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Stem Cells, Vol. 15, No. 2, 133-143, March 1997
© 1997 AlphaMed Press

The Broad Spectrum of Cytokine Gene Expression by Myoid Cells from the Human Marrow Microenvironment

Luc Sensebe, Marie Deschaseaux, Jian Li, Patrick Herve, Pierre Charbord

Laboratoire d'Etude de l'Hématopoièse, Etablissement de Transfusion Sanguine, Besançon, France

Key Words. Stromal cell • Smooth muscle cell • Stem cell • Interleukin • Growth factor • Polymerase chain reaction • Marrow culture

Correspondence: Dr. P. Charbord, Directeur de Recherches INSERM, Etablissement de Transfusion Sanguine, 1, Boulevard A. Fleming, 25020 Besançon, France.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Nontransformed stromal colony-derived cell lines (CDCLs) consist of a pure stromal cell population that differentiates following a vascular smooth muscle cell repertoire, and whose in vivo counterpart is that of myoid cells found in adult and fetal human bone marrow cords. We studied the cytokine expression by reverse-transcriptase polymerase chain reaction (RT-PCR) from pooled fast-growing clones from 10 different bone marrow samples.

RT-PCR indicated that 30 cytokines (out of 42 studied) were expressed by CDCLs (20 after medium renewal and hydrocortisone renewal, three after addition of interleukin 1ß (IL-1ß) and seven in only part of the CDCL layers examined). The cytokines expressed comprised mediators known to be involved in the maintenance of early and late hematopoiesis (IL-1{alpha} and IL-ß, IL-6, IL-7, IL-8, IL-11 and IL-13; colony-stimulating factors, thrombopoietin, erythropoietin, stem cell factor, flt 3-ligand, hepatocyte cell growth factor, tumor necrosis factor {alpha}, leukemia inhibitory factor, transforming growth factors ß1 and ß3; and macrophage inflammatory protein 1{alpha}), angiogenic factors (fibroblast growth factors 1 and 2, vascular endothelial growth factor) and mediators whose usual target (and source) is the connective tissue-forming cells (platelet-derived growth factor A, epidermal growth factor, transforming growth factors {alpha} and ß2, oncostatin M and insulin-like growth factor 1), or neuronal cells (nerve growth factor). The cytokines not expressed were lymphokines (IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, and IL-12 and interferon {gamma}) or mediators synthesized by macrophages (inhibin, activin, platelet-derived growth factor B, and IL-1 receptor antagonist).

This study complements the description of the phenotype of the myoid cells, confirming that these cells are the marrow connective tissue-forming cells; moreover, this work suggests that stromal control of hematopoiesis is multifactorial and that myoid cells are involved in the control of marrow angiogenesis and innervation.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
Although widely recognized as cells crucial for the maintenance of multilineal hematopoiesis in humans [1], sheep [2], mice [3], birds [4] and fishes [5], stromal cells still remain poorly understood in terms of tissue derivation. This lack of knowledge results from several obstacles. In adherent layers of long-term marrow cultures, many cell types are distinguishable by morphology; however, beyond morphology, criteria of investigation are difficult to define. CD antigens did not provide major clues except for the fact that stromal cells were CD45 negative [6] and therefore clearly distinct from adherent macrophages, and probably not of hematopoietic origin. Some monoclonal antibodies recently appeared to be of interest [7, 8], although the recognized membrane antigens are not found solely on stromal cells, and the ultimate biochemical characterization of the antigens has not been performed. Due in part to the previous difficulties, purified populations of stromal cells were difficult to obtain and many teams resorted to the generation of continuous cell lines resulting from selection after passages or from transformation/ immortalization using viral vectors; both methods are susceptible to sensibly modifying the phenotype, leading to daughter-lines far removed from the parental cells [9]. We decided, therefore, to study intracellular antigens. Cytoskeletal proteins appeared to be of special interest since study of intermediate filaments, actin isoforms and actin-binding-proteins allowed us to define a precise stromal cell component from human long-term cultures (LTCs), that of vascular smooth muscle-like stromal cells [10]. Studies on extracellular matrix proteoglycans and adhesive glycoproteins [11] were in line with this description, as well as glycoproteins found in lysosomes of stromal cells [12, 13]. The vascular smooth muscle-like stromal population appears to be the major one in human LTCs. In murine cultures, a similar population, although less widespread, has also been described [14].

