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Stem Cells, Vol. 18, No. 5, 331-342, September 2000
© 2000 AlphaMed Press

Thrombopoietin and Chemokine mRNA Expression in Patient Post-Chemotherapy and In Vitro Cytokine-Treated Marrow Stromal Cell Layers

Gretchen N. Schwartza, Udai Kammulab, M. Kim Warrenc, Matthew K. Parkd, Xiao-Yi Yana, Francesco M. Marincolab, Ronald E. Gressa

a Department of Experimental Transplantation and Immunology, Medicine Branch and
b Surgery Branch, National Cancer Institute, Bethesda, Maryland, USA;
c Poietic Technologies, Inc., Gaithersburg, Maryland, USA;
d Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA

Key Words. Quantitative RT-PCR • Marrow stromal layers • Microenvironment • TaqMan • Chemokines • Thrombopoietin • Insulin-like growth factors

Gretchen N. Schwartz, Ph.D., Building 10, Room 12N226, Department of Experimental Transplantation and Immunology, Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892, USA. Telephone: 301-435-8641; Fax: 301-402-0172; e-mail: gnschwar{at}cpcug.org


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD34+ cells and megakaryocyte progenitors were lower in marrow from patients after hematological recovery from the first cycle of 5-fluorouracil, leucovorin, adriamycin, cyclophosphamide (FLAC) chemotherapy plus PIXY321 (GM-CSF/interleukin 3; IL-3 hybrid) than in FLAC + GM-CSF or pre-FLAC marrows. Marrow stromal layers, an in vitro model of the marrow microenvironment, express a combination of stimulatory and inhibitory factors that modulate hematopoietic progenitor cell proliferation and differentiation. The TaqMan assay and quantitative reverse transcriptase-polymerase chain reaction were used to measure monocyte chemoattractant protein-1 (MCP-1), melanoma stimulatory growth activity, and monokine inducible by interferon-{gamma} (Mig) (inhibitory chemokines for primitive or megakaryocyte progenitors) mRNA levels in in vitro PIXY and GM-CSF-treated and patient post-FLAC marrow stromal layers. Chemokine mRNA was increased after in vitro GM-CSF and to a lesser extent after PIXY treatment. MCP-1 mRNA levels were fivefold higher in FLAC + PIXY than in FLAC + GM-CSF layers, and Mig mRNA was elevated in FLAC + GM-CSF layers. Thrombopoietin (TPO), insulin-like growth factor I (IGF-I), and IGF-II (stimulatory factors for primitive and megakaryocyte progenitors) mRNA were also measured. TPO mRNA levels were 30% lower in GM-CSF and PIXY-pretreated than in control layers with no decrease in IGF mRNA. TPO mRNA in stromal layers of patients who developed grade 3 thrombocytopenia (platelets < 20 x 109/l) during the third cycle of FLAC was only 24% of levels in stromal layers of marrow from other post-FLAC patients. Results demonstrate that patient and in vitro treatment had modulatory effects on TPO and chemokine mRNA expression in marrow stromal layers.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The continuous replacement of blood cells in normal adults is provided by committed and primitive progenitor cells that are heterogeneous with respect to their self-renewal, proliferation, and differentiation capacities [1, 2]. Normally, hematopoietic progenitors reside in the marrow, and blood cell production is regulated by complex interactions of progenitor cells within the marrow microenvironment [3-5]. The marrow microenvironment is composed of multiple cell types that include fibroblastic and adventitial reticular cells, macrophages, adipocytes, and endothelial cells. A close association of these cells provides the niches, adhesion molecules, and other factors that regulate the proliferation and differentiation of hematopoietic progenitors [3, 4, 6].

The stromal cell layer, an adherent layer of cells that forms in marrow long-term cultures (LTC), simulates the marrow microenvironment [3, 4]. Marrow stromal layers support the production, proliferation, and differentiation of colony-forming units for granulocytes and macrophages (CFU-GM), BFU-E, and megakaryocyte progenitors from cocultured CD34+ cells [3, 7-12]. Similar to the in vivo microenvironment, multiple stimulatory and inhibitory factors that regulate the proliferation and differentiation of hematopoietic progenitors are expressed in marrow stromal layers [3, 10, 13-17]. Increased or reduced levels of some of those factors have been associated with the reduced capacity of marrow stromal layers to support in vitro hematopoiesis that is observed with some malignancies, viral infections, and after chemotherapy and marrow transplantation [7, 8, 11, 18, 19].

