Stem Cells http://www.stemcellsportal.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hauser, S. P.
Right arrow Articles by Lipschitz, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hauser, S. P.
Right arrow Articles by Lipschitz, D. A.
Stem Cells, Vol. 16, No. 4, 261-270, July 1998
© 1998 AlphaMed Press

Effects of Myelotoxic Agents on Cytokine Production in Murine Long-Term Bone Marrow Cultures

Simon P. Hauser, Markus C. Allewelt, David A. Lipschitz

Departments of Medicine, Physiology, and Biophysics, University of Arkansas for Medical Sciences, and Geriatric Research, Education and Clinical Center (GRECC), John L. McClellan Memorial Veterans Administration Hospital, Little Rock, Arkansas, USA

Key Words. Bone marrow culture • Stroma • Interleukin 6 • M-CSF • Methotrexate • Ceftazidime

Dr. Simon P. Hauser, Central Hematology Laboratory, Inselspital, University Hospital Bern, CH-3010 Bern, Switzerland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In long-term bone marrow cultures we studied the effect of the addition of the myelotoxic agents methotrexate (MTX) and ceftazidime (CEF) on the kinetics of cytokine production in the supernatant (SN) and on mRNA expression in the adherent stromal layer. In response to a medium change, a prompt and significant increase in colony-stimulating activity (CSA) and interleukin 6 (IL-6) concentrations in the SN occurred, peaking 12 h later. Two macrophage colony-stimulating factors (M-CSF) mRNA of 2.3 kb and 4 kb were identified. In response to the medium change, the 4.0-kb transcript increased significantly six h later. The 2.3-kb transcript expression was stronger than the 4-kb mRNA but did not cycle with medium change. At medium change, IL-6 mRNA was only minimally expressed; then a prompt increase occurred, which peaked six h later. The addition of 500 mg/ml (=915 µM) CEF to the culture caused a dose-dependent suppression of CSA and IL-6 supernatant concentrations and IL-6 and M-CSF mRNA expression. By contrast, 1 µM MTX had minimal effect on cytokine concentrations in the SN following medium change. mRNA expression was, however, suppressed. These results provide insights into the possible mechanisms whereby cytokines lead to increased myeloid cell proliferation following medium change. We also demonstrate that two myelotoxic agents have different effects on cytokine production. This information could be of value in developing rational approaches to the therapeutic use of cytokines in drug-induced neutropenia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytokines play a central regulatory role in hematopoiesis [1-5]. In murine long-term bone marrow cultures (LTBMC) the production of colony-stimulating activity (CSA), macrophage-colony stimulating factor (M-CSF), GM-CSF, G-CSF, interleukin 6 (IL-6), stem cell factor (c-kit ligand), and IL-3 by stromal cells has been reported [4, 6-12]. However, few studies have examined the kinetics of cytokine production in this culture system [8, 11-13]. A previous report showed a prompt increase in CSA concentrations following medium change which preceded increases in myeloid cell number. The addition of CSA by itself to culture, even without medium change, resulted in an increase in myeloid cell number similar to that seen with medium change [8]. This suggests that cytokines released in response to medium change are responsible for the resultant increase in myelopoiesis.

The addition to LTBMC of myelotoxic agents with very different mechanisms of action such as methotrexate (MTX) or the betalactam antibiotic ceftazidime (CEF), has been shown to cause a reversible dose-dependent suppression of myelopoiesis [14, 15]. In LTBMC, we have shown that CEF had significantly greater toxic effects on the marrow stroma than did MTX [16]. In this report, we have defined the effect of the addition to culture of either MTX or CEF on the production of CSA and IL-6 and stromal expression of M-CSF and IL-6 mRNA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Four- to six-month-old female C57BL/6 mice, obtained from Charles River Laboratories (Wilmington, MA), served as the source of bone marrow [15]. Cell numbers were determined using a model ZF Coulter counter (Coulter Electronics; Hialeah, FL).

LTBMC
The method employed for LTBMC was similar to that initially described by Dexter et al. [17]. To each flask, 2 x 106 bone marrow cells per ml culture medium were added. The culture medium consisted of 25% pretested horse serum (HS), 10% hydrocortisone hemisuccinate (10–6 M final concentration; Sigma Chemicals Company; St. Louis, MO), and 65% {alpha}-minimal essential medium (MEM). Cultures were incubated in a water-jacketed incubator at 33°C in an atmosphere containing 5% CO2 in humidified air. At weekly intervals, the supernatant was removed, the cells were recovered by centrifugation, and the total supernatant cell count was determined. The LTBMC was refed with 60% fresh medium and 40% cell-free supernatant removed at the time of medium change. The total supernatant culture medium was changed prior to measurement of CSA or IL-6 and replaced by fresh culture medium. At medium change, CSA and IL-6 were measured in the supernatant spent during the preceding week. At indicated times, cell-free supernatant was aliquoted and stored at -20°C for subsequent measurement of CSA and IL-6 [13]. Most studies described in this report used cultures initiated in sterile glass flaskettes (Nunc Inc.; Naperville, IL) as described previously [18]. In order to obtain larger numbers of stromal cells, LTBMC for RNA extraction were cultured in Corning tissue flasks (25 cm2; Corning Glass; Corning, NY).

