British 1oournal of Hacrnatologg, 1992. 8 1, 1-5
New insights into the regulation of megakaryocytopoiesis by haematopoietic and fibroblastic growth factors and transforming growth factor b1 c. H A N , S Y L V I A BELLUCCI,H A I Y. W A N A N D JACQUES P. C A E N Znstitut des Vaisseaux et du Sang, ZNSERM U 1 5 0 , Hdpital Lariboisitre, Paris, France, and *Jiangsu Znstitute of Haematology. Suzhou, China ZHONG
Received I 5 July 1 9 9 1 ; accepted for publication 13 December 1 9 9 1
Summary. Etrects of cytokines on murine megakaryocyte (MK) colony formation from either unfractionated marrow cells or purified early haematopoietic cells were studied. Recombinant interleukin-3 (IL3),interleukin-6 (IL6),granulocyte-macrophage colony-stimulating factor (GM-CSF). erythropoietin (Epo) and acidic and basic fibroblast growth factor (aFGF and bFGF) each was able to stimulate MK colony growth although they varied somewhat in their potential. IL6 and FGFs, in addition to their effect on MK colony growth, increased the size of individual MK. The combination of IL3 with IL6 or FGF resulted in an additive action. Monoclonal anti-IL6 antibody completely neutralized the activity of
mouse IL6 and FGFs but had no effect on human IL6, mouse IL3 and GM-CSF. When using purified lineage negative marrow cells. only IL3 and IL6 promoted MK colony formation. Transforming growth factor 81 (TGF-Bl) at 10200 pg/ml selectively inhibited IL3-induced MK colony formation, and at 0.2-0.5 ng/ml it still had no obvious effect on the activity of IL6 or GM-CSF but caused an inhibition of FGF-induced MK colony formation. These data suggest that differential mechanisms are involved in the regulation of megakaryocytopoiesis by IL3, IL6, FGFs and GM-CSF, and that TGF-/I1 negatively regulates MK development mainly by interfering with the action of IL3.
Megakaryocytopoiesis is a multi-step cellular and biological process which is regulated by a number of cytokines. During the early stage of megakaryocytopoiesis. megakaryocyte (MK) progenitor cells proliferate and differentiate toward MK in the presence of cytokines possessing MK colony-stimulating activity (MK-CSA). The maturation of immature MK to produce platelets also is a humorally regulated process. Cytokines capable of stimulating megakaryocytopoiesis include interleukin-3 (IL3). interleukin-6 (IL6). granulocytemacrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) as well as two uncharacterized factors, the MK-CSF and thrombopoietin (TPO) (Hoffman, 1989: Williams. 1490; Han et al. 199 l ). In addition, a role of basic fibroblast growth factor (bFGF) in haematopoiesis has been suggested (Gabblanelli et a/. 1990; Gallicchio et al, 1991). Recent studies have further shown that megakaryocytopoiesis is negatively controlled by transforming growth factor fl (Ishibashi ut ul, 1987; Solberg et a/. 1987; Mitjavila et ul. 1988), interferon-alpha and -gamma (Han et a!, 1987; Canser et a / . 1987). platelet factor 4 and its related proteins (Cewirtz uf al, 1989: Han et a/. 1990, 1991) and a
glycoprotein released by platelets (Dessypris et al. 1987). Among these inhibitors TGFB is the most potent since it acts at picomolar levels. While these factors affect the proliferation and maturation of MK and their progenitor cells, their action mechanisms and their potential relationships remain poorly understood. In the present study we analysed the effects of several cytokines including IL3, IL6, GM-CSF, Epo. FGF and TGFB alone or in combination on MK colony growth from either unfractionated bone marrow cells or purified lineage negative (Lin-) marrow cells. We demonstrated that these cytokines affect megakaryocytopoiesis by differential mechanisms. MATERIALS AND METHODS
Growthfactors and antibodies. Purified recombinant murine IL3, IL6 and GM-CSF, and rat antimouse IL3 and IL6 monoclonal antibodies (anti-IL3 and anti-IL6. respectively) were purchased from Genzyme Corporation. Recombinant human IL6 and purified human platelet-derived TGF-Bl were purchased from British Bio-Technology Inc.. U.K. Recombinant erythropoietin was provided by Amersham, France. Recombinant human acidic FGF (Mascarelli et a/,
Correspondence: Dr 2. C. Han. Institut des Vaisseaux et du Sang. Hdpital Lariboisiere. h Rue Guy-Patin. 7501 0 Paris. France.