In vivo studies on trephine bone marrow biopsies strongly suggest that the vascular smooth muscle-like stromal cell population is the in vitro counterpart of myoid cells found in hematopoietic marrow cords in fetus and adults [10, 15].

We isolated stromal cells with this phenotype by developing cell lines from stromal colonies (colony-derived cell lines or CDCLs) [16]. This population was obtained after a four-step purification procedure: A) establishment of primary layer from long-term marrow culture; B) generation of clones by seeding cells, from primary layer treated by trypsin, in methylcellulose in the presence of interleukin 1ß (IL-1ß) and tumor necrosis factor {alpha} (TNF-{alpha}); C) generation of lines by plucking 15 to 20 clones from the methylcellulose and returning these in liquid culture comprising LTC medium supplemented with fibroblast growth factor 2 (FGF2), and D) selection of fast-growing lines by passaging confluent adherent layers two to three times. These lines show a definite lifespan, clearly indicating that they are not transformed or "immortalized". However, some of the lines display sufficient proliferative potential (20 to 30 doublings) to obtain material available for subsequent studies. We have shown that CDCLs were able to support multilineal marrow hematopoiesis [16, 17], which emphasizes their value as material to study relationships between stroma and hematopoiesis.

Obtaining CDCLs enables us to evaluate the expression of proteins that may be involved in the functional role of the stroma. The major class of such proteins is cytokines, which are increasingly appearing as essential for hematopoiesis maintenance and other regulatory processes. The pattern of expression of a number of cytokines is presented in this work. The width of the repertoire is remarkable, probably related to the mesenchymal nature of the cells. Moreover, some of the cytokines described here have not been previously reported as actively transcribed in human stromal cells. This study complements the description of the myoid cell phenotype [18], confirming that these cells are the marrow connective tissue-forming cells, and suggests not only that stromal control of hematopoiesis is multifactorial, but also that myoid cells are involved in other processes such as marrow angiogenesis and innervation.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
LTCs and CDCLs
After informed consent, cells were obtained from patients undergoing thoracic surgery. LTCs were performed as previously described [19]. LTC medium (LTCM) consisted of McCoy's 5A medium supplemented with amino acids, vitamins, antibiotics, 12.5% (v/v) heat-inactivated fetal calf serum (FCS) (GIBCO; Paisley, UK) and horse serum (GIBCO), and 10–6 M hydrocortisone (Sigma; St Louis, MO). Cells were suspended in 8 ml medium (2.5 x 106 cells/ml) and incubated in 25 cm2 tissue culture flasks at 33°C and 4% CO2 in a fully humidified atmosphere. Cultures were half-depleted and refed with fresh medium each week.

CDCLs were set up as previously described [16]. Week 3 to week 6 primary layers were trypsinized and the recovered cells were washed and passaged through a 23 gauge needle to obtain a single-cell suspension. Cells were then suspended at a concentration of 2 to 5 x 104/ml in semisolid medium containing 20% (v/v) FCS, 30% Iscove's modified Dulbecco's medium (IMDM) (GIBCO), 10–6 M ß-mercaptoethanol (Sigma), 20 U/ml IL-1ß and 200 U/ml TNF-{alpha} (R&D Systems; Minneapolis, MN) with 1.25% (w/v) methylcellulose (Eastman-Kodak; Rochester, NY) in IMDM. The mixture was incubated at 37°C in a humidified 5% CO2 incubator. After two weeks, stromal cell colonies were plucked and cultured in LTCM supplemented with 10 ng/ml fibroblast growth factor 2 (FGF2) (Boehringer-Mannheim; Germany). As soon as the cell lines became 80% confluent, they were passaged into 25 cm2 tissue culture flasks. After two to three passages, layers from fast-growing clones were passaged into 25 cm2 tissue culture flasks and sacrificed for study of cytokine gene expression.

