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Stem Cells, Vol. 14, No. 4, 445-451, July 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

The Inhibitory Effect of Human Macrophage Inflammatory Protein-1{alpha} (LD78) on Acute Myeloid Leukemia Cells in Vitro

Nadezda Basara, Stanislava Stosic-Grujicic, Dijana Sefer, Zoran Ivanovic, Petar Antunovic, Pavle Milenkovic

Institute of Hematology, Clinical Center of Serbia, Institute for Biological Research "Sinisa Stankovic," Institute for Medical Research, Belgrade, Yugoslavia

Key Words. Acute myeloid leukemia • Macrophage inflammatory protein-1{alpha} • Cell proliferation • Leukemic progenitors • Inhibitory cytokines • Bone marrow • Peripheral blood

Dr. Nadezda Basara, Institute of Hematology, Koste Todorovica 2, 11000 Belgrade, Yugoslavia.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) has recently been shown to inhibit proliferation of immature hemopoietic progenitors. In addition, significant inhibition of early and mature leukemic progenitors in acute myeloid leukemia (AML) has been obtained with MIP-1{alpha}. We performed a study of 25 AML patients at diagnosis to evaluate the effect of a human homolog of MIP-1{alpha} (LD78) on bone marrow (BM) and peripheral blood (PB) leukemic progenitors (colony-forming unit-AML [CFU-AML]) and AML cell proliferation. A methylcellulose culture system was used for CFU-AML and incorporation of 3H-TdR for AML cell proliferation. We found that LD78 inhibits CFU-AML colony formation up to 100% for the BM in 14/16 samples studied with the average maximal inhibition of 62.7 ± 9.1% and up to 100% for the PB in 12/13 samples studied with the average maximal inhibition of 71.4 ± 9.9%. In addition to this, LD78 inhibited AML cell proliferation up to 60% for the BM in 10/18 samples studied with the average maximal inhibition of 17.8 ± 3.5%, and up to 87.1% for the PB cell proliferation in 10/16 samples studied with the average maximal inhibition of 27.5 ± 6.8%. Our results have shown that LD78 is more active on AML progenitors than on AML cell proliferation. Inhibition of the AML cells, although less than that of the progenitors, indicates that more limited activity of LD78 on more mature leukemic cells is present in AML.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
Acute myeloid leukemia (AML) is a clonal malignant disease of hemopoietic stem cells characterized by the impaired production of normal blood cells and the proliferation of abnormal and leukemic blast cells in the marrow [1]. The mechanisms involved in regulation of AML cell proliferation are not fully understood [2]. Leukemia cell proliferation is maintained by a small population of leukemia blast progenitors [3]. It has been shown that AML progenitor cells are responsive to well-known stimulatory molecules that regulate normal hemopoietic stem cell proliferation [3]. On the other hand, the regulatory role of inhibitors of stem cell proliferation on AML progenitors and AML cell proliferation has not yet been established [4, 5].

Recently, a stem cell proliferation inhibitor has been characterized [6] and found to be identical to macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), an 8 kDa molecular weight glycoprotein. MIP-1{alpha} inhibited proliferation of immature hemopoietic progenitors in normal bone marrow (BM) both in vitro and in vivo [7–10]. The inhibition of normal BM progenitors might therefore be used for protection of these cells from the cytotoxic drugs effective on leukemia cells. However, more recently it has been shown that MIP-1{alpha} also inhibits early and mature AML progenitors and prevents AML progenitors from entering the proliferative phase of the cell cycle, thus indicating possible clinical benefits in AML therapy [11].

The aim of our study was to investigate the effect of the peptide LD78, the human homolog of murine MIP-1{alpha}, which shows a 747 amino acid sequence homology with MIP-1{alpha} [12], on BM and peripheral blood (PB) AML progenitor cells, as measured by in vitro colony-forming units-AML (CFU-AML) growth. In addition, proliferative status of the BM and PB AML cells in the presence of LD78 was evaluated.


   Material and Methods
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 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Patients
BM and PB samples from 25 previously untreated adult AML patients were studied (Table 1Go). In all cases, informed consent was obtained from the patients. The study was approved by the Institutional Human Research Committee. The diagnosis and classification were established according to the French-American-British (FAB) leukemia classification criteria [13]. There were one M0, one M1, seven M2, five M3, seven M4, three M5 and one M6. Median age was 53 years (range from 16 to 70 years) and sex distribution was 12 males and 13 females (1:1.08 ratio).


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  		Table 1. Clinical characteristics of AML patients at diagnosis
 
The BM cells were aspirated from the sternum, while the PB cells were obtained after venipuncture, separated by lymphoprep centrifugation (Lymphoprep, density 1.077 g/cm3, Nyegaard; Oslo, Norway) washed and resuspended in Iscove's modified Dulbecco's medium (IMDM) (Gibco; Grand Island, NY).

