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Published online before print January 2, 2004
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T-lymphocytes. Response to isopentenyl pyrophosphate and TGF-ß1/IL-15
University Medical Clinic I, Department of Pneumology and Allergy/Immunology, Friedrich-Schiller-University Jena, Germany
1Correspondence: Pneumology, Medical Clinic I, Friedrich-Schiller-University Jena, Erlanger Allee 101, 07740 Jena, Germany. E-mail: grefachew.workalemahu{at}med.uni-jena.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T-lymphocytes are believed to play a role in maintaining the normal configuration of epithelial tissue. As little is known about the factors mediating this function, we addressed the question of whether T-lymphocytes produce fibroblast growth factor (FGF)-9 as well as two other growth factors associated with epithelial tissue reconstitution. Blood T cells isolated from healthy donors were grown in the presence of isopentenyl pyrophosphate (IPP) or transforming growth factor-ß1 (TGF-ß1)/interleukin-15 (IL-15) for 24 h and were assessed for the expression and synthesis of FGF-9, keratinocyte growth factor (KGF), and epidermal growth factor (EGF). Resting human T cells constitutively expressed KGF and FGF-9 mRNA but no EGF mRNA. In the presence of IPP, FGF-9 mRNA expression significantly increased in a dose-dependent manner, expression of KGF remained unaltered, and EGF mRNA could not be detected. In contrast to IPP, stimulation of the cells with TGF-ß1/IL-15 did not alter FGF-9 expression. Moreover, stimulation with anti-CD3 does not induce FGF-9 expression but triggers a high signal of interferon- mRNA. Western blot analysis of T cell lysates, prepared 4 days following stimulation with IPP, showed an increase of FGF-9 protein as compared with control cells. In conclusion, the results demonstrate for the first time that human blood and bronchoalveolar lavage T-lymphocytes are capable of expressing FGF-9. The data also provide novel evidence that immunoregulatory cells can synthesize FGF-9.
Key Words: growth factors • bacterial peptide • IPP
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T-lymphocytes play an important role in the immune surveillance on epithelial surfaces preventing tissue injury from toxic agents [1
] and enhancing host defense in various bacterial infections [2
]. For instance, T cell-deficient mice treated with bleomycin respond with a fulminant inflammatory reaction leading to a diffuse pulmonary fibrosis as compared with wild-type animals [3
]. In addition, mice deficient in T-lymphocytes develop more severe and extensive epithelial damage than control mice after inoculation with Nocardia asteroides or exposure to ozone [4
]. Further, in the absence of T cells, mice do not survive an infection with Listeria monocytogenes [5
] or Klebsiella pneumoniae [6
] bacteria, normally not lethal for immunologically intact animals. Following infection, the mice react with an exaggerated innate response to the microbes with high systemic levels of proinflammatory cytokines [interleukin (IL)-6, IL-12, interferon- (IFN-)] and an antigen-specific T cell response [5
]. Moreover, the absence of T-lymphocytes is associated with a reduction in epithelial cell turnover and a down-regulation of the expression of major histocompatibility complex class II molecules on epithelial cells [7
]. Therefore, in addition to their potential role in the host-defense response toward exogenous agents, these findings suggest that T-lymphocytes may play a critical role in maintaining an epithelial integrity via promoting epithelial growth. However, the mechanisms through which T-lymphocytes specifically respond to certain stimuli and control homeostasis of epithelial surfaces are not known to date.
Human T-lymphocytes produce a number of cytokines such as IFN- and tumor necrosis factor-
(TNF-) [8
9
10
]. In addition, human T cells express and synthesize connective tissue growth factor (CTGF) [11
], a growth factor which stimulates cell proliferation and extracellular matrix synthesis by connective tissue cells [12
]. Further, T-lymphocytes obtained from mice have been shown to produce keratinocyte growth factor (KGF) [13
], a cytokine associated with epithelial cell growth in vitro and in vivo [14
]. However, the signals potentially involved in promoting epithelial restitution through T-lymphocytes in humans have not yet been identified.
