basic fibroblast growth factor expression in human bone marrow and

Basic Fibroblast Growth Factor Expression in Human Bone Marrow and Peripheral Blood Cells

By Georg Brunner, Hiep Nguyen, Janice Gabrilove, Daniel B. Rifkin, and E. Lynette Wilson

We have shown previously that basic fibroblast growth factor (bFGF) is a mitogen for human bone marrow (BM) stromal cells and that bFGF stimulates myelopoiesis in primary BM cultures. In this article, we demonstrate the presence of bFGF in two cell lineages in human BM and peripheral blood as well as the deposition of bFGF into the extracellular matrix of BM stromal cell cultures. In immunofluorescence experiments on BM and peripheral blood smears, megakaryocytes and platelets stained strongly for bFGF, whereas weaker staining was observed in immature and mature cells of the granulocyte series. The presence of bFGF in platelets was confirmed by enzyme-linked im- munosorbent assay as well as by immunoprecipitation followed by immunoblotting. bFGF was synthesized by BM

EMATOPOIETIC CELL proliferation and differentia- H tion is regulated by specific cell-cell interactions as well as by a variety of growth Apart from the well- characterized colony-stimulating factors, basic fibroblast growth factor (bFGF), a multifunctional growth factor known to be involved in angiogenesis and wound repair,5 also acts as a potent modulator of hematopoie~is.6-~

In human primary bone marrow (BM) cultures, bFGF stimulates the formation of an adherent stromal cell layer and promotes hematopoietic cell de~elopment.~.’ bFGF has also been shown to enhance colony formation in vitro by primitive as well as lineage-committed hematopoietic progenitor cells in synergy with other hematopoietic growth fac- to r~ .* .~ However, neither endogenous bFGF production in BM nor its distribution in primary BM cell cultures have been reported to date.

In several cell types, intracellular bFGF has been found to be localized in the cytoplasm as well as in the nucleus and the nucleoli,’@I3 suggesting a direct effect of bFGF on gene transcription as has been noted for ribosomal gene expression.13 Although it is not yet known how bFGF, a protein lacking a signal sequence, is released by cells, extracellular bFGF is found deposited as a complex with heparan sulfate proteoglycans (HSPGs) in the extracellular matrix (ECM) and on the cell surface of endothelial cells and primary BM cultures. ECM-bound bFGF provides a reservoir of biologically active growth factor, which is capable of mediating long-term biologic effects. l 5 Biologically active bFGF-HSPG complexes are released from these storage sites by phosphatidylinositol-specific phospholipase C or by the serine proteinase, plasmin. I 6 7 l 7

In this article, we demonstrate the presence of bFGF in two cell lineages of the hematopoietic system, megakaryocytes/platelets and cells of the granulocyte series. In addition, bFGF was detected in cultured BM stromal cells that were also found to synthesize bFGF. These findings indicate a possible role for bFGF in the biologic function, proliferation, or differentiation of these cells. We also show bFGF binding to extracellular heparin-like molecules in BM stromal cell cultures. We propose that HSPGs o n the cell surface or in the ECM of stromal fibroblasts serve as a reservoir for bFGF

stromal cell cultures and was found either cell associated or localized in the nucleus and the nucleoli, and its location was dependent on the fixation procedure used. Addition of exogenous bFGF to stromal cells showed the presence of extracellular binding molecules for this cytokine. bFGF could be released from these sites by soluble heparin or phosphatidylinositol-specific phospholipase C. This study sup- ports the role of bFGF as a stromal cell mitogen and stim- ulator of myelopoiesis. The data indicate that the stromal cells produce bFGF and that their extracellular matrix can serve as a reservoir for this growth factor. In addition, the results suggest a possible involvement of bFGF in platelet function as well as in megakaryocytopoiesis. 0 1993 by The American Society of Hematology.

from which it can be released in a biologically active form either enzymatically16 or by soluble heparin-like molecules.

MATERIALS AND METHODS

Recombinant bFGF was a generous gift of Synergen Inc (Boulder, CO).” Protein A-Sepharose and protein G-Sepharose CL4B, aprotinin (9,900 kallikrein inhibitor units [KIU]/mg), phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin, Triton X- 100, saponin, hydrocortisone, heparin, p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and nitro blue tetrazolium (NBT) were obtained from the Sigma Chemical Co (St Louis, MO) and Nytran 0.2-pm nylon membranes from Schleicher and Schuell (Keene, NH). Heparin Sepharose CLdB was purchased from Pharmacia (Piscataway, NJ) and Gelvatol from Air Products and Chemicals (Allentown, PA). [35S]Protein-labeling mix containing [35S]methionine (77%; I , 170 Ci/mmol), [35S]cysteine (l8%), and other [35S]components (5%) was obtained from Du Pont-New England Nuclear (Wilmington, DE).

