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Cell Biology International (2003) 27, 739–745 (Printed in Great Britain)
Expression of hematopoietic inhibitory factors in mouse fibroblasts, macrophages and endothelial cells
La Mei Cheng, Yan Yan and Qi Ru Wang*
Experimental Hematology Laboratory, Xiang Ya Medical School, Central South University, Changsha 410078, China


Abstract

Pure bone marrow fibroblasts, macrophages and endothelial cells were cultured in Iscove-modified Dulbecco's medium. RT–PCR was used to determine the expression of inhibitory cytokine mRNAs in these cell types. Serum-free conditioned medium was collected from each cell type and ultrafiltration was performed with a centriprep 10. The retentate contained substances whose molecular weights were >10 kD, whilst the filtrate contained substances with molecular weights <10 kD. The effect of conditioned media and their components on colony forming unit-granulocyte-macrophage (CFU-GM) were investigated.

The results showed: (1) six cytokines, MIP-1α, MIP-2, TGF-β, TNF-α, IFN-γ and Tβ4, inhibited the growth of CFU-GM when murine WEHI-3 conditioned medium was added to the culture system as a source of colony stimulation. (2) The original endothelial cell conditioned medium (E-CM) did not affect the production of CFU-GM, but the >10 kD component of E-CM increased its production, and the <10 kD component decreased it. Both fibroblast conditioned medium (F-CM) and the >10 kD component of F-CM stimulated proliferation of CFU-GM, but the <10 kD component suppressed it. All three components of macrophage conditioned medium (M-CM) inhibited the growth of CFU-GM. (3) Expression of four of the mRNAs, namely MIP-2, TNF-α, INF-γ and Tβ4, was seen in all three types of stromal cells, while TGF-β mRNA was only seen in endothelial cells and macrophages, and MIP-1α mRNA in endothelial cells and fibroblasts. The inhibitors TGF-β, MIP-1α, and Tβ4have an inhibitory effect on the growth of CFU-GM, but TNF-α, INF-γ and MIP-2 do not.


Keywords: Bone marrow stromal cells, Conditioned medium, Granulocyte-macrophage colony forming unit, Hematopoietic inhibitory cytokines, Hematopoietic progenitor.

*Corresponding author. Tel.: +86-731-4805421; fax: +86-731-4471289


1 Introduction

Fibroblasts, endothelial cells, and macrophages are the major cellular components of hematopoietic stromal cells. In vitro studies have shown that stromal cells produce many cytokines, including stimulators and inhibitors that could regulate hematopoietic progenitor proliferative activity. Many stimulators produced by stromal cells have been characterized (Eaves et al., 1991; Guerriero et al., 1997; Li et al., 2000; Slanicka et al., 1998; Solanilla et al., 2000). However, knowledge of hematopoietic inhibitors is limited. TGF-β was the first inhibitor shown to be active in long-term culture systems (Cashman et al., 1990). More recently, chemokines have been found to be the active inhibitory components in media obtained from long-term cultures. The best-studied chemokine inhibitors of hematopoiesis are MIP-1α and MCPs (Bonnet et al., 1995; Cashman et al., 1998, 1999; Xu et al., 1999).

We have previously reported that bone marrow endothelial cells express the mRNA of TGF-β, MIP-2, IFN-γ and Tβ4 (Li et al., 2000). In this study, pure marrow-derived fibroblasts, endothelial cells and macrophages were used to further investigate the negative regulation of hematopoiesis by stromal cells. The expression of six hematopoietic inhibitory cytokines (MIP-2, TGF-β, IFN-γ, TNF-α, MIP-1α and Tβ4) in these three types of stromal cells, and the effect of stromal cell conditioned media on the growth of hematopoietic progenitors CFU-GM, were investigated.

2 Methods

2.1 Reagents

Inhibitors and their antibodies were from R&D Co. (Minneapolis, MN, USA). PCR primers were synthesized by Sigma (St Louis, MO, USA). RNasin, Rnasin-Oligo (dT)12–18, AMV-RTase, DEPC, dNTP, Taq DNA polymerase, and DNA markers were from Biotech Co. (Freiburg, Germany).