Fibroblast growth factor (FGF)-9 represents a 23-kDa protein expressed in human and rat central nervous systems (CNS) [15
, 16
], where it functions as a mitogen for rat brain astrocytes and oligodendrocyte progenitors in vitro [17
]. Further, expression of FGF-9 has been reported in the epithelium of tooth germs [18
] and prostate epithelium [19
]. Moreover, endometrial epithelial and stromal cells express high-affinity receptors for FGF-9, and treatment with FGF-9 induces cell proliferation in a dose-dependent manner [20
]. Collectively, these data suggest a role of FGF-9 in epithelial growth and homeostasis. As little is known about growth factors produced by T-lymphocytes, we addressed the question of whether human blood and bronchoalveolar lavage (BAL) T-lymphocytes express and synthesize FGF-9. In addition, we compared the results to the expression of the epidermal growth factor (EGF), KGF, as well as FGF-9 expression in ß T-lymphocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T-lymphocytes T-lymphocytes were obtained from the routine BAL of a patient suffering from hypereosinophilic syndrome and a healthy control subject who volunteered to participate in a current research project. The volunteer gave his written informed consent, and the Ethics Committee of the Friedrich-Schiller-University of Jena (Germany) approved the study.
Blood lymphocytes were separated by Ficoll density gradient centrifugation and blood, and BAL T cells were purified with a T cell receptor-/ microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) using the magnetic cell-sorting technique. The purity of isolated cells was >95%, and the viability was >97%. The median distribution (range) of the isolated T-lymphocyte subsets was as follows (data given are percentage of total T-lymphocytes; n=4): V1 7 (5–10), V2 74 (37–93), V3 3 (1–7), V9 75 (36–96).
Culture and stimulation of T-lymphocytes
Human T cells (5x105/0.5 ml) were cultured in RPMI-1640 medium (Cell Concepts GmbH, Umkirch, Germany) supplemented with 10% fetal calf serum (Gibco, Germany), 2 mM L-glutamine (Biochrom KG, Berlin, Germany), 100 IU/ml ampicillin, 100 ng/ml streptomycin, and 100 ng/ml gentamycin (Roche Molecular Biochemicals, Mannheim, Germany). Culture of cells was performed in the absence or presence of transforming growth factor (TGF)-ß1 (1 ng/ml)/IL-15 (10 ng/ml; Cell Concept, Umkirch, Germany) or alternatively, isopentenyl pyrophosphate (IPP; 0.5, 1, 2, 3, and 5 µg/ml; Sigma, Taufkirchen, Germany).
In a parallel experiment, the kinetic of FGF-9 expression induced by IPP (2 µg/ml) was analyzed by exposing human blood T-lymphocytes to anti-CD3 monoclonal antibody (mAb; 1 µg/ml; BD Bioscience, Heidelberg, Germany). Total RNA was isolated at different time intervals (0, 8, 16, 24, and 48 h), and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using FGF-9 and IFN- primers.