Anti-bFGF was obtained by immunizing rabbits with recombinant bFGF. Preimmune rabbit serum was used as a control. The IgG fractions of the rabbit sera were purified by affinity chromatography on protein G-Sepharose CL-4B. Synthetic peptides corresponding to the amino acid sequence of bFGF (amino acids 14- 23, 45-54, 80-88, 106-1 13, 115-123) were kindly provided by D. Schlesinger (New York, NY), and antisera to these peptides were obtained by immunizing rabbits. The murine hybridoma cell line 42F producing a monoclonal IgCl antibody to human bFGF was a

From the Department of Cell Biology and the Department of Sur- gery, New York University Medical Center, the Department of He- matology, Memorial Sloan-Kettering Cancer Center, New York, NY.

Submitted March 23, 1992; accepted October I, 1992. Supported by Grants from the Deutsche Forschungsgemeinschaft

(DFG, Br 1125/1-1). the National Institutes of Health (CA 49419, CA 34282, and CA 20194), the Benjamin Weiss Fund, the South Afvican National Cancer Association, and the University of Cape Town Staff Research Fund.

Address reprint requests to E. Lynette Wilson, PhD, Department of Cell Biology, New York University Medical Center, 550 First Ave, New York, NY 10016.

The publication costs of this article were defvayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

Materials.

Antibodies.

0 1993 by The American Society of Hematology. 0006-4971/93/8103-0019$3.00/0

Blood, Vol81, No 3 (February 1). 1993: pp 631-638 63 1

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632 BRUNNER ET AL

generous gift of L. Ossowski (New York, NY). The hybridoma cells were cultured in serum-free HL-1 medium (Ventrex, Portland, ME), and IgG was purified from conditioned medium by affinity chromatography on protein G-sepharose CL-4B. Irrelevant mouse IgG 1 (MOPC 2 1) was obtained from the Sigma Chemical Co and was used as a control. Goat anti-rabbit IgG antibodies conjugated to fluorescein isothiocyanate (FITC) and goat anti-rabbit or sheep anti-mouse IgG antibodies conjugated to alkaline phosphatase were purchased from Cappel (Durham, NC).

Human BM and peripheral blood cells were obtained from healthy adult volunteers following informed consent. BM and peripheral blood smears were prepared on glass slides (3 inch X l inch), air-dried, and stored at -20°C.

Primary BM cultures were established as follows. Buffy coat cells were seeded in alpha Modified Eagle's Medium (Flow Laboratories, McClean, VA) containing 12.5% (vol/vol) fetal calf serum (Intergen Co, New York, NY), 12.5% (vol/vol) horse serum (GIBCO, Grand Island, NY), mol/L hydrocortisone, I 0-4 mol/L 2-mercaptoethanol, 2 mmol/L L-glutamine, and antibiotics. After 3 to 4 weeks, an adherent stromal cell layer containing hematopoietic progenitor cells was obtained? Cells were passaged twice, resulting in cultures enriched in adherent fibroblast-like stromal cells.6 For immunofluorescence experiments, cells were cultured on circular glass coverslips (18 mm in diameter) placed in six-well culture dishes.

Platelets were isolated from peripheral blood samples drawn into 0.15% EDTA. The majority of white and red blood cells was removed by centrifugation for I O min at 300gat room temperature. To isolate the platelets, the pH of the supernatant was adjusted to 6.5 using 0. I mol/L citrate, and platelets were collected by centrifugation for I5 minutes at I ,300g at room temperature. This platelet-enriched preparation was usually contaminated with 0.02% to 0.2% white blood cells and 2.4% to 5.6% red blood cells. The platelet suspension was adjusted to IO9 or IO" platelets/mL in phosphate-buffered saline (PBS) containing the protease inhibitors, aprotinin (100 KIU/mL), and phenylmethylsulfonyl fluoride (PMSF) ( I mmol/L). Platelets were disrupted by freeze-thawing followed by sonication for 3 X 5 seconds on ice. The platelet sonicates were centrifuged for I O minutes at 12,OOOg and the supernatant used for either the enzyme-link im- munosorbent assay (ELISA) or the immunoprecipitation of bFGF. To determine whether bFGF from white and red blood cells could potentially be contributing to the bFGF noted in platelet extracts, the bFGF content of isolated red and white blood cells was measured by ELISA.

