anti-GM-CSF antibody. A concentration-dependent inhibitory effect of TNF-alpha on 3H-TdR incorporation into AML blasts was observed only when these cells were grown in the absence of GM-CSF. Finally, we show that human recombinant IFN-alpha is a potent inhibitor of AML cell proliferation in vitro.     

Inhibition of acute myelogenous leukemia blast proliferation by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors.

Interleukin-1 (IL-1) has recently been reported to play an important role in acute myelogenous leukemia (AML) blast proliferation. We therefore investigated the effect of soluble IL-1 receptors (sIL-1R) and IL-1 receptor antagonist (IL-1RA) on the growth of AML bone marrow blast progenitors from 25 patients. In the AML blast colony culture assay, sIL-1R and IL-1RA inhibited blast colony-forming cell replication in a dose-dependent fashion, at concentrations ranging from 10 to 500 ng/mL (sIL-1R) and 10 to 1,000 ng/mL (IL-1RA), and their inhibitory effect was partially reversed by IL-1 beta. A similar inhibitory effect was also noted with the use of anti-IL-1 beta neutralizing antibodies. When AML blast progenitors were grown either in the presence of fetal calf serum (FCS) alone or with one of the following: phytohemagglutinin leukocyte-conditioned medium (PHA-LCM), granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, interleukin-3 (IL-3), or stem cell factor (SCF), addition of 100 ng/mL sIL-1R or IL-1RA inhibited blast colony formation by 3% to 96% and 2% to 97%, respectively. In sharp contrast, neither of these IL-1-inhibitory molecules significantly inhibited proliferation of normal marrow hematopoietic progenitors. Lysates of 2 x 10(7) low-density AML marrow cells were tested for intrinsic IL-1 beta content using an enzyme-linked immunoadsorbant assay (ELISA). Samples from five of six patients showed high concentrations (ranging from 501 to 2,041 pg), whereas 2 x 10(7) cells from two normal marrow aspirates yielded 54.6 pg of IL-1 beta. AML blast colony-forming cells from all six patients were inhibited by sIL-1R, IL-1RA, or both. Incubation of nine samples of AML low-density cells with either sIL-1R or IL-1RA reduced GM-CSF concentrations in cell lysates, and supernatants from nine (P less than .01) and six samples (P less than .037), respectively, and G-CSF concentration in lysates from six of nine samples (P less than .03), and in supernatants from five of six samples (P less than .06) when studied by ELISAs. Our data implicate IL-1 in AML blast proliferation and suggest the potential benefits of using IL-1-inhibitory molecules in future therapies for AML.     

Interleukin-1 is one factor which regulates autocrine production of GM-CSF by the blast cells of acute myeloblastic leukaemia

The role of interleukin-1 (IL-1) as a regulator of the autonomous growth of the blast cells of acute myeloid leukaemia (AML) has been studied on samples isolated from 15 patients. In nine out of 10 patients with evidence of partial autonomous blast cell growth. IL-1 further stimulated cell growth in both suspension culture and in a clongenic assay. IL-1 also stimulated cell growth in two out of three cases with no evidence of autonomous growth whereas no additional stimulation was observed in two cases with totally autonomous growth. In blast cells stimulated by IL-1, synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) was increased and the proliferative response to IL-1 was inhibited by an antibody to GM-CSF. In all samples with evidence of autonomous growth, blast cell conditioned medium (BCCM) contained IL-1 activity (range 0.7-74.2 units ml) and a polyclonal antibody to IL-1 markedly inhibited autonomous growth in four samples. BCCM from three of these four samples contained GM-CSF, the synthesis of which was suppressed when BCCM was prepared in the presence of anti-IL-1. Our data suggests that endogenous IL-1 is an important factor in the regulation of the production of GM-CSF and hence of autonomous growth of AML blasts, but that other mechanisms regulating GM-CSF production may exist.     