RT-PCR
At indicated time points, total RNA was isolated using an RNA isolation kit (Roche Molecular Biochemicals, Mannheim, Germany) and was reverse-transcribed into single-stranded cDNA using a first-strand cDNA synthesis kit (Roche Molecular Biochemicals). Briefly, 2 µg total RNA were incubated with 1.6 µg oligo (dT)15 primer at 25°C for 10 min followed by a 1-h incubation at 42°C in the presence of 1x first-strand buffer, 12.5 mM MgCl2, 2.5 mM each deoxy-unspecified nucleoside 5'-triphosphate (dNTP), 50 U RNase inhibitor, 10 µg/ml gelatin, and 20 U avian myloblastosis RT/50 µl total volume. The DNA was subjected to PCR as follows: the PCR reaction mixture contained 1x PCR Gold buffer, 1.5 mM MgCl2 solution, 0.8 mM dNTP mixture, 0.2 µM each specific primer, and 1.25 U Amplitaq Gold DNA polymerase (Roche Molecular Biochemicals) in a total volume of 50 µl. One-tenth of the cDNA obtained was amplified in each PCR reaction using AmpliTaq Gold polymerase (Roche Molecular Biochemicals) in a BioRad PCR cycler (BioRad Laboratories GmbH, München, Germany). Thirty-five cycles of amplification were performed with an initial heating step of 10 min at 95°C followed by 94°C for 45 s, the primer-specific annealing temperature for 45 s, 72°C for 1 min, and final synthesis of 10 min at 72°C. The annealing temperature used for FGF-9, KGF, EGF, IFN-, and cyclophilin was 56°C, 50°C, 60°C, 52 °C, and 60°C, respectively. Specific primer sequences were selected from the gene bank and synthesized by BioTez Berlin-Buch GmbH (Berlin, Germany). FGF-9 sense primer 5'-GGCGTGGACAGTGGACTCTACCTC-3'; FGF-9 antisense primer 5'-TTCCCATCCAAGCCTCCATCATAC-3'; EGF sense primer 5'-TGATTTGCCCTGACTCTACTCCAC-3'; EGF antisense primer 5'-GGCCTGCGACTCCTCACATC-3'; KGF sense primer 5'-AATATTACCTGCTTACTCTTCGTT-3'; KGF antisense primer 5'-GTGGGCTGTTTTTGTTCTTT-3'; IFN- sense primer 5'-TTTGGGTTCTCTTGGCTGTTAC-3'; IFN- antisense primer 5'-CTTTTTCGCTTCCCTGTTTTAG-3'; cyclophilin sense primer 5'- CATCTGCACTGCCAAGACTG-3'; cyclophilin antisense primer 5'-CTGCAATCCAGCTAGGCATG-3'.
Finally, the amplified PCR products were separated on a 1.5% agarose gel and detected by ethidium bromide staining under UV transillumination. The intensity of gene expression was quantified by densitometry using Phoretix 1D software. The amount of each cDNA product was standardized relative to cyclophilin.
Western blot
T-lymphocytes cultured for 4 days with or without stimulus were harvested after centrifugation at 300 g, and the cell sediments were lysed for 30 min on ice in lysis buffer (20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, and 10 µg leupeptin; Sigma, Steinheim, Germany). The protein-containing supernatants were separated from cell debris by centrifugation at 14,000 g, and protein concentrations were determined spectrophotometrically. The lysates containing 30 µg total protein per sample were boiled for 4 min in 2x sodium dodecyl sulfate (SDS) sample buffer (Invitrogen, Karlsruhe, Germany) and were subjected to 4–20% gradient SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions. Using electroblotting, the fractionated proteins were transferred to nitrocellulose membrane (Invitrogen). Equal loading was verified by staining the nitrocellulose membrane with Ponceau S (Sigma, Steinheim, Germany). Membranes were blocked at room temperature for 2 h in blocking buffer [3% bovine serum albumin (BSA) containing 0.2% azide in Tris-buffered saline/Tween 20 (TBS-T)].
The nitrocellulose membranes were incubated overnight at 4°C with mouse anti-human FGF-9 mAb (R&D Systems, Wiesbaden-Nordenstadt, Germany) or goat anti-ß-actin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), each diluted 1:1000 in dilution buffer (1% BSA, 0.2% azide, in TBT-T, pH 7.4). After repeated washing with TBS-T (20 mM Tris-base, pH 7.6, 137 mM NaCl, 0.1% Tween 20), the blots were incubated with horseradish peroxidase-conjugated anti-mouse or anti-goat antibodies (Santa Cruz Biotechnology) at room temperature for 1 h and were developed using enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech, Freiburg, Germany). The chemiluminescence was measured using a luminescent image analyzer LAS-1000 (Fuji Photo Film GmbH, Duesseldorf, Germany) and was analyzed densitometrically using Phoretix 1D Advanced v4.01 software (Biostep, Jahnsdorf, Germany).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T-lymphocytes T-lymphocytes with TGF-ß1 and IL-15 has an additive effect on the expression of the cellular activation marker Eß7 integrin (CD103) [21
] and induction of CTGF [11
]. Therefore, in the present study, we initially incubated purified human blood T-lymphocytes with medium or TGF-ß1/IL-15 at a concentration of 1 ng/ml and 10 ng/ml, respectively, for 24 h. Total RNA was isolated, and the cellular mRNA signal for FGF-9, KGF, and EGF was assessed using RT-PCR. Cells cultured for 24 h in medium buffer expressed FGF-9 and KGF, but no signal could be detected for EGF (see Fig . 2A
2B
2C
, lane 1). Addition of TGF-ß1/IL-15 to the culture did not alter the cellular expression pattern for the three growth factors (Figs. 1
, lane 2, and 2 . A
2B
2C
, lane 2). In contrast to T-lymphocytes, a fibroblast cell line (Colo 849) expressed EGF and KGF (Fig. 2B
and 2C
, lane 8) but not FGF-9 (Fig. 2A
, lane 8), indicating a cell-specific expression of the growth factors investigated.