LD78
We have used recombinant human MIP-1{alpha} (R&D Systems; Minneapolis, MN), sometimes referred to as LD78 as described by Obary et al. [12]. MIP-1{alpha} and LD78 are interspecies homologs and, due to the high structural homology between the two genes encoding MIP-1{alpha} and LD78, the two terms are used interchangeably as described recently by Lord [14].

CFU-AML Assay
The technique of blast colony formation was used as previously described by Marie et al. [15]. Briefly, 2 x 104 T cell-depleted BM and PB cells in 0.1 ml of IMDM were plated in a culture mixture of methylcellulose (0.8%), 20% fetal calf serum (FCS) (Gibco) and 10% phytohemagglutinin-leukocyte conditioned medium in 96-multiwell culture plates, 0.3 ml per well (Becton-Dickinson; San Jose, CA). Aggregates of more than 20 cells were counted at day 7. The percentage of inhibition of CFU-AML growth in the presence of increasing concentrations (50-400 ng/ml of culture) of LD78 was calculated by comparison to the growth of these colonies in control plates without LD78.

Proliferation Assay
For the liquid proliferation system (spontaneous proliferation), cells were plated for 66 h in RPMI 1640-10% FCS in 96-well microplates at 5 x 104 BM cells/well, or 1 x 105 PB cells/well in the continuous presence of increasing concentrations of LD78 or in the absence of LD78. Spontaneous proliferation was determined by incorporation of 3H-TdR added 18 h before harvesting as already described [16]. Results were expressed as the mean cpm obtained in triplicate cultures or as the percentage of inhibition of control 3H-TdR incorporation obtained in the absence of LD78.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
It was apparent that the AML cells of 6 out of 22 patients studied (27%) did not form BM CFU-AML colonies, so the effect of LD78 in those cases could not be evaluated. Inhibition of BM CFU-AML growth with LD78 was obtained in 14/16 samples which formed colonies (Table 2Go). The overall frequency of BM CFU-AML varied from 9 to 114 colonies per 2 x 104 cells plated. However, the concentration of LD78 inducing maximal inhibition of BM CFU-AML growth varied. The maximal inhibition was most often obtained with 100 ng/ml and 200 ng/ml, but in the BM of patient No. 17 the inhibition was significant even at the smallest concentration of LD78 (25 ng/ml) (data not shown) used. The average maximal percent of inhibition of BM CFU-AML was 62.7 ± 9.1 (x ± SEM, for this and following quotations) when compared to the control cultures. The dose-response inhibition of CFU-AML growth with LD78 peaked at 100 or 200 ng/ml, with less inhibitory effect at higher doses in most of the samples studied.


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  		Table 2. Effect of LD78 on CFU-AML colony formation
 
PB CFU-AML were carried out for 18 AML samples and varied from 13-154 colonies per 2 x 104 cells. The inhibition of PB CFU-AML in the presence of LD78 varied up to 100% with an average maximal percentage of inhibition of 71.4 ± 9.9 when compared to control cultures and was usually most pronounced with the concentrations of 100 ng/ml and/or 200 ng/ml. In 5 out of 19 samples (26%) tested, CFU-AML did not grow.

To further clarify the role of LD78 in AML cell growth, BM and PB, AML cells from 18 patients were cultured in a liquid proliferation assay system with or without increasing concentrations of LD78. BM AML cell spontaneous 3H-TdR uptake varied from 78 to 13,260 cpm and was significantly inhibited in 10/18 samples, (over 10% in comparison with medium control) when LD78 was added (Table 3Go). The average maximal percent of inhibition of BM AML cell proliferation was 17.8 ± 4.4 when compared with medium control. PB AML cell spontaneous 3H-TdR uptake was measured in 16 AML samples and varied from 89 to 31,227 cpm. In the presence of LD78, spontaneous proliferation was inhibited in 10/16 samples of AML cells. The average maximal percent of inhibition of PB AML cell proliferation was 27.5 ± 6.8 when compared to medium control. Dose-dependent inhibition was observed in only two patients (no. 15 for the BM and no. 25 for both BM and PB spontaneous proliferation). In summary, the inhibitory effect of LD78 was significantly (p < 0.001) more pronounced on both BM and PB AML progenitors (62.7 ± 9.1 and 71.4 ± 9.9, respectively) when compared with more mature BM and PB leukemic cells ZASF (17.8 ± 3.5 and 27.5 ± 6.8, respectively).