Zhong C. Hun et aJ
1991) and bovine bFGF (Carlo Erba. Milan, Italy) were provided by Dr Y. Courtois, INSERM U 118, Paris. Purification of lineage negative (Lin-) bone marrow cells. 8-week-old BALB/c mice (IFFA CREW. France) were killed by cervical dislocation. The femurs were removed and the marrow expelled with 5 ml ofalpha medium (Eurobio. Paris). Cells were washed twice by centrifugation in alpha medium at 250 g for 10 min at 22°C. Bone marrow cells were layered on lymphocyte separation medium (Eurobio-Paris) to obtain light density cells. Lincells were isolated using published method (Spangrude et a/. 1988). Briefly, 1 x lox cells were incubated at 4OC in alpha medium plus 5% fetal calf serum with a cocktail of antibodies, the rat monoclonal antibody to mouse B cells (KA3-6B2). to mouse granulocytes (RA6-8c), to mouse macrophages (MI-70.15). to mouse Ly-2 (CD8a) and to mouse L3/T4 (CD4) (Caltag Laboratories, South San Francisco). Each antibody was used at 1 pg/l x 10h cells. After 30 min incubation the cells were washed twice, centrifuged, and resuspended in the same buffer to a density of 1 x 1OHcells/ml Magnetic beads coated with sheep anti-rat IgC antibody (Dynal, Norway) were added to the cell suspension at a bead to cell ratio of 40: 1 and then incubated for 30 min at 4OC. The cells were then magnetically separated with a magnetic particle concentrator (Dynal) and the Lin- cells obtained were washed and used for culture. Colony-forming cell assays. Megakaryocyte colony-forming cells were assayed using a plasma clot system (Han et a/. 1987. 1990), with slight modifications. For culture of marrow unfractionated cells, 2 x 1 O5 nucleated cells were cultured in at least triplicate in Petri'dishes ( 3 5 mm) in a total volume of 1 ml alpha medium containing 1%bovine serum albumin (BSA) (Sigma Chemical Co.. St Louis, Mo.), 10% citrated bovine plasma (Flow Laboratory, Paris), 1 x M 2-mercaptoethanol (Sigma), 0 . 3 4 mg CaClz and 1 5 u penicillin + 1 5 p g streptomycin with or without growth factors or antibodies. For Lin- marrow cells, 1 x lo4cells per well were plated in octuplicate in 24-well microplates in 0.2.5 ml culture medium as described above. After 7 d of culture the clots were dried in situ with filter papers, fixed with 1%paraformaldehyde and stained for acetylcholinesterase staining (Jackson, 1973) to detect megakaryocyte colonies. followed by haematoxylin staining for CFU-GM. Identification of colonies was performed as previously described (Han et al. 1990). A CFU-MK-derived colony was defined as a cluster of three or more cells and a mCFU-MKderived colony was defined as a MK colony mixed with other haematopoietic lineages. A CFIJ-GM-derived colony was defined as a cluster of at least 40 morphologically recognizable granulocyte-macrophages. MK cell diameter was measured using an eyepiece micrometer (Zeiss, Germany) and calculated by determining the geometric mean of two perpendicular diameters of each acetylcholinesterase-positivecell. At least 2 0 0 MK from each group were examined.