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T-lymphocytes [22
], the cells were then cultured with increasing concentrations of IPP (0.5–5 µg/ml) for 24 h. As demonstrated in Figures 1
and 2A
, lanes 3–7), IPP caused a concentration-dependent increase of FGF-9 mRNA with a maximal effect at 2 µg/ml. In contrast, no mRNA could be found with respect to EGF (Fig. 2C
, lanes 1–7), and expression of KGF remained unchanged (Fig. 2B
, lanes 1–7).
To assess whether epithelium-associated T cells also express FGF-9, T-lymphocytes obtained from the routine BAL of a subject suffering from hypereosinophilic syndrome and a healthy control subject were analyzed using RT-PCR. As depicted in Figure 3C
and 3D
, BAL T-lymphocytes revealed a similar FGF-9 mRNA expression pattern as blood T-lymphocytes (Fig. 3A)
. They constitutively expressed FGF-9 mRNA, which was up-regulated in the presence of IPP at a concentration of 2 µg/ml, and TGF-ß1/IL-15 had no effect. In contrast, constitutive FGF-9 expression in ß T-lymphocytes was not affected by the presence of TGF-ß1/IL-15 or IPP (Fig. 3B)
.
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T-lymphocytes use the CD3-mediated pathway for the expression of FGF-9 mRNA, the cells were cultured in the presence or absence of an anti-CD3 mAb up to 48 h and analyzed for the expression of FGF-9 and IFN-.
Upon anti-CD3 mAb stimulation alone, human T-lymphocytes showed no significant expression of FGF-9 mRNA over basal expression throughout the period studied (Fig. 4A
). In contrast, increased amounts of IFN- mRNA were produced by T cells when stimulated with anti-CD3 mAb or with IPP (Fig. 4B)
.
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T-lymphocytes were investigated for the production of the protein encoded for by FGF-9 mRNA in response to IPP. Supernatants from cell lysates and from stimulated cells of human T-lymphocytes cultured for 4 days were subjected to immunoblot analysis after SDS-PAGE electrophoresis. As depicted in Figure 5
, trace amounts of FGF-9 were detected in cells stimulated with TGF-ß1/IL-15 (Fig. 5A
, lane 2) as compared with the nonstimulated controls (Fig. 5A
, lane 1). In contrast, in T-lymphocytes cultured in the presence of IPP (2 µg/ml) for 4 days, an increased amount of FGF-9 protein could be detected (Fig. 5A
, lane 3). However, in supernatants of unstimulated T-lymphocytes or cells incubated with TGF-ß1/IL-15 or IPP, no FGF-9 protein could be detected (Fig. 5B)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T-lymphocytes obtained from blood and BAL constitutively express FGF-9 mRNA, which is up-regulated when the cells are exposed to the bacterial-derived, nonpeptide organophosphate IPP, and TGF-ß1/IL-15 and anti-CD3 had no effect on the growth factor expression. In addition, T-lymphocytes synthesize the FGF-9 protein. Second, as previously the expression of the growth factor has been detected in nerve, epithelial, and stromal tissue, the data show for the first time that FGF-9 can be produced by human cells that are associated with the immune system, namely T-lymphocytes and ß T-lymphocytes.