Previous results demonstrated that stromal layers of marrow obtained from patients after their blood cell recovery from the first cycle of 5-fluorouracil, leucovorin, adriamycin, cyclophosphamide (FLAC) chemotherapy produced fewer CFU-GM and BFU-E than normal donor stromal layers [7]. Similarly, fewer CFU-GM were detected from normal donor CD34+ cells cocultured with marrow stromal layers previously treated in vitro with GM-CSF or PIXY321 (a GM-CSF/interleukin 3 [IL-3] hybrid molecule) [7], two factors that were used to enhance blood cell recovery after FLAC chemotherapy [20]. Neutralizing antibody to tumor necrosis factor (TNF)-{alpha} partially abrogated the hematopoietic suppressive effects of some patients post-FLAC and in vitro GM-CSF-pretreated stromal layers but had no effect in PIXY-pretreated cultures [7]. The results suggest that factors in addition to the possible direct effects of TNF-{alpha} contributed to the hematopoietic suppressive effects of post-chemotherapy and in vitro cytokine-treated stromal layers.

Monocyte chemoattractant protein (MCP)-1 and monokine inducible by interferon-{gamma} (Mig) are two chemokines recently shown to inhibit the proliferation or delay cell cycle progression of primitive progenitors [10, 21, 22]. Gro-{alpha} is a chemokine shown to decrease colony numbers from the 32D murine myeloid progenitor cell line [23] and suppress megakaryocyte production from normal donor CD34+ cells (unpublished data). Macrophages, endothelial cells, and fibroblasts (three of the cell types that comprise the marrow microenvironment) are some of the cells that can be induced to produce Mig and Gro-{alpha} [24-28]. MCP-1 is produced in marrow stromal layers [10], and GM-CSF enhances MCP-1 mRNA expression in monocytes [25]. Thrombopoietin (TPO) and insulin-like growth factors (IGF) have stimulatory effects on committed and primitive hematopoietic progenitors [21, 29-34] and are produced in marrow stromal layers [13, 14, 35]. An increase in inhibitory chemokines and a decrease in stimulatory factors such as TPO and IGFs may be the mechanisms responsible for the hematopoietic suppressive effects of cytokine-pretreated and patient post-chemotherapy marrow stromal layers. Recently, the TaqMan assay and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were developed [36], and this methodology has been used for the quantitative assessment of cytokine mRNA expression in peripheral blood cells and other tissues, and to follow sequential gene regulation during differentiation [13, 37-41]. In the present report, the TaqMan assay and real-time quantitative RT-PCR were used to determine relative levels of MCP-1, Gro-{alpha}, Mig, IGFs, and TPO mRNA in patient postchemotherapy and in vitro-treated marrow stromal layers.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Source of Normal Donor and Post-Chemotherapy Marrow
Normal donors and patients were required to give informed consent as participants of National Cancer Institute Institution Review Board-approved protocols. Some normal donor marrows were also purchased from Poietic/Biowhittaker (Gaithersburg, MD). Prechemotherapy marrow was obtained from three patients with stage IV breast cancer prior to the start of FLAC chemotherapy. Post-FLAC marrow was obtained from patients after their blood cell recovery from the first cycle of FLAC + no cytokine (n = 1), FLAC + GM-CSF (n = 4), or FLAC + PIXY321 (n = 3) and at least two days after discontinuing cytokine administration [20].

Establishment of Marrow Stromal Cell Layers
Low density cells (<=1.077 g/cm3) were resuspended to 2 x 106 cells/ml in Myelocult H5100 LTC medium (Stem Cell Technologies; Vancouver, BC; http://www.stemcell.com) supplemented with 1 x 10–6 M hydrocortisone-21-hemisuccinate (Sigma; St. Louis, MO; www.sigma-aldrich.com). One ml was added to inside wells of Nunclon 24-well plates (Nunc; Naperville, IL; http://www.nalgenunc.com) and sterile water was added to outside wells for humidity. Cultures were incubated at 37°C with 5% CO2 and 95% room air. Ten days later and weekly thereafter, 500 µl of medium and nonadherent cells were removed and replaced with an equal volume of fresh LTC medium. After four weeks the established stromal cell layers were exposed to 15 Gy gamma radiation (1 Gy/min) from 137Cs Gammacell 40 irradiator (1 Gy/min) to kill residual progenitor cells.

In Vitro Treatment of Marrow Stromal Layers
One day after irradiation, stromal layers were washed and fresh medium was added. For cytokine pretreatment studies, 20 ng/ml of human recombinant GM-CSF, IL-3, or G-CSF (all provided by R&D Systems; Minneapolis, MN; http://www.rndsystems.com), or PIXY321 (Immunex; Seattle, WA; http://www.immunex.com), or an equivalent volume of Dulbecco's phosphate buffered saline (DPBS) + 0.1% bovine serum albumin were added to the stromal layer cultures. Two or three days later, the layers were washed at least three times to remove cytokine, and fresh Myelocult was added without factors. After an additional seven days the layers were processed for detection of target mRNAs. Some stromal layers were also stimulated with either 5 µg/ml E. coli 0127:B8 lipopolysaccharide ([LPS], Difco Labs; Detroit, MI), or 100 ng/ml (1 x 103 units/ml) interferon {gamma} (IFN-{gamma}) (R&D).