In Situ Immunofluorescence
CDCLs were grown on chamber slides as previously described [10]. The supernatant was discarded and the layer was washed twice with phosphate-buffered saline (PBS), fixed and permeabilized using absolute methanol for 30 min at 4°C. After fixation, the chambers were washed with PBS. The primary antibodies used in this study were: 1B10 (Sigma), antifibroblast-associated antigen [20], BMS-1-recognizing horse serum proteins endocytosed by stromal cells [12]; Vim 13.2 (Sigma) anti–vimentin; clone 33 (Sanbio-Monosan; Uden, The Netherlands) antidesmin; clone 1A4 (Sigma) anti-{alpha}SM-actin; antilaminin polyclonal antibody (Institut Pasteur; Lyon, France) and an anti-osteocalcin monoclonal antibody (kindly provided by Dr. P. Seguin, CIS Bio-International; Bagnols-sur-Cèze, France). The primary antibody was added at the appropriate concentration and the chamber slide was incubated for 30 min at 4°C. After three washes with PBS, the fluorochrome-conjugated secondary antibody was added. After another 30 min incubation at 4°C, the chambers were washed three times with PBS. The upper plastic structure of the chamber slide was removed. The slide, moistened with PBS, was examined using a microscope equipped for fluorescence (Leitz Aristoplan, Wild Leitz; Wetzlar, Germany) with objectives whose magnification and numerical apertures were x 25 and 0.75, x 63 and 1.4, and x 100 and 0.60 to 1.32.

Western Blots
Western blots were performed as previously described [10]. When a confluent layer from a primary culture or pooled CDCLs was obtained, cells were washed in PBS with EDTA (10nM), sonicated, and the amount of protein for each sample was measured using the bicinchoninic acid protein assay reagent from Pierce Chemical Company (Rockford, IL). For immunoblot studies using antibodies directed against {alpha}SM actin, vimentin, desmin and antigens recognized by the BMS-1 antibody, proteins were solubilized in sample buffer containing 2% (v/v) SDS, 100 mM tris (pH 8) and 2% (v/v) ß-mercaptoethanol, and heated at 60°C for 5 min. For immunoblot study of antigens recognized by the 1B10 antibody, proteins were solubilized in sample buffer (without ß-mercaptoethanol) and heated at 60°C for 5 min, since reducing agents and boiling abolish antigen reactivity [20]. Thirty micrograms of protein were loaded per lane. Samples were run on 10% polyacrylamide minigel (8.3 x 10.2 cm, thickness 0.75 mm) (Biorad Laboratories; Richmond, CA), at 40 mA current per slab for 30 min and transferred for 1 h at 100 V to nitrocellulose paper. After transfer, the free-binding sites were saturated by incubating the nitrocellulose paper with 5% (w/v) dried milk, 5% (w/v) bovine serum albumin (BSA) in tris buffer saline (TBS) with tween (0.1% v/v), at room temperature for 1 h. The paper was then incubated with the appropriate dilution of monoclonal antibody for 2 h at room temperature and washed in TBS with 0.1% tween, three times for 5 min. The paper was incubated with peroxidase-linked goat antimouse Ig (Amersham; Little Chalfont, UK) and diluted at 1/300 for 1 h at room temperature. Positive bands were revealed with diamino-benzidine peroxidase tablets from Sigma.

RNA Preparation and cDNA Synthesis
RNA preparation and cDNA synthesis were performed as previously described [21]. After one to three passages in flasks, the culture medium was discarded and replaced by fresh medium without hydrocortisone and without FGF2. After 24 h, 1 ng/ml IL-1ß was added in half the flasks. After 3 h, supernatants were discarded and RNA was extracted by the single-step guanidium isothiocyanate/phenol/chloroform extraction procedure. The quantity of RNA was estimated on an aliquot by spectrophotometry at 260 nm. Complementary DNA was produced by reverse transcription at 42°C for 60 min in a 20 µl reaction mixture containing 5 µg of total cellular RNA, 100 pM pd(N)6 (Pharmacia; Sweden) reverse transcriptase (RT) buffer (50 mM Tris-HCl pH 8.3; 3 mM MgCl2; 75 mM KCl; and 10 mM dithiothreitol), 1.25 mM of dATP, dCTP, dGTP, dTTP, 30 U of RNAse inhibitor (Boehringer-Mannheim) and 200 U of M-MLV RT (GIBCO BRL; Gaithersburg, MA). After heating at 95°C for 3 min, the cDNA was used for amplification. Each amplification was performed using a set for ß-actin and another for CD45 (Table 1Go) in parallel to the cytokine primer sets. Any cDNA not giving a suitable ß-actin amplification was discarded.