Human T-lymphocytes have been shown to secrete a number of cytokines such as IFN- and TNF- [8
9
10
], growth factors such as CTGF [11
], as well as a number of chemokines [23
]. The demonstration herein that T-lymphocytes constitutively express FGF-9 mRNA and synthesize the FGF-9 protein, both of which are significantly increased on exposure of the cells to the bacterial-derived, nonpeptide organophosphate IPP, adds another growth factor to the list of factors produced by T-lymphocytes.
There are several lines of evidence connecting FGF-9 with epithelial tissue. For instance, expression of FGF-9 has been reported in the epithelium of tooth germs [18
] and prostate epithelium [19
] and during wound-healing of the skin [24
]. Endometrial epithelial and stromal cells express high-affinity receptors of FGF-9 [20
]. In addition, treatment with FGF-9 causes endometrial cell proliferation [20
], suggesting that FGF-9 may play a role in epithelial cell replication and growth, although the cells involved in production of the growth factor are not known. Epithelial cells form a barrier between the internal and external environments. Like skin, the function of epithelial tissue essentially depends on the constant renewal of epithelial cells. As it has been shown that T-lymphocytes play a role in the maintenance of mucosal surfaces, the cells may accomplish the reconstitution of epithelia by virtue of FGF-9 release.
The FGF-9 expression and synthesis by T-lymphocytes observed herein are in contrast to two other growth factors also assessed in this study. First, EGF mRNA could not be detected in naive and stimulated cells. Second, KGF mRNA was found to be constitutively expressed but remained unaltered when cells were exposed to IPP or TGF-ß1/IL-15. The latter finding differs somewhat from data in a study with mice that demonstrate the expression of KGF by activated T cells obtained from skin and intestine, whereas lymphoid ß and T-lymphocytes showed no expression [13
], emphasizing a possible difference between human and mouse cells. However, it is not clear why KGF is expressed but is not increased following stimulation. Moreover, we cannot exclude the possibility that T-lymphocytes synthesize KGF or EGF under conditions different from those applied in this study.
T-lymphocytes play an important role in the host response to bacterial infection. A potential link between bacterial infection and T-lymphocytes is provided by IPP, a small phosphorylated, nonpeptide compound derived from the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis [22
]. IPP is found in the membrane of many pathogens and is not presented by professional antigen-presenting cells. However, IPP specifically activates a major subset of human T cells expressing the V9/V2 phenotype [25
] and induces the production of significant amounts of IFN- and TNF- [8
9
10
]. The data presented above support the role of IPP in activating T-lymphocytes of the V9/V2 phenotype and indicate that these small, phosphorylated, nonpeptide compounds may play an important role in igniting the host-immune response to intruding bacteria.
On substitution of IPP for TGF-ß1/IL-15 under the same incubation conditions, T cells failed to show an increase in FGF-9 expression above basal values. This observation is surprising considering that TGF-ß1 and IL-15 have been previously shown to induce the expression of the E (CD103) antigen [21
], which in conjunction with the integrin ß7chain, functions as a homing receptor for lymphocytes to epithelial tissues [26
]. In addition, TGF-ß1 and IL-15 stimulate human T-lymphocytes to express and synthesize CTGF [11
]. The reasons for the distinct response pattern of the cells to cytokines on one hand and IPP on the other are not known. However, one might speculate that T-lymphocytes are capable of responding distinctly to specific signals in a particular inflammatory microenvironment or following exposure to infectious agents as observed in other cells [27
, 28
].
The second important finding presented in this study is the production of FGF-9 by cells other than nerve, epidermal, and stromal tissue. FGF-9 is a 23-kDa protein, which is highly conserved in humans, rats, and mice [29 30 31 ] and belongs to the FGF family. FGF-9 has been originally identified as a glia-activating factor in culture supernatants of the human glioma cell line NMCG-1 [17 ] and is expressed in human and rat CNS [15 , 16 ] as well as motor neurons [32 ]. FGF-9 has been shown to act as a mitogen for rat brain astrocytes and oligodendrocyte progenitors [17 ]. In addition to its expression in nervous cells, FGF-9 has been shown to be expressed in epithelial and stromal tissues of the endometrium [20 ] and the prostate [19 ]. However, although FGF-9 expression has been restricted to structural tissues, a role for FGF-9 as a mediator within the immune network has not been reported to date. Thus, this finding sheds new light on a potential role of FGF-9 and the interaction between immune cells in the vicinity of epithelial surfaces. However, direct evidence for the epithelial growth-inducing activity of FGF-9 has yet to be provided.