2.2 Culture of pure stromal cells and collection of conditioned media

Murine bone marrow-derived endothelial cells were cultured and passaged as described previously (Wanget al., 1998). Marrow fibroblasts were purified over four passages, also as described previously (Yan et al., 1990). Macrophages were obtained from murine bone marrow by culturing in Iscove-modified Dulbecco's medium (IMDM) supplemented with 20% horse serum and 20% L929-CM. Following 24 h incubation at 37 °C in a humidified atmosphere of 5% CO2, the non-adherent cells were removed, and the adherent layer was washed twice with PBS and cultured for another 96 h. The remaining adherent cells were >95% homogeneous, as determined by staining for Alpha-napthyl acetate esterase. Bone marrow-derived macrophages (BMM) were determined by staining with Mac-1 antibody. These cells were then used on day five.

E-CM, F-CM and M-CM were collected from separate confluent culture flasks that had been incubated for 48 h without serum. Conditioned media from the three cell types were ultrafiltered through a Centriprep 10 with a 10 kD cut-off. The concentrated retentate was ultrafiltered twice more after being diluted with IMDM. Finally, substances with molecular weights >10 kD were concentrated five times (designated the >10 kD component) and substances <10 kD were diluted about 500 times in the retentate after filtering several times (designated the <10 kD component). All conditioned media, including original, >10 kD and <10 kD components, were stored at −20 °C before use.

2.3 Colony forming assay

For the CFU-GM assay, 20×104BMC were plated in semi-solid medium containing 0.3% agar, 25% HS and 50 μg/ml or 15% WEHI-3-CM. Triplicate cultures were incubated in a humidified atmosphere containing 5% CO2at 37 °C. Colonies were scored under an inverted microscope after 7 days' incubation.

2.4 Antibody neutralization

In order to study the role of six different inhibitors in the E-CM, the latter was pre-incubated with antibodies to these inhibitors. The >10 kD component of E-CM was pre-incubated with antibodies to TGF-β (5 μg/ml), TNF-α (5 μg/ml), or IFN-γ (5 μg/ml) for 30 min, whilst the <10 kD component was likewise pre-incubated with antibodies to MIP-1α (100 ng/ml), MIP-2 (100 ng/ml), and Tβ4(10% Tβ4antiserum; Huang and Wang, 2001). After pre-incubation, the >10 kD and <10 kD components were added to separate CFU-GM culture systems. To evaluate the effect of the antibodies alone on the formation of CFU-GM, IMDM pre-incubated with each antibody was also assayed.

2.5 Preparation of RNA and cDNA

BMEC, BMM and fibroblast layers were washed twice with PBS. Cells were collected, and lysed by extraction buffer (0.14 M NaCl, 1.5 mM MgCl2, 10 mM Tris–HCl, pH 8.6, 0.5%NP-40, 1 mM DTT, 1000 U/ml RNasin). Total cellular RNA was extracted. The concentration of RNA was measured by A260absorption. Five μg of total cellular RNA was reverse transcribed in the presence of 30 U AMV-RTase, 1.0 mM Oligo(dT)12–18, 20 U RNasin, and 1.0 mM dNTP in a total volume of 50 μl. The reaction was run at 42 °C for 2 h and 94 °C for 5 min. The mixture was then quickly chilled on ice.

2.6 PCR analysis

PCR primers were designed according to their sequence (Table 1). We first prepared PCR reaction stock solutions containing sterile H2O, 5×PCR buffer, 200 μmol dNTPs, 20 pmol of each primer and 2.5 U Taq DNA polymerase. Twenty-one μl stock solution was then aliquoted into each labeled 0.5 ml microcentrifuge tube, and 4 μl cDNA added. The mixture was overlaid with 75 μl paraffin oil and the PCR was performed in a DNA Thermal Cycler for 30 cycles; 50 s denaturation at 94 °C, 50 s annealing at 60 °C and 1 min extension at 72 °C, followed by a final extension for 10 min at 72 °C. Six μl PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide in 0.5% TBE running buffer. Molecular weight markers were run in parallel. PCR products were examined on the gel, and photographed using a UVP Image Store 5000 ultraviolet camera.


Table 1. Sequence of PCR primers, length of PCR products

Image

F: forward; R: reverse.

2.7. Statistical analysis

Results are expressed as means±SD, obtained from three experiments. The significance of differences between the means was determined using Student's t-test.