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  		Table 1. RT-PCR studies on cytokines with previously described primers
 
Polymerase Chain Reaction (PCR) Amplification and Detection of Products
Enzymatic amplification of the specific cDNA sequences by PCR was performed according to a modified procedure [21] with a DNA thermal cycler (Perkin Elmer Cetus; Norwalk, CT) in a final volume of 100 µl Tris-HCl/MgCl2 buffers, containing 1 U of Amplitaq DNA polymerase (Perkin Elmer Cetus) and 50 pM of sense and antisense primers of each cytokine. For each cytokine, the optimal conditions for PCR were determined: A) using buffers with different concentrations of Tris-HCl pH 8.4: 10, 20 or 40 mM and MgCl2: 1.25, 2.5, or 7.5 mM and B) PCR conditions (temperature, duration of each step, number of cycles) either previously published (Table 1Go) or selected according to primers and fragment sizes. For IL-3, epidermal growth factor (EGF) and transforming-growth factor (TGF) {alpha} transcripts, a second run of PCR (nested PCR) was performed. Ten microliters of the solution containing PCR products were then electrophoresed in a 2% (w/v) agarose gel in the presence of 0.5 µg/ml ethidium bromide. The size of the product ensured that the amplification was specific for each primer pair, as shown on the gel relative to known size standards.

Each pair of primers spanned intron-exon splice sites. Cytokine primers were either previously described (references are given in Table 1Go) or were selected and are given in Table 2Go. For each cytokine, positive and negative controls of PCR were performed using RNA from cells or tissues known to express or not the cytokine as listed on Tables 1 and 2GoGo. The cell lines were LD2, a human melanoma cell line, given by Dr. M. Lilly (VA Medical Center, Seattle, WA); 5637 and MiaPaCA, human bladder and pancreatic carcinoma cell lines; U937, a monocytic cell line; K562, a myeloid leukemia cell line; and MRC-5, a human fetal lung fibroblastic cell line, all purchased from American Tissue Culture Collection (Rockville, MA). Control tissues were provided by the Department of Pathology of the University Hospital of Besançon. Human PBMNCs were isolated using Ficoll-Hypaque density centrifugation. T lymphocytes were obtained after rosetting with sheep red cells and culturing for four days in RPMI-1640 with 10% FCS and 0.5% phytohemagglutinin (Difco; Detroit, MI). Human macrophages were cultured from cord blood cells for three to five weeks in RPMI supplemented with 20% FCS and 5 ng/ml GM-CSF (R & D Systems).


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  		Table 2. RT-PCR studies with cytokines whose primers were chosen for this work
 
For each cytokine, 10 different unstimulated and stimulated CDCL layers were studied. Five to 10 cDNA samples were generated from the mRNAs from one layer. Each cDNA sample allowed the detection of approximately 10 cytokines. In all cases, the PCR was performed twice. In case of negative result, another PCR was performed with double the amount of cDNA. If the result was still negative, a third PCR was done using another cDNA sample from the same mRNAs. A cytokine gene was considered unexpressed only when these three PCRs gave negative results.

For each cytokine, we measured the relative band intensity using densitometry (Biorad Gel D 1000 System). To take into account even slight differences in DNA load from one gel to another, the value for each band was related to that of the 600 bp band of the 100 bp DNA ladder run on the same gel. Relative band intensity for a given cytokine was expressed as the percentage of the most intense band, i.e., that of IL-6.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
Study of the Phenotype
We have already shown, by flow cytometry and immunofluorescence on cells grown on chamber slide, that CDCLs did not contain hematopoietic cells, and more specifically, macrophages [18]. Here, we confirm these data at the mRNA level by showing by RT-PCR that mRNA for CD45 was not detected in CDCLs in contrast to its regular detection in primary layers (Fig. 1Go).



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  		Figure 1. RT-PCR for CD45. RT-PCR was carried out on cells from confluent layers of primary cultures grown for four to six weeks (primary layers, PLs) and from confluent layers generated by stromal CDCLs (10 to 15 stromal colonies from PLs were pooled, grown in LTCM with FGF2, and passaged usually once before RNA extraction). Results for four experiments are shown. One may notice the expected 376 bp band in all PLs, in contrast to the absence of any detectable band in CDCL layers. M: nucleic acid standard 100 bp DNA ladder.

 
As previously described, stromal cells grown under these conditions follow a vascular smooth muscle differentiation pathway [18]. We confirm these data on the presently studied lines using in situ immunofluorescence and Western blotting.