Stromal cells grown on coverslips were fixed for 20 minutes at room temperature with freshly prepared 2% paraformaldehyde in PBS containing calcium and magnesium and washed with PBS. Subsequently, cells were permeabilized by incubation with 0.1% (wt/vol) Triton X-100 in PBS for 10 minutes at room temperature. After washing with PBS, paraformaldehyde was quenched by incubation with 50 mmol/L ammonium chloride in PBS for 10 minutes at room temperature. Alternatively, stromal cells as well as BM and peripheral blood smears were fixed at -20°C with methanol for 5 minutes followed by fixation with acetone for 2 minutes. Prior to fixation, the air-dried BM and peripheral blood smears were prewashed with PBS containing calcium and magnesium for 5 minutes at room temperature to remove excess red blood cells. Fixed cells were washed with PBS and stored at 4°C.

Before the immunofluorescence staining, nonspecific protein- binding sites were blocked by incubation with 5% (wt/vol) dry milk in PBS for 2 hours at 37°C. Fc receptor sites present on BM cells were saturated by incubation for 1 hour at 37°C with 20% (vol/vol) goat serum in PBS containing 1.5 mg/mL human IgG. For the immunofluorescence staining, cells were incubated for 1 hour at 37°C with polyclonal rabbit sera (diluted 1 : lO to 1:25 in PBS containing

Cells.

Indirect immunojluorescence staining.

5% dry milk) or purified rabbit IgG (50 pg/mL in PBS containing 5% dry milk) followed by incubation for 1 hour at 37°C with goat antirabbit IgG conjugated to FITC (1:25 to 150 in PBS containing 5% dry milk). After washing and air drying, cells were embedded in Gelvatol and examined using a Zeiss (New York, NY) Axiophot fluorescence microscope. Microphotographs were taken on Kodak TMAX 400 (Eastman Kodak Co, Rochester, NY).

Passaged stromal cells were incubated overnight at 37°C with [35S]protein-labeling mix (1 14 pCi/mL ['%]methionine) in methionine-free Dulbecco's Modified Eagle Me- dium (DMEM; GIBCO) supplemented with 5% fetal calf serum, 6 pg/mL of methionine, 25 mmol/L HEPES pH 7.2, 2 mmol/L L- glutamine, 1 mmol/L sodium pyruvate, mol/L hydrocortisone,

mol/L 2-mercaptoethanol, and antibiotics. Freshly isolated platelets were labeled with [35S]protein-labeling mix (1 14 pCi/mL [3SS]methionine) by tumbling overnight at room temperature in the medium described above, supplemented with 1 X acid citrate dextrose pH 5 and 2 mmol/L EDTA.

After metabolic labeling, cells were washed with PBS, containing I X acid citrate dextrose in the case of the platelets, and lysed for I O minutes at 37°C in I O mmol/L TRIS (hydroxymethyl) aminometh- ane (TRIS HCI) pH 7.5 containing 0.5% Triton X-100, 2 mol/L sodium chloride, 2 mmol/L PMSF, 200 KIU/mL of aprotinin, I O pg/mL of pepstatin, I O pg/mL of leupeptin, and 2 mmol/L EDTA. Lysates were cleared by centrifugation, dialysed extensively against PBS, and loaded onto a heparin-Sepharose column. The column was washed with PBS containing 0.5 mol/L sodium chloride, and bound [35S]proteins were eluted with PBS containing 2 mol/L sodium chloride. After dialysis against PBS, [35S]bFGF from platelet or BM stromal cell lysates was immunoprecipitated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography as described below.