There have been numerous examples of blood cells that differ functionally and phenotypically from the same cell type obtained from organ sites. It is generally believed that the difference is a consequence of the migration process from the circulation into the tissue or a result of specific environmental conditions [27
]. With respect to the FGF-9 expression in T-lymphocytes, however, no qualitative difference was observed between cells obtained from blood or BAL.
These findings appear to contrast with previously published data showing a difference between blood and epithelium-derived human T cells. Boismenu and Havran [13
] demonstrated in the mouse that activated T-lymphocytes obtained from murine skin and intestine expressed KGF, whereas the growth factor could not be found in naïve, quiescent cells. In contrast, the experiments presented herein suggest that human peripheral blood T-lymphocytes constitutively express KGF. The most likely explanation for this conflicting data may refer to species difference, a varied experimental set-up, and the different stimuli used in the experiments.
The data obtained in the present study demonstrate that T-lymphocytes also produce the FGF-9 mRNA translation product. However, this protein could only be detected in the lysed lymphocytes, as we failed to detect the protein in the supernatant of stimulated, intact cells. The reason why FGF-9 protein was found only in lysed lymphocytes is not immediately apparent. However, as it appears unlikely that a cell would produce and retain the protein within the cytoplasm, several explanations should be considered. First, the assay may not be sensitive enough to detect the protein in the cell supernatant. Second, once released by the cell, FGF-9 may be degraded by proteases within the pericellular space. Third, the T-lymphocytes may require a second signal ("second hit") for the secretion of FGF-9. Finally, the T cell may use FGF-9 in an autocrine manner internalizing the FGF-9/receptor complex.
There are a couple of limitations to our study, which should be taken into consideration when extrapolating the results. First, as cell lysates were used for the detection of the FGF-9 protein, we cannot definitively state whether the protein is actually secreted by the cell or remains contained within the cell. Second, we did not directly investigate the different subpopulations of T-lymphocytes responsible for the production of FGF-9. However, as T-lymphocytes expressing the V9/V2 phenotype specifically respond to IPP [33
], and this phenotype represented up to 93% of the cell preparations used in the experiments, it is likely that we preferentially detected the response of an individual T cell subpopulation.
Taken together, the results presented herein extend the spectrum of biological factors synthesized by T-lymphocytes to include FGF-9. As T cells are involved in maintaining epithelial integrity, and FGF-9 is detected in epithelial tissues, the release of this factor by T-lymphocytes may represent at least one mechanism through which the cells exert control over mucosal tissue and promote epithelial growth. In addition, this report is the first to demonstrate the production of FGF-9 in cells associated with the immune system. Overall, the results provide new insights into the potential mechanism by which T-lymphocytes exert their function in maintaining homeostasis of epithelial surfaces. Further investigations on the function of FGF-9 in epithelial physiology and pathophysiology are warranted.
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ACKNOWLEDGEMENTS |
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Received September 25, 2002; revised October 30, 2003; accepted December 1, 2003.
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T cells are activated by intermediates of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis J. Immunol. 166,3655-3658This article has been cited by other articles:
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23 kDa) could be observed after stimulation with IPP (lane 3). The ordinate demonstrates the ratio of FGF-9/ß-actin protein. Additionally, serum-free culture supernatants (B) from each sample were concentrated using a centricon centrifugal filter device, and the same amount of total protein was used for Western blot. FGF-9 protein was not found in the supernatant (lanes 1–3), indicating that FGF-9 was not secreted in the medium. Data shown are representative of four experiments. rFGF-9, Recombinant FGF-9 protein.