Source of Cells for Standard Curves for Relative Quantitation of Target mRNAs
HepG2 cells, a liver carcinoma cell line (ATCC; Rockville, MD) shown to constitutively produce TPO [42], were used as sources of TPO and GAPDH mRNA. Cells were maintained in enriched Iscove's modified Dulbecco's medium (IMDM) [43] with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone; Logan, UT) and subcultured every four to five days. Cells were collected for RNA isolation after three days of culture, at which time 155 ng/ml TPO was detectable in the culture supernatants (Quantikine enzyme-linked immunosorbent assay [ELISA], R&D Systems).

Human monocytes were used as a source of mRNA for chemokine standard curves (Table 1Go). For control mRNA, freshly elutriated human monocytes (5-8 x 108 total cells) were obtained from healthy volunteers from the Clinical Center of the National Institutes of Health. Cells were resuspended in DPBS and 107 cells/ml were layered onto Ficoll-Paque Plus solution (Amersham Pharmacia Biotech AB; Uppsala, Sweden; http://www.apbiotech.com). After centrifugation (700 g, for 20 min at 18-20°C) to remove red cells, the interface was removed and washed twice in DPBS and resuspended at 106 cells/ml in IMDM containing 10% type AB endotoxin-tested (<60 pg/ml), male human serum (Sigma) and 30 mg/ml Ampicillin. Cells (15 ml) were plated in 100 x 20 mm untreated, suspension culture dishes (Corning Glass Works; Corning, NY) and incubated at 37°C, 5% CO2 with 95% room air. Six days later, nonadherent cells were removed by washing twice with RPMI 1640 (Life Technologies; Rockville, MD; http://www.lifetech.com). By seven days, 99% of the cells were CD11c+/CD13+ as determined by flow cytometry. On day 7 supernatants were discarded and fresh media supplemented with either 1,000 U/ml IFN-{gamma} (Pharmingen; San Diego, CA; http://www.pharmingen.com) or 1 µg/ml E. coli 0127:B8 LPS. Eight hours later, cell layers were rinsed with DPBS, and RNA was isolated.


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  		Table 1. Source of RNA used for standard curves in TaqMan quantitative RT-PCR
 
RNA Isolation and cDNA Synthesis
Total RNA was isolated from HepG2, CMK, and marrow stromal cell layers using Stratagene RNA Isolation Kit (Strategene; La Jolla, CA) following the manufacturer's instructions. Briefly, cells were washed at least three times with DPBS and the cells were lysed with guanidine isothiocyanate solution. Lysates were vortexed vigorously, then immediately frozen on dry ice, and stored at –70°C. RNA was isolated by phenol-chloroform extraction followed by isopropanol precipitation and air-dried as outlined in kit instructions. RNA pellets were resuspended in 40 µl DPEC-H2O (Quality Biological, Inc. [QBI]; Gaithersburg, MD) and heated in Eppendorf Thermomixer 5436 (Brinkmann Instruments, Inc.; Westbury, NY; http://www.brinkmann.com) for 5 min at 55°C to solubilize RNA. For RNA quantitation, dilutions were made in TE buffer, pH 7.4 (QBI) and optical densities were measured in Beckman DU 600 spectrophotometer (Beckman; Fullerton, CA; http://www.brinkmann.com). A260/A280 ratios were >=1.9 and RNA was stored -70°C. RNA was obtained from monocyte cultures by adding 3 ml Trizol (Life Technologies) per plate and following the manufacturer's instructions.

Quantitative RT-PCR
First-strand cDNA synthesis was performed using TaqMan Gold RT-PCR Kit (Applied Biosystems of Perkin-Elmer [ABI-PE]; Foster City, CA; http://www.perkin-elmer.com) following manufacturer's instructions. Random hexamers were used as primers to transcribe 2 µg total RNA per 100 µl reaction, and RT reactions were performed in a GeneAmp PCR System 2400 thermocycler (ABI-PE). Real-time quantitative RT-PCR was performed using the TaqMan assay and PCR amplifications in ABI-PE prism 7700 Sequence Detection System as previously described [36]. Briefly, a solution of 2x TaqMan Universal PCR Master Mix (ABI-PE) containing primers and probes was prepared and aliquoted into individual MicroAmp Optical Tubes (ABI-PE) and cDNA was added to give a final volume of 50 µl. Conditions for PCR reactions included 2 min at 50°C, 10 min at 95°C, and 40 cycles of denaturation at 95°C for 15 sec, and annealing/extension at 60°C for 1 min. Threshold cycle (CT) during the exponential phase of amplification was determined by real-time monitoring of fluorescent emission after cleavage of sequence-specific probes by nuclease activity of taq polymerase. An increase in fluorescence is proportional to the amount of PCR product, and the PCR amplification cycle at which the reporter dye fluorescence passes a selected baseline is called the CT. Samples with low CT values have a high copy number and samples with high CT values have lower copy numbers. CT values were exported to Excel for calculations.