Secretion of interleukin-1 by acute myeloblastic leukemia cells in vitro induces endothelial cells to secrete colony stimulating factors

The interaction of acute myeloblastic leukemia (AML) cells with stromal cells was investigated by adding AML-conditioned media to cultures of human endothelial cells. This conditioned media contained factors that induced expression of both the granulocyte macrophage colony- stimulating factor (GM-CSF) and granulocyte CSF (G-CSF) genes and release of colony stimulating activity from endothelial cells. The conditioned media contained interleukin-1 (IL-1) bioactivity and the endothelial cell stimulatory activity was partially neutralized by anti- IL-1 antiserum. Constitutive expression of the IL-1-beta gene was detected in ten of 17 AML cases analyzed. These results suggest that the unregulated secretion of IL-1 by AML cells can induce stromal cells in vitro to overproduce CSFs. This could contribute to the unrestricted growth of AML cells.     

Divergent effects of interleukin-4 (IL-4) on the granulocyte colony-stimulating factor and IL-3-supported myeloid colony formation from normal and leukemic bone marrow cells.

Human recombinant interleukin-4 (IL-4) was studied for its effects on myeloid progenitor cells from normal and leukemic bone marrow cells in the presence and absence of additional growth factors. IL-4 itself did not support myeloid cluster or colony formation (CFU-GM). However, cultures supplied with IL-4 (300 U/mL) and IL-3 demonstrated a significant decline in myeloid colony numbers (CFU-GM) compared with the effects of IL-3 alone: (48 +/- 27 v 88 +/- 27 CFU-GM/10(5) MNC). In contrast, IL-4 augmented the G-CSF-supported CFU-GM: (80 +/- 31 v 148 +/- 52 CFU-GM/10(5) MNC). The effects of IL-4 were not mediated by accessory cells because similar results were obtained with and without T-cell, B-cell, or adherent depleted cell fractions. Morphologic analysis of clusters (day 7) and the colonies (day 14) demonstrated that IL-4 enhanced myeloid colony formation in the presence of G-CSF, whereas the cultures supplied with IL-3 and IL-4 did not show a lineage-restricted decline of CFU-GM. A heterogeneity in growth response was observed in the leukemic counterpart. With the 3H-thymidine proliferation assay, IL-4 augmented the G-CSF-induced proliferation of acute myeloid leukemic (AML) cells in 4 of the 12 cases, while the IL-3-supported proliferation was antagonized in 3 of the 12 cases. In the blast colony assay, IL-4 suppressed the IL-3-supported AML-CFU in the majority of cases, but enhanced the G-CSF stimulated AML-CFU in 3 of 6 cases. These data demonstrate divergent effects of IL-4 on the normal myeloid progenitor cell in the presence of IL-3 or G-CSF, while a variability in responsiveness is observed in the leukemic counterpart.     

Effects of soluble interleukin-1 receptor and tumor-necrosis factor receptor,respectively, on the IL-1- and TNF-α-induced DNA synthesis of acute myeloblastic leukemia blasts in vitro

Abstract: This study demonstrates that soluble interleukin-1 receptor and tumor necrosis factor receptor modulate their corresponding cytokine-induced DNA synthesis of acute myeloblastic leukemia (AML) blasts in a dose-dependent, bimodal fashion; at lower concentrations they enhanced, while at high concentrations they inhibited, the cytokine-mediated effects. Furthermore, the concentrations of endogenously produced IL-1β and TNF-a were found to be significantly (p<0.01) higher in supernatants of AML cells cultured in the presence of corresponding soluble receptors compared to their levels in supernatants of cells growing in the absence of these molecules. Our data might suggest that the attenuation of the spontaneous decay of IL-1β as well as TNF-a activities by soluble receptors may account for their ability to augment some of their effects.     

Induction of eosinophilic colonies by interleukin-5 on acute myeloblastic leukaemic cells

The effect of interleukin-5 (IL-5) on blast colony formation of acute myeloblastic leukaemia (AML) cells was studied. IL-5 showed a weak but significant colony stimulatory effect in one out of six cases studied. IL-5 induced eosinophilic colony growth in this case, and chromosomal and histochemical analyses strongly suggested that these eosinophilic colonies were derived from leukaemic blast progenitor cells. To our knowledge, this is the first report that IL-5 induces eosinophilic colonies on fresh AML cells. A possible induction of differentiation of AML blast cells by IL-5 is suggested.     