Immunofluorescence studies are shown in Figure 2Go. All cells contain intermediate filaments of vimentin (Fig. 2AGo) and microfilaments of {alpha}SM actin (Fig. 2BGo); in addition, all cells permeabilized by methanol are strongly labeled by an antilaminin polyclonal antibody (Fig. 2CGo), indicating the widespread presence of this adhesive glycoprotein within the cytoplasm.



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  		Figure 2. Immunofluorescence studies. Cells from CDCLs grown in flasks were passaged onto chamber slides as described in Materials and Methods. When the layer was confluent, cells were fixed and permeabilized with absolute methanol before being labeled with different antibodies. A) Anti-vimentin (clone Vim 13.2). Notice coarse intermediate filaments clearly delineating the nucleus of some of the stromal cells. B) Anti-{alpha}SM actin (clone 1A4). Notice thin and linear microfilaments. C) Anti-laminin (polyclonal antibody). Notice intracellular material in the cytoplasm of all stromal cells. In some cells, very fine granules can be observed.

 
Western blotting studies are shown in Figure 3Go, where for each marker, molecule comparison is made between primary layer (lane 1) and CDCLs (lane 2). Vimentin appears as several closely related bands, the upper one being 58 kDa; for identical protein loads (30 µg/lane), there is no major difference between primary layer (lane A1) and CDCLs (lane A2). {alpha}SM actin appears as a unique 43 kDa band in either primary layer (lane B1) or CDCLs (lane B2), where the {alpha}SM actin content is somewhat higher. Desmin is not detected in either primary layer or CDCLs (lanes C1 and C2). BMS-1 is a monoclonal antibody that recognizes a sugar moiety specific for horse serum proteins that are endocytosed by marrow stromal cells and cells from smooth muscle cell lines [12]. Stromal cells from primary layers pick up more intensely horse serum proteins (ranging from 20 kDa to more than 200) (lane D1) than those from CDCLs, where predominant bands at 220, 105, 82, 77 and 58 kDa are visible (lane D2). 1B10 is a monoclonal antibody that recognizes proteins from lysosomes and membranes of myofibroblasts [20]. Stromal cells from CDCLs contain larger amounts of such proteins (with molecular weights in the range of 20-45 kDa) (lane E2) than stromal cells from the primary layer, where antigens are barely detectable (lane E1).



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  		Figure 3. Western blotting studies. Proteins were extracted from cells from primary layer (lanes 1) or CDCLs (lanes 2). SDS-PAGE and Western blots were performed as described in Materials and Methods. 30 µg of protein were loaded per lane. Immunoblots are shown for: A) Anti-vimentin, B) Anti-{alpha}SM actin, C) Anti-desmin, D) BMS-1 and E) 1B10. Molecular weight markers are indicated on the left-hand side of the figure. Arrows on the right-hand side indicate the two major bands at 45 kDa and 38 kDa revealed by 1B10.

 
We also found (data not shown) expression in cells from CDCLs of metavinculin, calponin, h-caldesmon and smooth muscle myosin heavy chains, as previously reported [18]. Detection using anti-von Willebrand factor and anti-CD31 antibodies was negative, indicating the lack of endothelial cells. Using a monoclonal antibody directed against osteocalcin, we found rare and weakly positive cells within CDCL layers, which may indicate osteoblastic differentiation in some cells [44], although vascular smooth muscle cells from human aortic intima were also reported as able to synthesize bone-associated proteins [45], and those from bovine aorta able to yield nodule-forming clones with osteoblastic features and, in particular, the capacity to synthesize osteocalcin [46].

In conclusion, these data validate the complex multistep procedure to grow the cells, insuring that subsequent studies were made using a specific homogeneous population of marrow stromal cells whose predominant, if not exclusive, differentiation pathway was that described for vascular smooth muscle cells.

The Pattern of Cytokine Expression

Some Cytokines are Constitutively Expressed by CDCL Layers   A cytokine was considered constitutively expressed when it was detected without stimulation by IL-1ß in 9 out of 10 of the CDCL layers. The 20 cytokines constitutively expressed are shown on Figure 4Go. These are IL-1{alpha}, IL-1ß, IL-6, IL-7 and IL-8; monocytic and granulomonocytic colony-stimulating factors (M-CSF and GM-CSF); flt-3 ligand; thrombopoietin (TPO); leukemia inhibitory factor (LIF); fibroblast-growth-factors (FGF) 1 and 2; VEGF; platelet-derived growth factor (PDGF) A; and transforming growth factor {alpha} (TGF-{alpha}). We found also constitutive expression of negative regulators, TGF-ß1 and ß3 and MIP 1{alpha}. Eventually, we found constitutive expression of nerve growth factor (NGF) and hepatocyte growth factor (HGF).