GAPDH was used as an internal control gene, primers and probe were from GAPDH Control Kit (ABI-PE). Primers and fluorogenic probes for TPO, MCP-1, Gro-{alpha}, Mig, IGF-I, and IGF-II were designed using Primer Express Software (ABI-PE, version 1.0) so that either the forward primer or the probe spanned an intron-exon junction (Table 2Go). Probes were synthesized with 6-FAM as a reporter dye at the 5' end and TAMRA as a quencher dye at the 3' end; primers and probes were prepared by ABI. Concentrations of primers and probes were optimized and calculations for relative quantitation were performed as outlined in User Bulletin #2: ABI Prism 7700 Sequence Detection System.


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  		Table 2. Primer pairs and fluorogenic probes used in TaqMan PCRa
 
Relative Quantitation of Target mRNAs
Relative quantitation of TPO and GAPDH in marrow stromal layers was determined from standard curves established from PCR amplifications of serial dilutions of cDNA from HepG2 cells (Fig. 1Go). Cells from a normal donor's stromal layers were used to establish standard curves for IGF-I and IGF-II (Table 1Go). Relative quantitation for the chemokines was determined from standard curves established from PCR amplifications of cDNA from IFN-{gamma}-stimulated monocytes for Mig, and LPS-stimulated monocytes for MCP-1 and Gro-{alpha} (Table 1Go). The values were then normalized to values obtained for GAPDH and then expressed relative to normalized values from nonstimulated monocyte cultures. After 40 PCR amplification cycles, there was no significant increase in signal above baseline for the target mRNAs in nontranscribed RNA. In some runs, a positive signal for GAPDH was observed; however, the expression was less than 0.5% of levels detected in cDNA from marrow stromal cell layers (data not shown).



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  		Figure 1. Example of standard curves for TPO and GAPDH mRNA expression. PCR amplifications were performed on serial dilutions of cDNA prepared from total RNA isolated from the HepG2 liver carcinoma cell line. Values are from duplicate wells at each dilution. Amplifications for TPO and GAPDH were performed in separate tubes.

 
Detection of Megakaryocyte Production from CD34+ Cells
To quantitate production of megakaryocytes from CD34+ cells, the cells were placed in liquid culture with growth factors to stimulate proliferation and lineage commitment. CD34+ cells were seeded into 96-well plates at 500, 1,000, and 2,000 cells per well in serum-substituted medium supplemented with 2 ng/ml IL-3 and 20 ng/ml c-kit ligand (R&D Systems). TPO was not commercially available at the time patient cells were assayed. After 10 days of culture, megakaryocyte-lineage cells were identified by expression of GP IIb/IIIa (CD41a) and quantitated using an ELISA as previously described [32, 44, 45]. Absorbance values for GP IIb/IIIa expression obtained after proliferation of 2,000 CD34+ cells were normalized to the number of CD34+ cells per ml of unseparated marrow aspirate.

Statistics
Student's two-tail t-test was used to determine significant differences between different treatment groups. Groups were considered to be significantly different for p < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of in Vitro Treatments on Chemokine mRNA Levels in Marrow Stromal Layers
Real-time quantitative RT-PCR was used to assess the effects of in vitro treatments on MCP-1, Gro-{alpha}, or Mig mRNA levels in normal donor marrow stromal layers. To characterize possible changes in chemokine mRNA levels in marrow stromal layers, the effect of in vitro LPS and IFN-{gamma} treatment on chemokine expression in marrow stromal layers was investigated. MCP-1 and Gro-{alpha} mRNA levels were increased by 2- to 20-fold and 10-fold, respectively, in response to LPS stimulation (Table 3Go). MCP-1 mRNA levels were two- to sixfold higher in IFN-{gamma}-treated stromal layers than in DPBS control cultures. MCP-1 protein concentrations in stromal layer culture supernatants were also increased in response to IFN-{gamma} treatment (Table 3Go, footnote e). In contrast, no significant increase in Gro-{alpha} mRNA levels or protein concentration was observed with IFN-{gamma} treatment. IFN-{gamma} induced a 2- to 4-log increase in Mig mRNA that was evident within 7 h (Table 4Go). By 24 h, mRNA levels had decreased in both DPBS-control and IFN-{gamma}-stimulated stromal layers; however, mRNA levels in IFN-{gamma} stimulated stromal layers were still significantly elevated. The results demonstrate that in addition to MCP-1 and Gro-{alpha} mRNA, Mig mRNA was also detectable in marrow stromal layers, and that elevated mRNA levels were still detectable 24 h after stimulation.