3. Results

3.1. Effects of inhibitors on the colony formation of CFU-GM

MIP-1α, MIP-2, TGF-β, TNF-α, IFN-γ, and Tβ4were added separately to CFU-GM culture systems. The different inhibitors were used at concentrations chosen according to previously published data: MIP-2 at 100 ng/ml, TNF-α at 25 ng/ml, TGF-β at 2 ng/ml, IFN-γ at 25 ng/ml, MIP-1α at 100 ng/ml, Tβ4at 10−9M. The results showed that Tβ4inhibited the growth of CFU-GM in semi-solid agar culture when GM-CSF or WEHI-3-CM were used as colony stimulators. However, the other five inhibitory cytokines showed various effects on CFU-GM when different stimulators were used in the culture system. When rmGM-CSF was used as a source of colony stimulating activity, these five inhibitory cytokines enhanced the growth of CFU-GM. When conditioned medium from the WEHI-3 cell line was used, they inhibited its growth (Fig. 1).



Full-size image (11K) - Opens new windowFull-size image (11K)


2.7 Statistical analysis

Results are expressed as means±SD, obtained from three experiments. The significance of differences between the means was determined using Student's t-test.

3 Results

3.1 Effects of inhibitors on the colony formation of CFU-GM

MIP-1α, MIP-2, TGF-β, TNF-α, IFN-γ, and Tβ4were added separately to CFU-GM culture systems. The different inhibitors were used at concentrations chosen according to previously published data: MIP-2 at 100 ng/ml, TNF-α at 25 ng/ml, TGF-β at 2 ng/ml, IFN-γ at 25 ng/ml, MIP-1α at 100 ng/ml, Tβ4at 10−9M. The results showed that Tβ4inhibited the growth of CFU-GM in semi-solid agar culture when GM-CSF or WEHI-3-CM were used as colony stimulators. However, the other five inhibitory cytokines showed various effects on CFU-GM when different stimulators were used in the culture system. When rmGM-CSF was used as a source of colony stimulating activity, these five inhibitory cytokines enhanced the growth of CFU-GM. When conditioned medium from the WEHI-3 cell line was used, they inhibited its growth (Fig. 1).


Fig. 1

Effects of six inhibitory cytokines (MIP-2, TGF-β, IFN-γ, TNF-α, MIP-1α and Tβ4) on colony formation of CFU-GM. Results are expressed as mean percentage ±SD of control for three experiments. (A) Colonies stimulated by GM-CSF (B) Colonies stimulated by WEHI-3-CM. Compared with control, *P<0.05, **P<0.01, ***P<0.001.


3.2 Effects of conditioned media from three types of stromal cells on the growth of CFU-GM

Conditioned media from pure bone marrow-derived fibroblasts, endothelial cells and macrophages were collected and divided by ultrafiltration into the concentrated retentate (>10 kD) and the filtrate (<10 kD). The addition of original E-CM to the CFU-GM culture system did not affect its growth. Addition of the >10 kD component of E-CM resulted in a significant increase in CFU-GM numbers, while the <10 kD component gave a significant decrease. The addition of original F-CM and the >10 kD component of F-CM stimulated the growth of CFU-GM, but the <10 kD component inhibited its growth. The addition of M-CM and its ultrafiltration components to the CFU-GM culture system all caused a significant decrease in the CFU-GM numbers (Fig. 2).


Fig. 2

Effects of three types of stromal cell conditioned media and their ultrafiltration components on the growth of CFU-GM. (A) E-CM and its ultrafiltration components. (B) F-CM and its ultrafiltration components. (C) M-CM and its ultrafiltration components. Each bar indicates the mean±SD for colony numbers in three experiments. Compared with control, *P<0.05, **P<0.01, ***P<0.001.


3.3 Biological activity assay of inhibitors in conditioned medium

E-CM pre-treated with antibodies to the six inhibitors was used to study their biological activity in conditioned medium. The >10 kD component of E-CM was used to study the roles of TGF-β, IFN-γ and TNF-α, whose molecular weights exceed 10 kD. The <10 kD component of E-CM was used to study the roles of MIP-2, MIP-1α and Tβ4, whose molecular weights are below 10 kD. After pre-incubating the >10 kD component with antibodies to TGF-β, IFN-γ and TNF-α, and pre-incubating the <10 kD component with antibodies to MIP-2, MIP-1α and Tβ4, their effects on colony formation of CFU-GM were assayed. To evaluate the effects of the antibodies alone on colony formation, control media were also separately pre-treated with the same antibodies. The results showed that adding antibodies alone to the culture system did not influence the growth of CFU-GM.