Protein A-Sepharose CL-4B beads were prewashed with PBS and coated with IgG by incubation with rabbit anti-bFGF antiserum (45 pL/100 pL of packed beads) or preimmune serum, respectively, for 1 hour at 37"C.I6 The supematants of platelet sonicates (see above) were first precleared by incubation for 4 hours at 4°C with beads coated with preimmune IgG. bFGF was then precipitated from the precleared supernatant by overnight incubation at 4°C with beads coated with anti-bFGF IgG. The beads containing the immunoprecipitates were washed with PBS, boiled in reducing SDS-PAGE sample buffer, and analyzed for the presence of bFGF by immunoblotting (see below). After immunoprecipitation of [35S]labeled bFGF from platelet or BM stromal cell lysates the beads were washed with 20 mmol/L TRIS HCI pH 7.5 containing 1% Triton X-100, 0.05% Tween 20, 0.1% SDS, 0.3 mol/L NaCI, I O mmol/L EDTA, I O p/mL of aprotinin, and 1 mmol/L PMSF. The immunoprecipitated proteins were analyzed by SDS-PAGE followed by autoradiography.

bFGF immunoprecipitates (see above) were subjected to SDS-PAGEI9 in a 20% gel under reducing conditions. Proteins were transferred for 6 hours at 5 to 10°C (70 V, 500 to 600 mA) onto 0.22-pm nylon membranes according to Towbin et aLZ0 Unoccupied protein-binding sites on the membrane were saturated by incubation with 5% dry milk in PBS for 1 hour. After overnight incubation with the bFGF-specific mouse monoclonal antibody (MoAb) 42F ( I O pg/mL), bound antibodies were detected by incubation for I hour with sheep anti-mouse IgG conjugated to alkaline phosphatase (diluted 1:5,000 in PBS containing 5% dry milk). Irrel- evant mouse IgG I (MOPC 2 1) was used as a control. Alkaline phosphatase activity was visualized using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) as substrates. All incubations were performed at room temperature.

Immulon 96-well plates (Dynatech, Burling- ton, MA) were coated overnight at 4°C with 100 pL/well of mono-

Metabolic labeling of bFGF.

Immunoprecipitation.

Immunoblotting.

Sandwich ELZSA.




bFGF IN HUMAN BONE MARROW 633

clonal anti-bFGF antibody 42F (10 pglmL in 0.05 mol/L sodium bicarbonate buffer pH 9.6). ARer saturation of nonspecific protein- binding sites by incubation with 5% dry milk in PBS (200 pL/well) for 6 hours at 37°C. bFGF-containing samples (100 pL/well) were incubated in the antibodycoated wells overnight at 4°C. After washing with PBS, antibody-bound bFGF was detected by incubation for I hour at 37°C with rabbit polyclonal anti-bFGF IgG (10 pglmL in PBS containing 5% dry milk; 100 pL/well). This was followed by incubation for I hour at 37°C with 100 pL/well of antirabbit IgG coupled to alkaline phosphatase (diluted 1:1O,OOO in PBS containing 5% dry milk). The enzymatic color reaction of the alkaline phosphatase was developed by addition of 200 pL/well of the substrate pnitrophenyl phosphate ( 1 m@mL in 9.7% [vol/vol] diethanolamine HCI buffer pH 9.8 containing 0.5 mmol/L magnesium chloride). The optical density at 405 nm was measured after I or 3 hours. Using recombinant human bFGF, a linear standard curve was obtained and the detection limit was estimated to be approximately 50 pgl mL of bFGF.

RESULTS

Detection of bFGF in BM-derived hematopoielic cells. BM smears were prepared from freshly taken samples and immunofluorescence staining for bFGF was performed as described in Materials and Methods.

Megakaryocytes, which could be identified morphologically in phase contrast microscopy by their size and multilobulated nuclei (Fig IA), stained positively for bFGF using rabbit anti- bFGF antiserum (Fig I B). Similarly, bFGF was detected in megakaryocytes present in primary human BM cultures (not shown). Bright staining for bFGF was also observed in BM- derived platelets (Fig ID), whereas immature and mature cells of the granulocyte series were more weakly stained (Fig 1 E; this microphotograph was taken with a longer exposure time than Fig 1 B and ID and therefore appears equivalently fluorescent). Preimmune rabbit serum did not result in significant staining of any of the BM cells (Fig IC and IF) . No

Fig 1. Indirect immunofluo- & rescence staining for bFGF in hematopoietic cells derived from human EM. EM smears were fixed with cold methanol/acetone and stained with anti-bFGF antiserum ( 1 : l O ) (B, D, E) or preimmune rabbit serum (1 :lo) (C, F) followed by antirabbit lgG- FlTC (1 :50) (see Materials and Methods). (A) phase contrast photomicrograph of a megakaryocyte; (E, C) megakaryocyte; (D) platelets; (E) granulocytes; (F) negative control of a BM smear containing platelets and granulocytes. Ears: 50 pm.

specific staining was observed aRer removal of bFGF-specific antibodies from the anti-bFGF antiserum by bFGF affinity chromatography (results not shown).