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  		Table 3. Effect of LPS and IFN-{gamma} on MCP-1 and Gro-{alpha} mRNA levels in marrow stromal layers
 

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  		Table 4. Effect of in vitro IFN-{gamma} treatment on Mig mRNA levels in normal donor marrow stromal layers
 
The effect of in vitro GM-CSF and PIXY pretreatment on chemokine protein and mRNA levels in marrow stromal layers was investigated. In a preliminary study, chemokine mRNA levels were measured in stromal layers after 7 h in the presence of either DPBS, GM-CSF, or PIXY. Increased mRNA levels for MCP-1 in GM-CSF and PIXY-treated layers were fourfold higher than in DPBS control layers. Mig mRNA levels were 40-fold higher in GM-CSF and 10-fold higher PIXY-treated layers (Fig. 2AGo). A twofold increase in Gro-{alpha} mRNA was observed with GM-CSF, but not PIXY. Relative chemokine mRNA levels were also determined in marrow stromal layers after the cytokines were washed out and fresh medium added without the addition of cytokines. Seven hours after media change, MCP-1 and Gro-{alpha} mRNA levels were elevated in GM-CSF, but not PIXY, pretreated layers (data not shown). Twenty-four hours after a media change, MCP-1 mRNA levels in stromal layers from a different donor were elevated in both GM-CSF and PIXY-pretreated layers and no increase was observed for Gro-{alpha} (Fig. 2BGo). Similar results were observed 24 h after media change for stromal layers of marrow from another donor (data not shown). Mig mRNA levels were 2.5-fold higher in GM-CSF-pretreated layers than in DPBS control or PIXY-pretreated layers. Concentrations of MCP-1 and Gro-{alpha} in culture supernatants from marrow stromal layers treated with either GM-CSF or PIXY were approximately 1.2- to 1.5-fold higher than in DPBS control (data not shown). No ELISA was available for Mig. These results demonstrate that in vitro, GM-CSF and PIXY pretreatment promoted an increase in expression and production of multiple hematopoietic suppressive chemokines.



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  		Figure 2. Effect of in vitro GM-CSF and PIXY321 treatment on chemokine mRNA expression in normal donor marrow stromal layers. Stromal layers established from normal donor marrow were incubated with 20 ng/ml of GM-CSF or PIXY321 or equivalent volume of DPBS. A) 7 h after addition of factors cells were lysed for RNA isolation and cDNA synthesis (i.e., Stro05) or B) cytokines were washed out three days later and fresh medium without factors was added. Medium was replaced after an additional week. Twenty-four hours later cells were lysed for RNA isolation and cDNA synthesis (i.e., Stro07). PCR amplifications were run on cDNA from 50 ng total RNA for MCP-1 and Gro-{alpha}, and 308 ng for Mig. Amplifications for chemokines and GAPDH were performed in separate tubes. Relative expression in marrow stromal layers was determined from standard curves and then normalized to values obtained for GAPDH. The normalized values were then divided by normalized values from nonstimulated peripheral blood monocyte cultures. Values are the mean ± SD calculated from duplicate wells each for chemokine and GAPDH.

 
Effect of in Vitro Treatments on TPO and IGF mRNA Levels in Marrow Stromal Layers
Real-time quantitative RT-PCR was used to assess the effects of in vitro treatments on TPO, IGF-I, and IGF-II on mRNA levels in normal donor marrow stromal layers. Time after addition of fresh medium or LPS stimulation had little effect on relative levels of TPO mRNA in individual donor marrow stromal layers (Table 5Go). The effect of in vitro cytokine treatment was also evaluated. Marrow stromal cell layers were incubated with either DPBS as a control, GM-CSF, or PIXY321. Three days later the cytokines were washed out and replaced with fresh media without cytokines. Twenty-four hours after a later medium change, the cells were lysed for RNA isolation. TPO mRNA levels in stromal layers established from three out of four different normal donor marrows were approximately 30% lower than in DPBS-control layers (Fig. 3AGo). In contrast, no effect of cytokine pretreatment was observed for IGF-I (data not shown) or IGF-II mRNA levels (Fig. 3BGo). These results demonstrate that prior exposure of marrow stromal layers to GM-CSF or PIXY321 was associated with a reduction in TPO mRNA, and this reduction was detectable 24 h after fresh medium without factors was added.