The >10kD component pre-treated with anti-TGF-β markedly increased its stimulatory effect on CFU-GM, but the >10 kD component pre-incubated with anti-IFN-γ and TNF-α had no effect. This indicates that, with regards to the >10 kD component of E-CM, TGF-β alone exerts a significant suppressive effect on the formation of CFU-GM. The <10 kD component pre-incubated with anti-MIP-1α and Tβ4significantly reduced its inhibitory effect on CFU-GM, but the <10 kD component pre-incubated with anti-MIP-2 had no effect. This indicates that, with regards to the <10 kD component of E-CM, MIP-1α and Tβ4, but not MIP-2, exert a significant suppressive effect on the formation of CFU-GM (Fig. 3).


Fig. 3

The effects of (A) >10kD component of E-CM pre-incubated with antibodies to TNF-α, IFN-γ and TGF-β, and (B) <10kD component of E-CM pre-incubated with antibodies to MIP-1α, Tβ4, and MIP-2 on the growth of CFU-GM. Each bar indicates the mean±SD of three experiments. Compared with control, *P<0.05.


3.4 Expression of inhibitory cytokine mRNA

RT–PCR analysis showed that endothelial cells expressed specific mRNAs for all six inhibitors, fibroblasts expressed mRNAs for all except TGF-β, and macrophages expressed mRNAs for all except MIP-1α (Fig. 4). No amplified fragment caused by DNA contamination was detected in any experiment.


Fig. 4

mRNAs expression. (A) Bone marrow-derived endothelial cells; (B) Fibroblasts; (C) Bone marrow-derived macrophages. Lane 1: Marker (2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); Lane 2: β-actin; Lane 3: MIP-2; Lane 4: TGF-β; Lane 5: IFN-γ; Lane 6: TNF-α; Lane 7: MIP-1a; Lane 8: Tβ4.


4 Discussion

In this study, hematopoietic inhibitors from pure fibroblasts, endothelial cells and macrophages were investigated. To elucidate the nature and effect of negative regulation by cytokines on stromal cells, serum-free conditioned media from these three types of pure stromal cells were collected. The effects of stromal cell-conditioned media and their ultrafiltration components on CFU-GM formation were investigated. The results showed that E-CM had no significant influence on the growth of CFU-GM. F-CM, >10 kD component of F-CM and >10 kD component of E-CM stimulated the growth of CFU-GM, whilst <10 kD component of E-CM, <10 kD component of F-CM, as well as M-CM and its >10 kD and <10 kD components all inhibited its growth.

Using a colony forming assay, we detected the effects of MIP-1α, MIP-2, TGF-β, Tβ4, IFN-γ and TNF-α, which are thought to be inhibitory cytokines, on the growth of CFU-GM. Our results showed that Tβ4had an inhibitory effect on CFU-GM when GM-CSF or WEHI3-CM were used as colony growth stimulators. However, the other five inhibitory cytokines revealed contrasting effects when different stimulators were used. When rmGM-CSF was used as a source of colony stimulating activity, they enhanced the growth of CFU-GM, but when conditioned medium from WEHI-3was used as a stimulator, they inhibited growth. The effects of these cytokines on CFU-GM formation are approximately in agreement with the effect of MIP-1α on CFU-GM formation. The authors have previously reported that MIP-1α enhances the formation of CFU-GM when M- or GM-CSF are used as colony stimulators, but suppresses its growth when early acting hematopoietic stimulators, such as SCF or G-CSF are used (Broxmeyer et al., 1990). Since stromalcells secrete early acting hematopoietic stimulators, such as SCF, G-CSF, IL-6, and IL-1, they may have an inhibitory effect on CFU-GM growth when the six inhibitory cytokines occur in F-CM, E-CM or M-CM.