RESULTS Ejjiict of haeniatopoietic growth factors on colony formation The results obtained from the experiments using unfrac-
Table 1. Effects of cytokines on the growth of CFU-MK and CFIJ-GM
(2x 1 0 i cells plated
h f 1
111f12 l 2 2 f I4
12f2 22f3 29f 3 28f2
llf3 25*3 28f4 30f 3
1 ng 5 ng* 1 0 ng
13f2 2hf2 22f 3
1 ng 10 ng 2 0 ng*
5 0 ng
Ilk2 18k3 2Yf3 24f3
13f4 22f 3 23f3 21f2
1 ng 10 ng 2 0 ng" 5 0 ng
IOf2 21f3 28f2 27f3
14f 3 25&2 22h3 21 f2
0.5 u 1 u'
lOf2 llf2 9f2
if1 hf2 8f2
7f2 8f2 5f2 6f2 4f 1
if 3 hf2
Interleukin- 3 10 u 50 u 100 u* 150u Interleukin-h 1 ng 1 0 ng 20 ng* 50 ng
TGF-p 1 10 Pg 100 Pg 500 pg 1000 pg 5000 pg
h f 1
5f2 h f l
Data represent the mean fSEM of triplicate determinations from three separate experiments. * indicates the optimal concentration for inducing megakaryocyte development ( P < O . O 5 compared to control).
tionated marrow cells were summarized in Table 1. In control cultures containing no added growth factors, the average of MK colonies was 5.1 f 0 . 4 MK colonies/dish. When various concentrations of mouse IL3, GM-CSF. human IL6, Epo and bovine FGFs were added to cultures, a n increase in MK colony number was observed for each of them. Optimal concentration of these factors for stimulating MK colony formation was seen at 100 u/ml for IL3.20 ng/ml for IL6 and FGFs, 5 ng/ml
Cytokine Regulation of Megakaryocytopoiesis
Table 11. Etfect of cytokines at their optimal concentration alone or in combination on megakaryocyte ( M K ) development in vitro
( 2 x l o 5 cells) ~~
No addition 11.3 IL6 (;M-CSF ZIFCF hPGF
0 4f1 0 3fl 0 0 if1 5f2 4f 1 h+ I
11.3 +aFGF 11,3 + hFGF IL3+CM-CSF I I h + aFGF Il,h t hFCF Ilh +GM-CSF aFCF + GM-CSF hFGF CM-CSF
0 2 f1 4f1 5 f1
5f1 81419' 28f5' 26f3* 27=t3* 3Of3* 125f 10' 113f12' 118*13*
76f9* 31 f3* 29f3' 33f4' 31f4' 29f3'
MK diameter MKs/colonyt (rim) CFU-CM 3.4fl 4.3fl 3.hfl 4.2f1 4.lf2 4.2fl 6.3f2* 6.5+2* 6.1+2* 5.4f2' 4.0f 1 4.2fl 4.5f2 4.7f1 4.1&1
21 f 2 22f 3 29f 3* 21f3 28f4' 2 7 f 3' 27f2* 26f4' 2 5 f 3* 21f3 28+2* 29f 3* 2 7 f 3* 28k.4' 2 7 f 3"
6f2 1 1 1 f 12*
2 9 f 7* 69 f8' 17f3' 2 0 f 3* 109fll' 98 f9* 8 7 f 11' 187f17' 3 1 f 3* 28f4' 108 f7' lllf9* 121f11'
Data represent the mean fSEM of triplicate determinations from three to live separate experiments. *indicates P < 0 . 0 5 compared to No addition as determined by Student's t test. tall MK colonies were examined.