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  		Figure 4. RT-PCR for cytokines constitutively expressed in CDCL layers. RT-PCR was carried out using optimal conditions as described in the text. 1) IL-1{alpha} (408 bp). 2) IL-1ß (263 bp). 3) IL-6 (610 bp). 4) IL-7 (361 bp). 5) IL-8 (183 bp). 6) GM-CSF (165 bp). 7) M-CSF (221 bp). 8) flt3-ligand (390 bp). 9) thrombopoietin (129 bp). 10) LIF (427 bp). M, nucleic acid standard 100 bp DNA ladder. 11) FGF1 (489 bp). 12) FGF2 (349 bp). 13) VEGF (645 bp). 14) PDGF- A (225 bp). 15) TGF-{alpha} (nested PCR-237 bp) - M, nucleic acid standard 100 bp DNA ladder. 16) TGF-ß1 (350 bp). 17) TGF-ß3 (247 bp). 18) MIP-1{alpha} (169 bp). 19) HGF (348 bp). 20) NGF (263 bp). M, nucleic acid standard 100 bp DNA ladder. Notice that three gels performed with slightly different migration rates. The molecular weights given on the right-hand side of the figure apply strictly to the last gel (lanes 16 to 20).

 

Some Cytokines are Induced by IL-1 ß in CDCL Layers   A cytokine was considered induced when detected without IL-1ß stimulation in few, if any, layers but detected after IL-1ß stimulation in 9 out of 10 layers. The three induced cytokines are shown on Figure 5Go. These are G-CSF, stem cell factor (SCF) and TNF{alpha}.



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  		Figure 5. RT-PCR for cytokines induced by IL-1ß stimulation in CDCL layers. 24 h after medium renewal (fresh medium without hydrocortisone and FGF2), in half of CDCL layers 1 ng/ml of Il-1ß was added; 3 h after RNA was extracted from CDCL layers with (+) or without (-) stimulation. 1) G-CSF (-). 2) G-CSF (+) (355 bp). 3) SCF (-). 4) SCF (+) (294 bp). 5) TNF{alpha} (-). 6) TNF{alpha} (+) (325 bp). M, nucleic acid standard 100 bp DNA ladder.

 

Some Cytokines are Expressed in a Fraction of the CDCL Layers without Induction by IL-1ß   Seven cytokines were expressed only in three to seven out of the 10 CDCL layers tested, and no significant increase in expression was induced when adding IL-1ß. These are IL-11 and -13, erythropoietin (EPO), oncostatin M, insulin-like growth factor (IGF) 1, TGF-ß2 and EGF. This heterogeneity in cytokine expression is shown for EPO in Figure 6Go.



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  		Figure 6. RT-PCR for EPO in CDCL layers. 27 h after medium renewal without hydrocortisone and FGF2, in four CDCL layer cultures (passages 2 or 3), RNA was extracted. 1) EPO-positive control (liver)(146bp). 2) CDCL 1: negative. 3) CDCL 2: negative. 4) CDCL 3: positive. 5) CDCL 4: positive. M, nucleic acid standard 100 bp DNA ladder.

 

Some Cytokines are Never Detected in CDCL Layers   Twelve cytokines were never detected (as shown in Fig. 7Go). These are IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, and IL-12 (IL-12 p40 was never detected in contrast to the ubiquitously expressed IL-12 p35); activin and inhibin; IFN-{gamma}; and PDGF-B. The IL-1 receptor antagonist (IL-1RA) was also not detected. Additional studies have shown that IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12 and IFN-{gamma} are expressed by activated T lymphocytes and that activin, inhibin PDGF-B and IL-1RA are expressed by cord blood macrophages (data not shown).