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  		Table 5. Effect of media change and LPS stimulation on TPO mRNA expression
 


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  		Figure 3. Effect of in vitro cytokine treatment on TPO and IGF-II mRNA expression in normal donor marrow stromal cell layers. Marrow stromal layers were incubated with 20 ng/ml of GM-CSF, PIXY321, or equivalent volume of DPBS. Two or three days later the layers were washed at least three times to remove cytokine, and fresh Myelocult was added without factors. After an additional seven days the layers were processed for detection of target mRNAs. PCR amplifications were run on cDNA from 428 ng total RNA for TPO and 200 ng total RNA for IGF-II. Amplifications for TPO and GAPDH were performed in separate tubes. Relative expression of TPO in marrow stromal layers was determined from the standard curve and then normalized to values obtained for GAPDH. Values are the mean ± SD calculated from duplicate wells each for TPO and GAPDH. *p < 0.05.

 
Effect of Patient Treatment on Chemokine and TPO mRNA Levels in Marrow Stromal Layers
Marrow obtained from patients before FLAC chemotherapy and after their hematological recovery from the first or second cycle of FLAC was enriched for CD34+ cells and then cultured for detection of megakaryocyte production. Absorbance values for expression of the platelet glycoproteins GP IIb/IIIa were used as a measure of megakaryocyte production from progenitors. Absorbance values after the proliferation of 2,000 CD34+ cells were not significantly different between the patient treatment groups (Table 6Go). However, due to a reduced number of CD34+ cells in FLAC + PIXY marrows, absorbance values normalized to the number of CD34+ cells/ml were only approximately one-third of the values in FLAC + GM-CSF and FLAC + no cytokine marrows. Similar lower numbers of CFU-GM and possibly LTC-initiating cells associated with reduced numbers of CD34+ cells were also observed in FLAC + PIXY marrows [7, 46]. The results suggest that recovery of CD34+ cells and progenitors was delayed or suppressed in FLAC + PIXY marrows.


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  		Table 6. Megakaryocyte production from patient CD34+ cells
 
Levels of mRNA were compared in stromal layers of marrow from FLAC + GM-CSF and FLAC + PIXY-treated patients. Stromal layers were established from marrow obtained from patients after their hematological recovery from the first cycle of FLAC chemotherapy and from normal donors. Stromal layer cells were lysed 24 h after a media change and the samples frozen until assay. Total RNA and cDNA from patient and normal donor samples frozen at the same time were prepared together. After PCR, stromal layer target mRNA measurements were normalized to GAPDH and then compared to levels from normal donor stromal layers (Fig. 4Go). MCP-1 mRNA levels were approximately fivefold higher in FLAC + PIXY stromal layers than in FLAC + GM-CSF stromal layers (p = 0.007) and Mig mRNA was higher in FLAC + GM-CSF layers. The mRNA levels for Gro-{alpha}, TPO, IGF-I (data not shown), and IGF-II were not significantly different in FLAC + GM-CSF and FLAC + PIXY layers. One FLAC + PIXY patient developed grade 4 thrombocytopenia (platelets less than 10 x 109/l) and two FLAC + PIXY patients and one FLAC + GM-CSF-treated patient developed grade 3 thrombocytopenia (platelets less than 20 x 109/l) during cycle 3 of FLAC chemotherapy. Normalized values for TPO mRNA expression from those four patient marrow stromal layers were 31 ± 11 x 10–4 (mean ± standard error [SE]) or only 24% of the 131 ± 31 x 10–4 (n = 4) values (p = 0.02) in stromal layers of marrow from patients who maintained platelet counts greater than 20 x 109/l during three cycles of FLAC chemotherapy. Even though patient sample numbers were small, the results indicate that the TaqMan assay and quantitative RT-PCR provide a sensitive assay for detection of differences in cytokine mRNA levels in marrow stromal layers associated with patient treatment.