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  		Table 3. Effect of LD78 on spontaneous AML cell proliferation
 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
The results presented demonstrate the inhibitory effect of LD78 on AML cell growth of both BM- and PB-derived progenitors probably mediated by an effect on AML cell proliferation. This suggests that the normal inhibitory control mechanisms mediated by LD78 are still intact in AML progenitor cells. The observed inhibitory effect of LD78 in CFU-AML growth was not related to the FAB criteria for classification of AML. Furthermore, in the same FAB group of patients, the inhibitory effect was not present in all cases tested.

An inhibitory effect of recombinant human MIP-1{alpha} on AML progenitors was also recently described [11] using other available culture techniques. The difference in degree of inhibition between the results obtained in our study and those obtained in the study of Ferrajoli et al. [11], who found less pronounced inhibition of AML progenitors in the presence of MIP-1{alpha} (up to 79% in their study versus up to 100% in ours), could be ascribed to the different molecules used for the study: recombinant human MIP-1{alpha} versus LD78, higher concentrations of MIP-1{alpha} in their study (up to 1,600 µg/ml) compared to our study (up to 400 ng/ml) and finally, different culture techniques.

Proliferation of hemopoietic progenitors is influenced by a balance of stimulatory and inhibitory effects of defined hemopoietic growth factors and cytokines [7, 10, 15]. The overall effects of these molecules on the in vitro growth of hemopoietic progenitors are the consequence of combined effects on cell growth. MIP-1{alpha} was recently described as a proliferation inhibitor [6] which is shown to act at the level of CFU-spleen (CFU-S) and CFU-Type A of the murine stem cell compartment in vitro and in vivo [7, 10, 17, 18]. In addition, MIP-1{alpha} is able to protect the multipotent progenitor CFU-S population from the effect of S-phase specific agents such as hydroxyurea [10] and cytosine arabinoside.

Broxmeyer et al. [8, 19] demonstrated the activity of MIP-1{alpha} on normal human progenitors in vitro. According to their results, it seems that MIP-1{alpha} is inactive on more mature lineage-restricted progenitor cells. Furthermore, MIP-1{alpha} seems to be a bidirectional regulator of hemopoietic progenitor growth whose effect is dependent on other growth factors present in the culture and the maturation stage of progenitors [20]. This may in part explain the heterogeneity in the effect of LD78, on CFU-AML growth observed here and in the difference in degree of inhibition on spontaneous AML cell proliferation and CFU-AML growth originating from the same patient. The mechanisms of the effect of LD78 on normal progenitor cells as well as on leukemic cells are not clear. It has been shown recently [21] that the effect of MIP-1{alpha} is direct on single-plated CD34+ cells from human BM and that it is not mediated via contaminating accessory cells. In contrast, the effect of MIP-1{alpha} on the proliferation of T lymphocytes seems to be mediated in part by the inhibition of interleukin 2 production [22]. Thus, elucidation of the precise mechanism of action of MIP-1{alpha} will require further studies.

Problems with eventual polymerization or aggregation of LD78 [18] may possibly influence the dose accuracy interpretation and therefore the results obtained. However, we obtained a clear-cut inhibition of CFU-AML colony growth and BM or PB cell proliferation in the presence of LD78 in the majority of the samples tested. The wild form of the molecule (LD78) is now available as a nonaggregating mutant form with the code name BB-10010 [23]. It is possible that a more homogenous effect on AML cells might be demonstrated with a nonaggregating form of LD78.

The inhibitory effect of LD78 observed in our study on AML leukemic progenitors and cell proliferation is unlike the effects seen with chronic myeloid leukemic progenitors. Eaves et al. [24] have recently shown that the response of normal primitive hemopoietic cells and primitive chronic myeloid leukemic cells is different. The cycling of normal BM hemopoietic cells may be inhibited by the action of MIP-1{alpha}, while primitive chronic myeloid leukemic cells show unresponsiveness to MIP-1{alpha}.

The question of the mechanisms of the overgrowth of AML cells to normal marrow cells in vivo is not yet solved. One explanation is that AML cells have become unresponsive to inhibitors of normal hematopoiesis, thus gaining a growth advantage. The effect of inhibitory factors in leukemic cells has been investigated only to a limited degree [11, 24, 25]. This study has shown that LD78 is more active on AML progenitors than on AML cell proliferation, suggesting that the limited activities of LD78 on more mature leukemic cells are present in AML, contrary to its effects in CML [25].


   Acknowledgments
 
The authors are grateful to Dr. B.I. Lord at the Paterson Institute for Cancer Research, Manchester, United Kingdom, for constructive discussion. This work was supported in part by grants from the Ministry of Science and Technology of Serbia.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

  1. Lichtman MA, Henderson ES. Acute myelogenous leukemia. In: Williams WJ, Beutler E, Erslev AJ et al., eds. Hematology. New York: McGraw-Hill Inc.,1990:251-272.