In vitro effects of insulin-like growth factor-1 (IGF-1) on proliferation and constitutive cytokine secretion by acute myelogenous leukemia blasts

The effects of insulin-like growth factor 1 (IGF-1) on autonomous proliferation, cytokine-dependent proliferation and constitutive cytokine secretion by acute myelogenous leukemia (AML) blasts were investigated using serum-free in vitro conditions. IGF-1 enhanced AML blast proliferation independent of the presence of other exogenous cytokines only for 2 of 21 patients, but for 10 additional patients IGF-1 altered blast proliferation in the presence of certain exogenous cytokines or cytokine combinations. IGF-1 had minor effects on AML blast cytokine secretion only for a subset of the patients (decreased levels for 1 patient, increased levels for 7 patients). Our in vitro observations indicate that IGF-1 can modulate AML blast proliferation and/or cytokine secretion for a subset of patients.     

Comparative effects of G-CSF, GM-CSF and IL-3 on cytosine arabinoside- and daunorubicin-mediated cytotoxicity of acute myeloid leukemia cells and normal myeloid progenitors.

We carried out an in vitro study on the combined effects of three CSF (G-CSF, GM-CSF and IL-3) plus the cycle-specific chemotherapeutic drugs [cytosine arabinoside (Ara-C) and daunorubicin (DNR)] on the proliferation and cytotoxicity of blasts and clonogenic cells (CFU-AML) in the AML-193 cell line, in AML patients and in normal bone marrow CFU-GM. The number of surviving blasts and/or DNA synthesis in blasts treated with CSF plus Ara-C or DNR was greater than those treated without CSF in the AML-193 cell line, and in some AML patients. On the other hand, the Ara-C- and DNR-mediated cytotoxicity of CFU-AML was not abrogated by CSF in any instance, but rather, it was significantly enhanced by all the CSF in the majority of instances. Although the enhancement was clearer when Ara-C was used, compared with DNR, there were no significant differences among the enhancing effects of the CSF. Under the same culture conditions as those for CFU-AML, all of the CSF significantly enhanced the Ara-C-mediated cytotoxicity of day 7 normal CFU-GM, although to a lesser extent than in CFU-AML. However, none of the CSF significantly affected the Ara-C-mediated cytotoxicity of day 14 normal CFU-GM or the DNR-mediated cytotoxicity of day 7 or day 14 normal CFU-GM. These results suggest that in the selection of a strategy entailing combined use of cycle-specific drugs plus CSF to increase the antileukemic effectiveness of chemotherapy in AML, G-CSF is preferable to GM-CSF or IL-3, since it has fewer potential clinical side effects, and that, furthermore, DNR may be as useful as Ara-C.     

Interleukin-1 stimulates proliferation of acute myeloblastic leukemia cells by induction of granulocyte-macrophage colony-stimulating factor release

In this study, we further established the role of interleukin-1 (IL-1) alpha and IL-1 beta as regulators of proliferation of acute myeloid leukemia (AML) cells. IL-1 stimulated tritiated thymidine (3H-TdR) uptake of AML cells in 13 of 28 cases. Cytogenetic analysis confirmed the leukemic clonality of the IL-1-stimulated cells. Most likely, IL-1 exerted these stimulative effects directly on AML blast cells because IL-1 effectively induced 3H-TdR uptake of CD34-positive AML blasts (separated following cell sorting). Furthermore, adherent cell-depleted AML samples of three patients were more effectively stimulated than nondepleted AML fractions. Cluster and colony formation from adherent cell depleted AML samples could also be stimulated with IL-1, ie, in seven of ten cases analyzed. Subsequent experiments indicated that IL-1 stimulation depended on the release of GM-CSF because (1) induction of DNA synthesis of AML cells by IL-1 could be abrogated with antigranulocyte-macrophage colony-stimulating factor (GM-CSF) antibody, (2) conditioned media (CM) prepared from IL-1 stimulated AML blasts (adherent cell depleted) could stimulate the proliferation of purified normal bone marrow progenitors whereas supernatants from nonstimulated AML blasts did not, and (3) GM-CSF was demonstrated in IL-1/AML-CM with a specific radioimmunoassay, while GM-CSF was not detectable in nonstimulated supernatants. In one case of AML showing significant 3H- TdR uptake in the absence of CSFs, this spontaneous DNA synthesis was found to depend on autocrine IL-1 beta release as it could be suppressed with anti-IL-1 beta antibody or anti-GM-CSF. The blockade by anti-IL-1 beta could be overcome by the addition of high concentrations of IL-1 beta as well as GM-CSF. Thus, in this particular case, endogenously produced IL-1 beta had stimulated the release of GM-CSF which resulted in GM-CSF-dependent proliferation. The results indicate that GM-CSF production by AML blasts is often regulated by IL-1 rather than being constitutive.     