Similar staining patterns for bFGF were obtained using purified anti-bFGF IgG as well as with two of five anti-bFGF peptide antisera (not shown). Because platelets are known to contain a variety ofgrowth factors.2' we tested the anti-bFGF antibodies used for immunofluorescence staining for possible cross-reactivity with other growth factors. In ELlSA or immunoblotting experiments, neither affinity-purified anti- bFGF lgG nor rabbit anti-bFGF antiserum cross-reacted significantly with acidic FGF, Kaposi's sarcoma FGF, plateletderived growth factor, epidermal growth factor, or transforming growth factor B (results not shown), confirming the specificity of the anti-bFGF antibodies used.

Because bFGF was found in megakaryocytes, platelets, and cells of the granulocyte series in the BM, we examined peripheral blood cells for the presence of this growth factor. Blood smears were prepared from freshly taken samples of peripheral blood and immunofluorescence staining for bFGF was performed as described in Materials and Methods.

As noted in the BM, the platelets found in peripheral blood stained strongly for bFGF using anti-bFGF antiserum (Fig 2A). No significant staining was observed with preimmune rabbit serum (Fig 2B). When the platelets were incubated with the antiserum before fixation, no significant staining was observed (results not shown). This indicates that bFGF was not associated with the extracellular leaflet of the platelet plasma membrane. bFGF was also evident in peripheral blood granulocytes (results not shown). These cells stained more weakly than those in the BM, indicating a lesser bFGF content.

Delection of bFGF in peripheral blood cells.

The results obtained with BM and peripheral blood smears




BRUNNER ET AL 634

demonstrate the presence of bFGF in at least two cell lineages of the hematopoietic system. The most prominent staining for bFGF was observed in megakaryocytes and platelets, whereas lesser amounts of bFGF were present in cells of the granulocyte series.

bFGF quantifrcation in platelet sonicates. To confirm the

kDa

bFGF -

1 2 3 4

- 70.3 - 44.1 - 27.9

-18.1

Fig 3. lmmunoblotting of bFGF from platelet sonicates. bFGF was precipitated from the cleared supematant (5 mL) of platelet sonicates (1 0'' platelet/mL) using protein A-Sepharose beads coated with polyclonal rabbit anti-bFGF lgG. The bFGF precipitates (lanes 2 and 4) or 10 ng of a bFGF standard (lanes 1 and 3) were subjected to SDS-PAGE in a 20% gel under reducing conditions and transferred electrophoretically onto nylon membranes. bFGF was detected by incubation with the monoclonal mouse anti-bFGF lgGl 42F (1 0 pg/mL) (lanes 1 and 2) followed by incubation with sheep antimouse IgG conjugated to alkaline phosphatase (1 :5,000). In- cubation with irrelevant monoclonal mouse lgGl (MOPC 21) re- vealed cross-reactivity of the sheep antimouse lgG with the rabbit lgG used for the precipitation (lanes 2 and 4; lgG heavy chain at approximately 50 Kd, light chain at 25 Kd). The positions and the relative molecular mass of marker proteins is indicated in the right panel.

Fig 2. Indirect immunofluorescence staining for bFGF in human peripheral blood cells. Blood smears were fixed and stained with anti-bFGF antiserum (1:25) (A) or preimmune rabbit serum (1:25) (B) as described in the legend of Fig l. (A) platelets; (B) blood smear containing plate-lets. Bar: 50 pm.

identification of bFGF in platelets, we examined platelet sonicates for bFGF using the biochemical techniques of immunoprecipitation and immunoblotting. Platelets ( lO''/mL) were disrupted by freeze-thawing followed by sonication. bFGF was precipitated from the cleared sonicate supernatant by the addition of protein A-Sepharose beads coated with rabbit polyclonal anti-bFGF IgG. The precipitates were analyzed for the presence of bFGF by SDS-PAGE followed by immunoblotting using the mouse monoclonal anti-bFGF antibody 42F (see Materials and Methods). As shown in Fig 3, bFGF was precipitated from platelet sonicates and was detected as an 18-Kd band by immunoblotting (Fig 3, lane 2) at the same position as standard bFGF (Fig 3, lane 1). The additional staining in the high molecular weight range was caused by cross-reactivity of the anti-mouse IgG antibodies used for immunoblotting with the rabbit IgG used for precipitation because it was also observed with irrelevant mouse IgGl (Fig 3, lane 4).