  2. McCulloch EA. Regulatory mechanism affecting the blast stem cells of acute myeloblastic leukemia. J Cell Physiol 1986:4(suppl):27–33.

  3. Löwenberg B, Touw I. Hematopoietic growth factors and their receptors in acute leukemia. Blood 1993;81:281–292.[Free Full Text]

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  6. Graham GJ, Wright EG, Hewick R et al. Identification and characterization of an inhibitor of haemopoietic stem cell proliferation. Nature 1990;6265:442–444.

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  8. Broxmeyer HE, Sherry B, Lu L et al. Enhancing and suppressing effects of recombinant murine macrophage inflammatory proteins on colony formation in vitro by bone marrow myeloid progenitor cells. Blood 1990;76:1110–1116.[Abstract/Free Full Text]

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  10. Lord BI, Dexter TM, Clements JM et al. Macrophage-inflammatory protein protects multipotent hematopoietic cells from the cytotoxic effects of hydroxyurea in vivo. Blood 1992;79:2605–2609.[Abstract/Free Full Text]

  11. Ferrajoli A, Talpaz M, Zipf TF et al. Inhibition of acute myelogenous leukemia progenitor proliferation by macrophage inflammatory protein 1-alpha. Leukemia 1994;8:798–805.[Medline]

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  13. Benett JM, Catovsky D, Daniel MTH et al. Proposed revised criteria for the classification of the acute leukemias. French-American-British (FAB) Cooperative group. Ann Int Med 1985;103:620–625.

  14. Lord BI. MIP-1{alpha}: biological and clinical perspectives. Forum 1995;5:125–141.

  15. Marie JP, Zittoun R, Thevenin D et al. In vitro culture of clonogenic leukemic cells in acute myeloid leukemia; growth pattern and drug sensitivity. Br J Haematol 1983;55:427–432.[Medline]

  16. Stosic-Grujicic S, Basara N, Milenkovic P et al. Modulation of acute myeloblastic leukemia (AML) cell proliferation and blast colony formation by antisense oligomer for IL-1 beta converting enzyme (ICE) and IL-1 receptor antagonist (IL-1ra). J Chemother 1995;7:67–70.

  17. Quesniaux VFJ, Graham GJ, Pragnell I et al. Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1{alpha} on long-term reconstituting stem cells in vivo. Blood 1993;81:1497–1504.[Abstract/Free Full Text]

  18. Cooper S, Mantel C, Broxmeyer HE. Myelosuppressive effects in vivo with very low dosages of monomeric recombinant murine macrophage inflammatory protein-1. Exp Hematol 1994;22:186–193.[Medline]

  19. Broxmeyer HE, Sherry B, Cooper S et al. Comparative analysis of the human macrophage inflammatory protein family of cytokines (chemokines) on proliferation of human myeloid progenitor cells. J Immunol 1993;150:3448–3458.[Abstract]

  20. Keller JR, Barteimez SH, Sitnicka E et al. Distinct and overlapping direct effects of macrophage inflammatory protein-1{alpha} and transforming growth factor ß on hematopoietic progenitor/stem cell growth. Blood 1994;84:2175–2181.[Abstract/Free Full Text]

  21. Lu L, Xiao M, Grisby S et al. Comparative effects of suppressive cytokines on isolated single CD34+++ stem/progenitor cells from human bone marrow and umbilical cord blood plated with and without serum. Exp Hematol 1993;21:1442–1446.[Medline]

  22. Zhou Z, Kim YJ, Pollok K et al. Macrophage inflammatory protein-1{alpha} rapidly modulates its receptors and inhibits the anti-CD3 mAb mediated proliferation of T-lymphocytes. J Immunol 1993;151:4333–4341.[Abstract]

  23. Lord BI, Woolford LB, Wood LM et al. Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein-1{alpha}. Blood 1995;85:3412–3415.[Abstract/Free Full Text]

  24. Eaves CJ, Cashman JD, Wolpe SD et al. Unresponsiveness of primitive chronic myeloid leukemia cells to macrophage inflammatory protein 1{alpha}, an inhibitor of primitive normal hematopoietic cells. Proc Natl Acad Sci USA 1993;90:12015–12019.[Abstract/Free Full Text]

  25. Holyoake TL, Freshney MG, Sproul AM et al. Contrasting effects of rhMIP-1{alpha} and TGF-ß1 on chronic myeloid leukemia in vitro. STEM CELLS 1993;11(suppl 3):122–128.

Received December 5, 1995; Revision received January 25, 1996. accepted for publication March 7, 1996.




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