Effects of interleukin-4 and interleukin-6 on the proliferation of CD34+ and CD34- blasts from acute myelogenous leukemia.

We studied the effects of interleukin-4 (IL-4) and IL-6 on the growth of leukemic blasts from 40 patients with acute myelogenous leukemia (AML). Patients were selected on the basis of negativity for a series of B-cell antigens including CD10 and CD19. Twenty-one cases were CD34-positive (CD34+) (greater than 15% of blasts) and the remaining 19 were CD34-negative (CD34-) (less than 3% of blasts). IL-4 alone (100 U/ml) could stimulate either DNA synthesis (with greater than 2.0 stimulation index) or leukemic blast colony formation in 24 of 40 AML patients. In the presence of other growth factors, IL-4 showed divergent effects on IL-3-, granulocyte-macrophage colony-stimulating factor-, granulocyte colony-stimulating factor-, or erythropoietin-dependent colony formation. These effects of IL-4 were observed in both CD34+ and CD34- AML cases. IL-6 (100 U/mL) alone could not stimulate DNA synthesis and blast colony formation except for one CD34+ case. On the other hand, IL-6 showed synergistic effects on IL-3- and IL-4-dependent blast colony formation in 10 of 12 and 7 of 9 CD34+ AML cases, respectively. Among CD34- AML cases, such synergism was seen only in 1 of 12 cases for IL-3-dependent colony formation and in 3 of 7 cases for IL-4-dependent colony formation. The divergent effect of IL-4 and the synergistic effect of IL-6 were also observed in purified CD34+ leukemic blast populations, indicating that these phenomena are not mediated by accessory cells. The present study suggests that IL-4, alone or in combination with other growth factors, has divergent effects on the growth of AML progenitors irrespective of the CD34 expression, and that IL-6 acts synergistically with IL-3 or IL-4 on the growth of leukemic progenitors preferentially in CD34+ AML.     

Effect of mast cell growth factor on clonogenic blast cell growth in acute myelogenous leukemia

 The effect of the mast cell growth factor (MGF), also known as stem cell factor, steel factor, and kit ligand, alone or in combination with other GFs on clonogenic blast cell growth in 23 patients with acute myeloblastic leukemia (AML) was investigated. MGF alone enhanced colony formation by about 35%, being clearly stimulatory (>20% increase in colony numbers) in nine patients. The additive effect of MGF on colony growth was observed in combination with interleukin-3 (IL-3). Preincubation of the cells with MGF in suspension did not sensitize them to the effect of IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, or IL-4 in a clonogenic cell culture assay. Although almost all the blast cell samples expressed the c-kit the receptor for MGF, at the mRNA and/or the protein level, the cells did not necessarily respond to exogenous MGF. On the other hand, blast cells were able to respond to exogenous MGF even when the cells themselves expressed MGF. Neither the expression of MGF nor the response of blast cells to exogenous MGF was related to the capability of the cells to form colonies spontaneously. In conclusion, MGF alone, but especially combined with IL-3, was a potent growth factor for clonogenic blast cells in AML. Autocrine production of MGF by AML blast cells analyzed at the mRNA level was not related to autonomous growth of the cells. Received: 5 December 1995 / Accepted: 2 May 1996     

The soluble form of interleukin-6 receptor modulates cell proliferation by acute myeloblastic leukemia blast cells