For the quantification of bFGF in platelet sonicates ( lo9 platelets/mL), we used a sandwich ELISA as described in Materials and Methods. The bFGF concentration in the sonicate supernatant of platelets isolated from five individuals ranged between 3.4 and 12.7 ng/mL (6.2 f 1.0 ng/mL) (Table I ) . The bFGF detected was not associated with contaminating white or red blood cells because a sonicate of a mixture of these cells (2.8 to 3.1 X IO6 white blood cells and 0.7 to 1. I X IO8 red blood cells/mL) did not contain detectable amounts (40 pg/mL) of this factor (results not shown). Assuming a

Table 1. bFGF in Platelets

Platelet Preparation' bFGF (ng/mUt

1 3.4 2 2.1 2 5.4 * 0.7 3 5.7 2 1.1 4 7.3 * 1.0 5 12.7 + 1.7

Platelet-enriched preparations (1 Oe platelets/mL) of five individuals were sonicated and the cleared supematants were assayed for the presence of bFGF by ELISA.

t Values denote the mean values & SEM of one to two experiments performed in duplicate.





platelet number in peripheral blood of 2.5 X 10' platelets/ mL:2 the concentration of platelet-associated bFGF in the circulation would amount to approximately I .6 ng/mL. The bFGF found in platelets was biologically active as it modu- lated the plasminogen activator activity of BM stromal cells (data not shown).

The above data confirm the presence of bFGF in platelets and suggest that platelets might represent a source of biologically active bFGF. Metabolic labeling experiments failed to detect bFGF in platelet lysates indicating that peripheral blood platelets may not synthesize bFGF or that the amount of metabolically labeled bFGF produced by these cells was not detectable by the techniques used.

We reported previously that bFGF is found in primary human BM cultures and that it is deposited into the ECM of the BM stromal cells.I6 Because bFGF was detected in immunoblotting of extracts of passaged BM cultures consisting mainly of stromal fibroblasts (unpublished results, May I99 l ) , we examined whether the stromal cells themselves produced bFGF in vitro. For these studies, primary BM cultures were passaged twice and examined for bFGF expression by immunofluorescence as well as by metabolic labeling with [3sS]methionine/cysteine.

We noted that stromal cell cultures, fixed with paraformaldehyde and permeabilized with Triton X- 100, contained bFGF. In these cultures, the bFGF was found primarily in the nucleus and the nucleoli of the cells (Fig 4A). The ex- tranuclear cell-associated staining was considerably weaker. This could have been on account of the loss of bFGF from the paraformaldehyde-fixed cells as this procedure does not result in complete fi~ation.~' The staining proved to be specific because no significant staining was observed with preimmune rabbit serum (Fig 4C). In addition, the nuclear staining obtained with anti-bFGF antiserum was competed for by ex- ogenously added bFGF (Fig 4B). However, the overall cellular staining was slightly enhanced in this experiment. Because bFGF-antibody complexes, formed by addition of exogenous bFGF to the antiserum, are known to bind to bFGF-binding HSPGS'~.'' and had not been removed before the experiment, binding of these complexes to nonsaturable ECM-binding sites for bFGF might account for the increased cellular staining. However, no specific staining was observed when bFGF- specific antibodies were removed from the anti-bFGF antiserum by bFGF affinity chromatography.

To demonstrate bFGF production by stromal cells, cells were metabolically labeled with [3SS]methionine/cysteine. Heparin-binding proteins were isolated by affinity chromatography on heparin-Sepharose and examined for the presence of labeled bFGF by immunoprecipitation followed by SDS-PAGE and autoradiography. Lysates of metabolically labeled stromal cells contained ["SIbFGF (Fig 5, lane I ) demonstrating bFGF synthesis in stromal cells.

We have shown previously that bFGF is deposited in primary BM cultures on the cell surface or in the ECM as a complex with HSPGS.'~ Biologically active bFGF can be released by phosphatidylinositol-specific phospholipase C or plasmin, and bFGF binding can be competed for by soluble heparin. To examine whether these bFGF-binding HSPGs are expressed by passaged stromal cell cultures, we added

hFGF expression in BM stromal cells.