for GM-CSF and 1 u/ml for Epo. TGF-PI alone had little effect on MK colony formation. At their optimal concentration. 11.3 was more potent than IL6. GM-CSF and FGFs in promoting MK development (Table 11). IL3 and GM-CSF have found to be able to induce the formation of mCFU-MK-derived colonies. IL3 induced more mCFU-MK-derived colonies than did GM-CSF. The combination of IL3 with IL6 or FGFs resulted in a significant increase in the number of MK colonies compared to these factors alone. This additive action was found not only in the number of MK colonies but also in the size of MK colonies. When combining these factors, an increase of MK colony size was observed mainly in the IIh- or FGF-containing cultures. Furthermore, the addition of IL6 or FGFs into cultures caused a significant increase in MK diameter. one of the parameters of MK maturation (Table 11). The antibody neutralizing experiments were then performed in order to determine the action mechanisms of these factors. Table I11 shows that anti-113 neutralized the activity of mouse IL3 but not the other factors. In contrast, a n t i 4 6 neutraliied the activity of mouse IL6 and FGFs but had no effect on the action of human IL6 and mouse IL3. Culture experiments using purified [,in- marrow cells were subsequently performed to determine whether these cytokines influence MK colony formation in the absence of lineage positive cells which may produce growth factors affecting MK development. The results (Table IV) show that only IL3 and 1Lh significantly promoted MK colony formation from Lin- marrow cells. GM-CSF. Epo or FGF alone did not induce significant MK colony growth. TGF-fl has been known to be able to inhibit MK colony formation (Ishibashi et ul. 19 87: Solberg et a / , 1987: Mitjavila et al, 1988). However, the mechanism of the action of TGF-/3
Table Ill. EKect of the neutralizing anti-IL3 and -1L6 antibodies on the
action of cytokines in stimulating megakaryocytopoiesis
No. of megakaryocyte coloniesi2 x 1 Oi cells
None IL3 Mouse ILh Human ILh aFGF hFGF CM-CSF
5fl 92f7 31f3 29f4 28f3 30f2 27f2
6f2 93f8 5f2 30f 3 5f2 7f2 31 f 3
6fl 8f2 30* 3 28f2 30f 5 28+4 29&3
Murine bone marrow cells were cultured in quadruplicate for 7 d without or with mouse IL3 ( 1 0 0 u/ml). mouse or human IL6 (20 ng/ ml). mouse GM-CSF ( 5 ng/ml). aFGF or hFGF ( 2 0 ng/ml). Rat antiIL6 and anti-113 monoclonal antibodies were added at 5 pg/ml respectively.Data represent the mean fSEM of determinations from two separate experiments.
remains poorly understood. If one considers potential mechanisms for TGF-8 action, the most proximate would include inhibition of MK progenitor cell proliferation stimulated by factors possessing MK-CSA such a s IL3. IL6. GM-CSF. FGFs and Epo. We attempted to assess this hypothesis in several ways. First. we tested the effect of TGF-P1 on MK colony growth promoted by these factors a t their optimal concentration. As can be seen in Fig 1, the inhibitory effect of TGF-jl was dose-dependent and factor-selective. At concentrations as low as 10-100 pg/ml. TGF-PI significantly inhibited MK
Zhong C. Hun et a1
colony formation induced by IL3 but not by the other factors. TGF-Bl at 200-500 pg/ml inhibited FGF promoted MK colony formation but still had no effect on the activity of GMCSF and IL6. However, TGF-/?I at 1or 5 ng/ml caused 45% or 67% inhibition of GM-CSF promoted MK colony formation, respectively. The addition of TGF-B1 at concentrations of 100-5000 pg/ml into IL6 or Epo-containing cultures did not result in any significant change in MK colony growth. We then tested the effect of TGF-Bl on cytokine induced MK colony formation from Lin cells. It was found that TGFfll only inhibited the action of IL3 (Table IV).
Table IV. Etrect of cytokines on megakaryocyte colony growth from purified lineage negative (Lin-) bone marrow cells Megakaryocyte colonies/l O4 Lincells Factor
Medium IL 3 IL6 IL3/IL6 CM-CSF
0.5f0.2 8fl.l* 5f1.2' 25 f 1.9' 1.3f0.5 2.1 5 0 . 8 1.1 f 0 . 5 14.lf0.4
With TGF-/l 0
ot 4zk0.7 12 f 2 . l - t 1.2f.0.2 2.1 f0.6 0 0
Data are the mean fSEM of a typical experiment, each performed in octuplicate. The concentration ofthe factors was at 100 U/ml for IL3. 20 ng/ml for IL6.5 ng/ml for GM-CSF. 1 U/ml for Epo and 20 ng/ml for FGF. *Different from cultures without factor (P