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  		Figure 4. Expression of mRNA in post-FLAC chemotherapy marrow stromal layers. Stromal layers were established from marrow obtained from patients after their blood cell recovery from the first cycle of FLAC chemotherapy. The layers were lysed for RNA isolation and cDNA synthesis 24 h after media change. PCR was performed on cDNA from 50 ng total RNA for MCP-1 and Gro-{alpha}, 308 ng total RNA for Mig, 428 ng of total RNA for TPO, and 100 ng total RNA for IGF-II. Amplifications for target mRNAs of interest and GAPDH were performed in separate tubes. Values were normalized to GAPDH and then compared to average values from normal donor and pre-FLAC stromal layers. Values are the mean ± SE from five normal donors, 1 FLAC + no cytokine, 4 FLAC + GM-CSF, and 4 FLAC + PIXY marrow stromal layers, except 2 FLAC + GM-CSF for Mig. *By Student's two-tail t-test values from FLAC + GM-CSF stromal layers were significantly different (p < 0.05) from FLAC + PIXY layers.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results in the present and previous reports [7, 46] demonstrated that committed and possibly primitive hematopoietic progenitor numbers were lower in patient marrow after hematological recovery from the first or second cycle of FLAC chemotherapy plus PIXY321 (a GM-CSF/IL-3 hybrid molecule) than in patient FLAC + GM-CSF or pre-FLAC marrows. Real-time quantitative RT-PCR was used to investigate possible differences in mRNA expression of the stimulatory factors TPO, IGF-I, and IGF-II and of the inhibitory chemokines, MCP-1, Gro-{alpha}, and Mig in marrow stromal layers after in vitro GM-CSF or PIXY treatment or in vivo FLAC chemotherapy.

MCP-1, Gro-{alpha}, and Mig mRNA were detectable in normal donor marrow stromal layers. MCP-1 and Gro-{alpha} protein was also detectable in culture medium from the layers (no ELISA was available for Mig). MCP-1 mRNA and protein in cultures of human and murine marrow stromal layers have been reported by others [10, 16]. There are no known reports of Gro-{alpha}, Mig mRNA or protein measured in cultures of marrow stromal layers; however, expression of both chemokines can be induced in macrophages, fibroblasts, and endothelial cells [24-28], similar cell types found in primary stromal layers. LPS and IFN-{gamma} have been shown to promote increases in MCP-1 mRNA levels in monocytes, fibroblasts, and a murine marrow stromal cell line [16, 25]. In the present report, LPS and IFN-{gamma} promoted a 2- to 20-fold increase in MCP-1 mRNA levels within 7 h, and levels were higher after 24 h. As reported for other cells [24, 25], LPS promoted an increase in Gro-{alpha} mRNA and protein levels in marrow stromal layers. IFN-{gamma} has not been shown to induce Gro-{alpha} mRNA [24, 25] and in the present report, IFN-{gamma} had no effect on Gro-{alpha} mRNA or protein expression in marrow stromal layers. Similar to results observed for monocytes/macrophages and monocytic cell lines [26], IFN-{gamma} induced a rapid and dramatic two- to four-log increase in Mig mRNA in normal donor marrow stromal layers. These results are the first to demonstrate that Mig mRNA can be induced in primary marrow stromal layers. The results also demonstrate that chemokine mRNA levels were increased in marrow stromal layers in response to LPS and IFN-{gamma} stimulation, as reported for other cells.

GM-CSF and IL-3 have been shown to induce increases in mRNA or protein levels of hematopoietic suppressive factors. Some of these factors include TNF-{alpha}, macrophage inflammatory protein (MIP)-1{alpha}, and MCP-1 [7, 25, 47]. In the present report, GM-CSF and PIXY promoted an early increase in MCP-1 mRNA levels in marrow stromal layers and elevated levels were still evident a week after the factors had been washed out. There was an increase in Gro-{alpha} mRNA levels in response to GM-CSF, but not PIXY, even though protein concentrations were increased in response to both factors. Mig mRNA expression was increased in response to GM-CSF and to a lesser extent with PIXY. Jarmin et al. [47] observed that the stimulatory effects of GM-CSF and IL-3 on levels of MIP-1{alpha} mRNA expression in murine marrow macrophages were concentration-dependent and that increases in MCP-1 mRNA expression were different for similar concentrations of IL-3 and GM-CSF. The differences in Gro-{alpha} and Mig mRNA levels in response to GM-CSF and PIXY may have been related to concentration effects. Elevated chemokine mRNA expression in marrow stromal layers was still evident even after GM-CSF and PIXY were no longer present. Jarmin et al. [47] demonstrated that increased MIP-1{alpha} mRNA expression was a direct effect of GM-CSF on transcription and might be one mechanism for the early stimulatory effects of GM-CSF and PIXY on chemokine mRNA induction in marrow stromal layers. Previous work demonstrated that LPS stimulation induced the release of two- to sixfold higher concentrations of TNF-{alpha} in cultures of GM-CSF and PIXY-pretreated stromal layers than in control cultures [7]. TNF-{alpha} alone or in combination with other factors can induce MCP-1, Gro-{alpha}, and Mig expression [16, 24, 25, 27], and thus increased chemokine expression in GM-CSF and PIXY-pretreated layers may be in response to TNF-{alpha}.