Fig 4. Indirect immunofluorescence staining for bFGF in passaged BM stromal cells. Primary human BM cuttures were passaged twice and stromal cells were cultured on 18-mm glass coverslips. Cells were fixed with 2% paraformaldehyde and permeabilized with 0.1 % Triton X-1 00 as described in Materials and Methods. Cells were stained for bFGF using rabbit anti-bFGF antiserum (1 : lo) (A, B) or rabbit preimmune serum (1: lO) (C) and goat anti-rabbit IgG- FlTC (1 :50). For the experiment shown in panel B, the anti-bFGF antiserum was preincubated for 30 minutes at 37°C with 1 pg/mL of bFGF and used for the staining procedure. Bar: 50 pm.

exogenous bFGF to the cultures and studied bFGF binding and release by heparin or phospholipase C. For these experiments, cells were preincubated with heparin to remove ex- tracellularly bound endogenous bFGF.

Fixation of heparin-pretreated stromal cells with methanol and acetone resulted in weak cell-associated staining for bFGF (Fig 6A) in contrast to the nuclear staining noted after para-




636 BRUNNER ET AL

100.6 - 7182-

28.6 -

18.3-

1 2 Fig 5. Demonstration of metabolically labeled bFGF in BM

stromal cells. Stromal cells were metabolically labeled with [3sS]methionine/cysteine and bFGF was isolated by heparin sepharose affinity chromatography and immunoprecipitation. [3sS]Proteins precipitated with rabbk anti-bFGF lgG (lane 1) or preimmune rabbit lgG (lane 2) were analyzed by SDS-PAGE followed by autoradiography.

formaldehyde fixation. The reason for the different staining patterns is not known. The cross-linking of proteins by paraformaldehyde or their precipitation by methanol/acetone could result in differential protein preservation in various cellular compartments. When cells were preincubated for 1 hour at 37°C with exogenous bFGF (1 pg/mL), the cellular bFGF staining was significantly enhanced (Fig 6B). No significant staining was seen with preimmune rabbit serum (Fig 6C). This increased staining was caused by bFGF binding to extracellular binding sites on the stromal cell surface and/or in the ECM, as treatment with heparin (Fig 6D) or phos-

pholipase C (Fig 6E) both resulted in significantly reduced staining, indicating that bound bFGF was partially removed by these procedures.

These results indicate that BM stromal cells produce bFGF and that heparin-like bFGF-binding sites on the cell surface and/or in the ECM are available for complexing the endog- enously produced cytokine. The majority of these binding sites appears to consist of the phosphatidylinositol-linked bFGF-binding HSFG characterized previously.'6

DISCUSSION

bFGF is a multifunctional growth factor that is a mitogen for human BM stromal cells6 and also stimulates hemato- po ie~ i s .~ -~ The experiments indicating that bFGF may stim- ulate progenitor cell growth have been performed in vitro in long-term BM cultures7 and on freshly isolated primitive progenitor There are no reports concerning the production or cellular distribution of this factor within the hematopoietic system. We have, therefore, examined the cells of human BM and peripheral blood for expression of bFGF in vivo. We have also studied expression and distribution of this factor in vitro in passaged human BM stromal cell cultures.

Immunofluorescence staining of cells from human BM or from human peripheral blood indicated that bFGF was pre- dominantly located in platelets and megakaryocytes (Figs 1 and 2). Lesser amounts of bFGF were observed in cells of the granulocyte lineage (Fig 1). The staining proved to be bFGF-specific by the following criteria: (1) No significant staining was observed with preimmune rabbit serum or purified irrelevant IgG. (2) No significant staining was observed after removal of bFGF-specific antibodies from the antiserum by bFGF affinity chromatography. (3) The anti-bFGF antibody preparations used did not cross-react with other growth factors (acidic FGF, Kaposi's FGF, plateletderived growth factor, epidermal growth factor, transforming growth factor- 8) in ELISA or immunoblotting experiments. (4) Similar staining patterns were obtained using different anti-bFGF antibody preparations such as antiserum to the intact recombinant bFGF molecule, purified IgG fractions thereof, or antisera to synthetic peptides corresponding to various regions of the bFGF molecule. bFGF was not detected in platelets stained prior to fixation, making it unlikely that this factor was absorbed onto the external surface of the platelet. It has been reported that certain plasma proteins present in platelet a-granules such as fibrinogen, albumin, and IgG are not synthesized by BM megakaryocytes but are endocytosed from the c i r~ula t ion .~~ On the other hand, however, platelets are known to contain stable megakaryocytederived mRNAZS and to synthesize proteins de novo?6 Although bFGF was not detected by metabolic labeling of platelets, bFGF production in platelets cannot be excluded because the protein synthetic capacity of platelets was reported to decrease with agingF7 Therefore, the presumably low biosynthetic capacity of isolated platelets may not yield detectable amounts of labeled bFGF.