In this study, we reported that bone marrow endothelial cells express MIP-1α, MIP-2, TGF-β, Tβ4, IFN-γ and TNF-α mRNA. Endothelial cells also secrete hematopoietic stimulators such as SCF, G-CSF, GM-CSF, IL-6, IL-1, and IL-11, whose molecular weights are more than 10 kD. These hematopoietic stimulators occur in the >10 kD component of E-CM. TGF-β, TNF-α and IFN-γ, also >10 kD, are hematopoietic inhibitors, and also occur in the >10 kD component of E-CM. According to the antibody neutralization experiments, of these three inhibitors, only TGF-β had an inhibitory effect on CFU-GM. MIP-1α, Tβ4and MIP-2, all of which are hematopoietic inhibitors with molecular weights less than 10 kD, occur in the <10 kD component of E-CM. According to the antibody neutralization experiments, of these three inhibitors, only MIP-1α and Tβ4had an inhibitory effect on the growth of CFU-GM.

As mentioned above, many stimulators and inhibitors are present in E-CM. However, hematopoietic stimulators and inhibitors may occur in equilibrium in E-CM, which may be the reason that E-CM had no significant influence on the growth of CFU-GM. Hematopoietic stimulators may be predominant in the >10 kD component of E-CM, whilst inhibitors may be predominant in the <10 kD component. This may explain why the >10 kD component of E-CM stimulated proliferation of hematopoietic progenitors, while the <10 kD component inhibited it. Recently, several authors have reported that endothelial cells or endothelial cell conditioned medium were more readily able to expand hematopoietic stem cells than other stromal cells (Cheng and Wang, 2002; Chute et al., 2002; Davis and Lee, 1997). We propose that TGF-β, which is produced by endothelial cells but not by fibroblasts, may be at least partly responsible for this effect in E-CM. Inhibitor(s) could protect hematopoietic stem cells from excessive differentiation in their expansive system (Cheng and Wang, 2002).

Fibroblasts are able to secrete various cytokines, such as GM-CSF, G-CSF, SCF, M-CSF, LIA, HGF, bFGF, IL-6, IL-11, IL-8, RANTES, MIP1and MCP (Brouty-Boye et al., 1998; Denburg, 1995; Imokawa et al., 1998; Karagenc and Petitte, 2000). Besides SCF, GM-CSF, G-CSF, M-CSF, IL-6 and IL-11 being supporters of hematopoiesis, LIA, HGF and bFGF are also hematopoietic stimulators. In this study, we detected the presence of mRNA for MIP-2, MIP-1α, TNF-α, Tβ4and IFN-γ in fibroblasts. However, hematopoietic stimulators may predominate in F-CM and the >10 kD component of F-CM. This may explain why they both stimulated formation of CFU-GM. The presence of MIP-1α and Tβ4in the <10 kD component of F-CM may be the reason that it inhibited formation of CFU-GM.

Macrophages are one of the constituents of the hematopoietic microenvironment. In long-term bone marrow cultures in vitro, macrophages are part of the stromal layer (Allen and Dexter, 1983; Mori et al., 1990). Early evidence suggested that macrophages played an important role in the regulation of hematopoiesis. Although BMM can produce growth-stimulating cytokines such as SCF, M-CSF, G-CSF, EPO and IL-6 (Kurland et al., 1978), it also produces growth inhibitory cytokines such as TNF-α, IFN-γ, TGF-β and PGE (Rossi et al., 1980). After induction, macrophages can produce MCP-1, MIP-1α and MCP-3 (Jarmin et al., 1999). In this study, we detected the presence of mRNA for TGFβ, TNF-α, IFN-γ, MIP2and Tβ4in bone marrow-derived macrophages. It is clear from our data and that of others, that the inhibitors could be the main cytokines in M-CM. This explains why M-CM and its >10 kD and <10 kD components are able to inhibit CFU-GM formation.

We have reported here that fibroblasts, endothelial cells and macrophages express different inhibitors. Among these inhibitors, TGF-β, Tβ4and MIP-1α have inhibitory effects on the growth of CFU-GM, whereas TNF-α, IFN-γ and MIP-2 do not. E-CM, F-CM, M-CM and their >10 kD and <10 kD components contain stimulators and inhibitors, and exert different effects on the growth of progenitors. Our investigation of the inhibitors expressed in three types of pure stromal cells may be useful for the expansion of hematopoietic stem/progenitor cells by stromal cell layers or stromal cell conditioned medium, and also aid understanding of the mechanisms by which stromal cells regulate hematopoiesis.

Acknowledgements

This work was supported by the National Natural Sciences Foundation of China (Nos. 39970092 and 30030070).

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Received 18 June 2002/1 April 2003; accepted 25 May 2003

doi:10.1016/S1065-6995(03)00144-6


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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