Results demonstrate that GM-CSF and PIXY, cytokines used to enhance patient blood cell recovery after FLAC chemotherapy [20], promoted an increase in expression of the hematopoietic suppressive chemokines MIP-1{alpha} and IL-8 (data not shown) in addition to MCP-1, Gro-{alpha}, and Mig. Broxmeyer et al. [22] demonstrated that picogram amounts of multiple chemokines were sufficient to significantly decrease CFU-GM and BFU-E colony numbers from CD34+ cells. Small increases in concentrations of several chemokines in the marrow might be one possible cause for the hematopoietic suppressive effects of patient chemotherapy and cytokine treatment. In the present report, chemokine mRNA levels were measured in stromal layers of marrow obtained from patients after their hematological recovery from the first cycle of FLAC chemotherapy. Gro-{alpha} mRNA levels were not significantly different in patient FLAC + GM-CSF and FLAC + PIXY-pretreated layers. Mig mRNA levels were approximately fourfold higher in FLAC + GM-CSF than in FLAC + PIXY stromal layers. It is of interest to note a similar enhancement was observed in normal donor stromal layer pretreated with GM-CSF. MCP-1 mRNA was approximately fivefold higher in FLAC + PIXY than in FLAC + GM-CSF layers. Recent studies demonstrated that increasing the amount of MCP-1 in LTC inhibited the proliferation of primitive progenitors [10]. It is not known whether the increased levels of MCP-1 mRNA in FLAC + PIXY stromal layers were associated with increased production of MCP-1 sufficient to suppress the recovery of progenitors in FLAC + PIXY marrows.

TPO and IGF-II mRNA levels were also measured in GM-CSF and PIXY-pretreated and patient postchemotherapy stromal layers. Similar to results by others [13], TPO mRNA was detectable in normal donor marrow stromal layers. TPO mRNA expression in GM-CSF and PIXY-pretreated marrow stromal layers was approximately 50% of levels in DPBS control layers. IGF-II mRNA expression was similar in control and pretreated layers. TPO mRNA levels in patient FLAC + GM-CSF and FLAC + PIXY stromal layers were not significantly different. However, TPO mRNA expression in stromal layers of marrow from patients who developed grade 3 thrombocytopenia (platelets less than 20 x 109/l) during the third cycle of FLAC was only 25% of levels detected in stromal layers of marrow from patients who had higher platelet counts. No difference was observed for IGF-I, IGF-II, or chemokine mRNA expression relative to platelet counts. Results of others demonstrated that TPO levels were either maintained or increased in stromal layers of marrow obtained during thrombocytopenia that developed after chemotherapy or transplantation [48]. In the present report, stromal layers were established from marrow obtained after blood counts had recovered and before the next cycle of FLAC chemotherapy. Due to the small sample size, it is not clear whether low TPO mRNA levels reflect either a decrease in TPO mRNA expression or a failure of a compensatory increase in response to demand after chemotherapy. Two possible causes for reduced expression of TPO mRNA in GM-CSF and PIXY-treated and some post-chemotherapy stromal layers include either a loss of TPO-producing cells or decreased production per cell. Others demonstrated that reduced TPO mRNA in liver cirrhosis was due to a combination of a loss of TPO-producing liver parenchymal cells as well as decreased production in the surviving cells [49]. Cell separation studies, in conjunction with quantitative RT-PCR for expression of TPO and phenotypic markers that differentiate cell types, might be a useful approach to further assess the mechanisms and sequential changes responsible for reduced TPO mRNA in post-chemotherapy marrow stromal layers.

Quantitative RT-PCR for measuring TPO, IGF-II, IGF-I, MCP-1, Gro-{alpha}, and Mig mRNA levels in marrow stromal layers was performed using the TaqMan assay and ABI Prism 7700 sequence detection system [36]. Similar to results by others [36, 37, 39, 41] mRNA determinations were linear over 3- to 6-log dilutions, thus providing a sensitive assay for detection of low levels of a specific target mRNA such as TPO and Mig. The preliminary results in this report suggest that changes in the production of stimulatory and inhibitory factors in the marrow microenvironment may be one cause for impaired marrow function and delayed blood cell recoveries observed after chemotherapy and hematopoietic cell transplantation. Recent studies by Kummula et al. (personal communication) demonstrated that with the TaqMan assay and real-time quantitative RT-PCR, small changes in cytokine levels could be reproducibly detected in fine needle aspirates from melanoma tumors. Use of this methodology provides an approach for comparing cytokine levels in cultured stromal layers and marrow aspirates. Analysis of marrow directly without culturing should better reflect changes in the balance of negative and positive regulators produced by the marrow microenvironment that could be a cause for atypical marrow function observed after chemotherapy and autologous marrow or blood stem cell transplantation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received September 8, 1999; accepted for publication July 27, 2000.



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