The presence of bFGF in platelets could also be demon- strated by immunoblotting experiments (Fig 3). Owing to the low concentration of bFGF in platelet sonicates and the lim-





__

Fig 6. Detection of bFGF binding to BM stromal cells by indirect immunofluorescence staining. Human BM stromal cells grown on glass coverslips were pretreated for 30 minutes at 37°C with 10 pg/mL of heparin in serum-free medium in order to remove endogenous bFGF bound to extracellular sites in the cultures. Cells were then processed as follows: (A) Control: incubated for 1 hour at 37°C with serum- free medium without addition of exogenous bFGF; (B-E) incubation for 1 hour at 37°C with 1 rg/mL of bFGF in serum-free medium; (0) addition of exogenous bFGF followed by incubation with 10 rg/mL of heparin for a further 1 hour at 37°C; (E) addition of exogenous bFGF followed by incubation with phosphatidylinositol-specific phospholipase C (0.1 pm/mL) for a further 1 hour at 37°C. Cells were fixed and stained with anti-bFGF antiserum (1 :25) (A, E, D, E) or preimmune serum (1 :25) (C) and goat antirabbit IgG-FITC (1 :25). Bar: 50 pm.

ited sensitivity of immunoblotting, bFGF had to be enriched by immunoprecipitation for it to be detected in immunoblotting experiments. The amount of bFGF in platelet sonicates was quantified by a sandwich ELISA using platelets isolated from fresh blood samples of five individuals. The average concentration was determined to be 6.2 ng/109 platelets (Table I ) . Assuming a platelet density of 2.5 X IO8 platelets/mL of peripheral the amount of platelet- associated bFGF within the circulation is approximately 1.6 ng/mL. These results suggest that platelets might be consid- ered as a potentially relevant source for this growth factor in the circulation.

Passaged cultures of BM stromal cells stained positively for bFGF in immunofluorescence experiments (Fig 4). These cells also produced bFGF as shown by metabolic labeling experiments (Fig 5). We have reported previously that bFGF is a potent mitogen for human BM stromal cells: Thus, bFGF could act as an autocrine growth factor in these cells as has been reported for endothelial cells.28 We have noted that bFGF was found in the nucleus and nucleoli of permeabilized stromal cells. This could have functional significance, as it has been shown in other cell types that bFGF enters the nucleus'@'3 and regulates gene expre~sion.'~

We have previously noted that in primary human BM cultures, bFGF is deposited on the cell surface or in the ECM.I6 These binding sites were identified as HSPGs and binding could be competed for by heparin. In this article, we demonstrate that passaged BM adherent cell layers, consisting mainly of fibroblast-like stromal cells, express extracellular heparin-like binding sites for bFGF (Fig 6). This suggests that stromal cells in BM cultures produce bFGF and generate a reservoir of this growth factor by depositing bFGF into their microenvironment as a complex with HSPGs. bFGF can be released from these storage sites by soluble heparin-like mol-

ecules (Fig 6D), by phosphatidylinositol-specific phospholipase C (Fig 6E), or by the serine proteinase plasmin.I6

These results demonstrate bFGF expression by two human hematopoietic cell lineages in vivo, megakaryocytes/platelets and cells of the granulocyte series, as well as by human BM stromal cells in vitro. In addition, the data suggest that BM stromal cells serve as a reservoir for bFGF in human primary BM cultures providing biologically active bFGF-HSPG complexes. These complexes might act in an autocrine and/or paracrine fashion to support stromal and hematopoietic progenitor cell growth. Experiments are currently in progress to delineate the role of bFGF in granulopoiesis and megakaryocytopoiesis.

ACKNOWLEDGMENT

The authors thank Drs L. Ossowski for supplying the hybridoma cell line 42F and J. Thomas for helpful advice with the fluorescence microscopy.

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1993 81: 631-638

G Brunner, H Nguyen, J Gabrilove, DB Rifkin and EL Wilson peripheral blood cellsBasic fibroblast growth factor expression in human bone marrow and

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