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  		Figure 7. RT-PCR for cytokines never expressed in CDCL layers. RNA extraction from CDCL layers and positive controls and RT-PCR were performed as described in the text. 1) IL-1RA CDCL. 2) IL-1RA control (492 bp). 3) IL-2 CDCL. 4) IL-2 control (457 bp). 5) IL-3 CDCL. 6) IL-3 control (148 bp). 7) IL-4 CDCL. 8) IL-4 control (345 bp). 9) IL-5 CDCL. 10) IL-5 control (293 bp). 11) IL-9 CDCL. 12) IL-9 control (355 bp). 13) IL-10 CDCL. 14) IL-10 control (204 bp). 15) IL-12 p40 CDCL. 16) IL-12 p40 control (267 bp). M, nucleic acid 100 bp DNA ladder. 17) IFN-{gamma} CDCL. 18) IFN-{gamma} control (234 bp). 19) inhibin CDCL. 20) inhibin control (746 bp). 21) activin CDCL. 22) activin control (1072 bp). 23) PDGF-B CDCL. 24) PDGF-B control (217 bp). M, nucleic acid 100 bp DNA ladder. Notice that two gels performed with slightly different migration rates. The molecular weights given on the right-hand side apply strictly to the second gel.

 
The data on cytokine expression are summarized in Table 3Go, where, in addition, the relative intensity is given as a percentage of the cytokine most intensively expressed, i.e., IL-6. High expression (>50% of that for IL-6) was observed for IL-1{alpha}, TPO, LIF, FGF1 and 2, TGF-ß1, HGF, NGF and SCF. Intermediate expression (15% to 50% of that for IL-6) was observed for IL-7, IL-8, flt3 ligand, PDGF-A, MIP-1{alpha}, G-CSF, TNF{alpha}, IL-11, OSM and TGF-ß2. Low expression (<15% of that for IL-6) was observed for GM-CSF, M-CSF, TGF-ß3, IL-13 and EPO. The high expression obtained for TGF-{alpha} and EGF is likely due to the nested PCR and cannot therefore be compared to that of the other cytokines; a similar remark can be made for IL-1ß since in this case the number of cycles was superior to that used for the other cytokines (40 instead of 35). On the contrary, the intermediate expression observed for VEGF and IGF-1 might be slightly underevaluated since the number of cycles for these two cytokines was 33. These data indicate differential expression within the panel of cytokines studied. However, we must emphasize that each intensity of expression is related not only to the amount of cytokine mRNA, but also to the technical procedure used for each cytokine. Such densitometric evaluation cannot therefore replace true quantitative estimation.


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  		Table 3. The pattern of cytokine gene expression by CDCLs (densitometric value expressed as % of the most intensely expressed cytokine, i.e., IL-6)
 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
This study indicates that a specific component of the human marrow microenvironment, that of myoid cells generated in vitro from CDCLs, expresses, at the mRNA level, a very large range of cytokines, growth factors and negative regulators.

As we demonstrate here and in a previous study [18], cells from CDCLs are mesenchymal cells whose predominant, if not exclusive, differentiation pathway is that of vascular smooth muscle cells. Early markers for this line of differentiation comprise vimentin and {alpha}SM actin, but not desmin, allowing definition of the cells as VA myofibroblasts following the classification of Sappino et al. [47]. In addition, early markers comprise lysosomal proteins, recognized by BMS-1 as reported for marrow stromal cells from primary culture and smooth muscle cells grown in LTCM [12], and recognized by 1B10 as reported for cultures of myofibroblasts [20]. Early markers also comprise laminin observed in all cells, mainly within the cytoplasm, within secretory granules usually not resolved at the light microscopy level. Late markers comprise metavinculin, calponin, h-caldesmon and smooth muscle myosin heavy chain (usually SM-1). Osteocalcin, detected in some cells, may indicate osteoblastic differentiation [44], although its expression is not incompatible with a vascular smooth muscle phenotype [45, 46].

From data in the literature [48-50] and from our previous experience using LTC [21], we selected three conditions for detection. The RNA was extracted 27 h after medium renewal and hydrocortisone withdrawal (the medium at this stage was LTCM comprising FCS and horse serum, but deprived of hydrocortisone and not supplemented with FGF2) and, for half the cultures, 3 h after addition of IL-1ß.