 As interleukin-6 (IL-6) has been shown to have diverse effects on blast cell growth in acute myeloblastic leukemia (AML), and as a soluble (s) form of IL-6 receptor (IL-6R) agonizes IL-6 effects in many cell types, we investigated whether sIL-6R was able to modulate clonogenic blast cell growth in AML. The proliferation responses of eight autonomously growing AML cell lines and eight primary AML blast cell samples were compared with their IL-6 and sIL-6R expression. Only three of the 16 AML samples were influenced by IL-6, two of them being stimulated and one inhibited by it. The sIL-6R-induced responses were more frequent, however, and, in contrast to those by IL-6, always stimulatory: clonogenic cell growth in six of the 16 AML samples was stimulated by sIL-6R treatment. All the cell lines and four of the seven primary blast cell samples analyzed expressed IL-6, and the expression was associated with unresponsiveness to exogenous IL-6. sIL-6R was also frequently expressed by AML cells: only one of the samples was negative for it. However, there was no correlation between sIL-6R expression and the responsiveness of cells to exogenous sIL-6R. The work presented here shows that sIL-6R is able to stimulate blast cell growth in AML. As AML blast cells are provided by exogenous IL-6 and sIL-6R in a bone marrow environment, and as many of them also express IL-6 and sIL-6R themselves in vitro, it is possible that signaling through the IL-6/sIL-6R system plays a role in maintaining their growth also in vivo. Received: February 20, 1998 / Accepted: November 26, 1998     

Human marrow stromal cells: response to interleukin-6 (IL-6) and control of IL-6 expression

Production of interleukin-6 (IL-6) by marrow stromal cells from human long-term marrow cultures and from stromal cells transformed with simian virus 40 was examined. As with other cultured mesenchymal cells, unstimulated stromal cells produced undetectable amounts of IL-6 mRNA when assayed by Northern blots. However, within 30 minutes after exposure of transformed marrow stromal cells to the inflammatory mediators, recombinant human interleukin-1 alpha (IL-1 alpha) or recombinant human tumor necrosis factor alpha (TNF alpha), significant increases in IL-6 expression were observed. The time course of IL-6 mRNA upregulation in transformed marrow stromal cells with IL-1 alpha and TNF alpha differed: The maximal response to TNF alpha was observed at 30 minutes whereas that to IL-1 alpha occurred at 8 hours. Although IL-6 at a concentration of 500 U/mL was inhibitory to adherent transformed marrow stromal cell proliferation, a concentration- dependent stimulation of anchorage-independent colony growth was observed when the cells were plated in semisolid medium with IL-6. The stromal cell colony-stimulating effect of IL-6 was abrogated by a neutralizing antibody to IL-6. Moreover, the heteroserum with anti-IL-6 activity and two anti-IL-6 monoclonal antibodies partially blocked autonomous and IL-1 alpha-induced colony formation, suggesting that colony formation by transformed marrow stromal cells may require IL-6. Clonal-transformed stromal cell lines were derived from the anchorage- independent stromal cell colonies. Both IL-6 mRNA and protein were constitutively produced at high levels. The addition of IL-6 to either long-term marrow culture adherent cells or transformed marrow stromal cells downregulated the expression of collagen I, a major stromal cell matrix protein. Thus, IL-6 affects proliferation of stromal cells and influences their production of extracellular matrix, suggesting that IL- 6 may have indirect as well as direct influences on hematopoietic cell proliferation.     

Effects of recombinant interleukin 4 on the growth and differentiation of blast progenitors stimulated with G-CSF, GM-CSF and IL-3 from acute myeloblastic leukaemia patients

The effects of human recombinant interleukin 4 (rIL-4) on the growth of leukaemic blast progenitors were investigated. Cells obtained from 20 acute myeloblastic leukaemia (AML) patients were evaluated using the blast colony assay in methylcellulose and suspension cultures. While rIL-4 by itself did not show any colony stimulatory activity in the blast colony assay, it suppressed the blast colony formation in methylcellulose stimulated with G-CSF, GM-CSF or IL-3 in 14 patients. In another six patients, rIL-4 enhanced blast colony growth in four patients or did not show any significant effect with any CSF in two patients. In suspension cultures of 12 cases studied, the effects of rIL-4 on the clonogenic cell recoveries were essentially similar to the results of the blast colony assay in each case. In three patients, rIL-4 augmented the differentiation of the leukaemic cells to monocyte lineage. Further, the clinical outcome was significantly different between the patients whose blast progenitors were stimulated by rIL-4 and the patients whose blast progenitors were suppressed by rIL-4 (P less than 0.05); three out of four patients in the former group failed in achieving complete remission (CR), while 12 out of 14 patients in the latter group achieved CR. The results show that the effects of IL-4 on leukaemic blast progenitors were diverse and the responsiveness to IL-4 may be correlated with the prognoses of the patients.