These conditions of stimulation have to be emphasized, as well as the different selection steps required to grow CDCLs (a period of three to six weeks of LTC, the growth of stromal colonies for two weeks in the presence of both IL-1ß and TNF-{alpha} and, eventually, the growth in LTCM with FGF2 of stromal cell lines for three to six weeks comprising two to three passages). Our data indicate the potential of cytokine expression by vascular smooth muscle-like stromal cells grown and stimulated under these specific conditions in vitro, which may not reflect the steady state or even the stimulated state in vivo, although it is probably indicative of what some myoid cells achieve in steady state or under conditions of stress. On the other hand, negative results strongly suggest that some cytokines are not produced by myoid cells, being specific for other cell populations such as resident macrophages or T or B lymphocytes, or stromal cells still poorly defined.

Most cytokines (20 out of 30) were detected after medium renewal and hydrocortisone withdrawal; these were considered as "constitutively" expressed, which, as stated in the previous paragraph, does not imply in vivo steady-state conditions. Three cytokines required addition of IL-1ß. Seven cytokines were detected in only some of the CDCL layers, which suggests that conditions of detection were not appropriate; addition of TNF-{alpha}, irradiation or treatment by cytotoxic drugs might have induced the expression of these cytokines in all layers. Eventually, twelve cytokines were not detected in any layers, which suggests that they were not actively transcribed due to the very nature of the cells studied.

The generation of continuous stromal lines has made it possible to determine which cytokines in the marrow microenvironment (human or mouse) were of stromal origin [51]. Moreover, IL-6 [52], IL-7 [53], IL-11 [54] and SCF [55] have been cloned from stromal cell lines. The use in this study of well-characterized myoid cells generated by CDCL enables a more precise definition of the phenotype according to cytokine expression. This study indicates that the spectrum of cytokine gene expression is remarkably large (30 out of 42 cytokines studied), which might be due to the mesenchymal origin of the cells, although there is, to our knowledge, no reference study. Moreover, to our knowledge, the expression of a number of cytokines has not been as yet described as actively transcribed in human stromal cells: TPO, EPO, FGF1, VEGF, PDGF-A, NGF, TGF-{alpha}, ß2 and ß3, EGF, oncostatin M and IGF-I.

The very width of cytokine expression and the expression of some specific cytokines (PDGF-A, TGF-{alpha}, FGF1 and 2, IGF-1 and EGF) strongly suggest that myoid cells are the marrow connective tissue-forming cells which reinforce data on their pattern of synthesis of extracellular matrix components [18]. Moreover, the sole expression of PDGF-A and not PDGF-B stresses the similarity between myoid cell and vascular smooth muscle cells from the aortic intima, since the A isoform appears to be specific for cultured smooth muscle cells from the intima [56]. The expression of a very large number of cytokines involved in the maintenance of early and late hematopoiesis (IL-1, IL-6, IL-7, IL-8, IL-11, and IL-13; CSFs; flt3 ligand; LIF; SCF; TPO; EPO; TNF-{alpha}; MIP 1{alpha}; and TGF-ß1 and ß3) supports data on hematopoiesis maintenance by CDCLs [16, 17] and suggests that stromal control of hematopoiesis is a multifactorial process. The expression of angiogenic factors FGF1 and 2, VEGF, TGF-ß2 and {alpha} and that of NGF suggest that myoid cells are also involved in the control of marrow angiogenesis and innervation.

Also informative is a review of the cytokines that were not detected in our study. Even after the multistep selection procedure for CDCLs and various stimulatory stimuli, we never detected expression of IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, and IL-12; IFN-{gamma}; activin; inhibin; PDGF-B; and IL-1RA. It is well known, and we have confirmed (data not shown), that IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12 and IFN-{gamma} are true lymphokines. Activin, inhibin, PDGF-B and IL-1RA are, in our experience, expressed by macrophages. The fact that stromal cells grown under other conditions or stromal cell lines express activin and inhibin or IL-1RA [33, 57] suggests that we have selected here a specific, well-characterized, and nontransformed cell population within the stroma.

In conclusion, this study may serve as a reference when evaluating the pattern of expression of cytokine genes from other cells from the marrow microenvironment (endothelial cells, macrophages and lymphocytes), and underscores the potential functional role of a number of cytokines shown here to be of stromal origin.


   Acknowledgments
 
Work supported by grants from INSERM (Contrat de Recherche n° 950401), from Fonds d'Organisation pour la Recherche en Transfusion Sanguine and from Direction des Etudes et Recherche (DRET n° 92.132).


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

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accepted for publication on August 11, 1996.




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