CSA
CSA was measured by assessing colony number when 5 x 104 fresh murine bone marrow cells were cultured for colony forming units-granulocyte/macrophage (CFU-GM) in the presence of 10% cell-free supernatant from the LTBMC, as described by Worton et al. [19]. We expressed CSA values as units/ml (U/ml), where one unit corresponded to a single colony. For each experiment, a positive control culture with maximum colony growth was set up using lung-conditioned medium as a source for colony growth factors [20].

IL-6 Bioassay
IL-6 levels in the culture supernatants were determined using the factor-dependent mouse-mouse 7TD1 hybridoma cell line (Batch # F-10658; American Type Culture Collection; Rockville, MD) [21] and the MTT colorimetric assay for cell growth and survival [22]. Briefly, 4 x 103 7TD1 cells/well of a 96-well flat-bottom microtiter plate were cultured with a series of dilutions of LTBMC supernatants in a final volume of 100 ml colorless RPMI 1640 (Sigma), supplemented with 5 x 0.5 mM of 2-mercaptoethanol (Sigma), and 10% fetal bovine serum ([FBS], Sigma, Cell Culture), as reported recently [13].

RNA Extraction and Northern Blot Hybridization
After removal of the culture medium and three washes with ice-cold phosphate-buffered saline (PBS), total cellular RNA was extracted from the adherent whole stroma of three T-25 flasks per sample using lysis in guanidinium-isothiocyanate, followed by acid phenol-chloroform-isoamylalcohol extraction and precipitation in ethanol-sodium acetate [23]. Total RNA (20 µg/line) was electrophoresed on denaturing formaldehyde agarose gels followed by capillary transfer to a Zeta-Probe Blotting membrane (BioRad; Richmond, CA), and crosslinked with 1.2 Joules UV light (UV Stradalinker 1800, Stratagene; LaJolla, CA).

The oligonucleotide probe for murine M-CSF, IL-6, and beta-actin were synthetic 30-base cDNAs purchased from R&D Systems (Minneapolis, MN). Labeling with [alpha-32P]-dCTP (3000 Ci/mM; New England Nuclear; Boston, MA) at the 3' end and Northern hybridization [24] were done using a modification of a protocol designed in the laboratories of British Bio-Technology Ltd. (Oxford, UK) and provided by R&D Systems. Briefly, a mixture of 4 µl (40 ng) oligonucleotide, 5.3 µl distilled DEPC water, 4 µl of 5 x TdT tailing buffer (BRL Life Technologies; Grand Island, NY), 0.7 µl TdT (10.5 U; BRL), and 6 µl [alpha-32P]-dCTP (10 µCi/ml, specific activity 3000 Ci/mM) were reacted at 37°C in a water bath for 90 min. Subsequently, 5 µl EDTA (0.1 molar) were added to stop the reaction. The labeled probe was purified in a G-25 Sephadex column (Quick Spin Columns; Boehringer Mannheim; Indianapolis, IN).

The prehybridization for three h, and hybridization for 18-20 h at 54.5°C for M-CSF, at 53.3°C for IL-6, and at 60.0°C for beta-actin were performed in 1 M sodium phosphate buffer, pH 7.2, containing 1% sodium dodecyl sulfate (SDS), 5 x Denhardt's solution (Sigma), 1 mM EDTA, and 50 µg/ml sonicated salmon sperm DNA (Sigma). The concentration of radioactivity during hybridization ranged from one to two million cpm/ml (>3 x 108 cpm/µg specific activity). Subsequently, the filters were washed in prewarmed 6 x standard saline citrate (SSC) + 0.1% SDS twice at hybridization temperature for 15 min, rinsed in 2 x SSC + 0.1% SDS, exposed to Storage Phosphor Screen (Molecular Dynamics; Sunnyvale, CA), and scanned and analyzed using the PhosphorImager and MD ImageQuant Software, Version 3.3 (Molecular Dynamics).

Myelotoxic Agents
Ceftazidime (MF C22H22N6O7S2, FW 546.57) was purchased as preservative-free powder (Tazidime®; Eli Lilly; Indianapolis, IN), and prepared as described [15]. Methotrexate (MF C20H22N8O5, FW 454.4) was purchased as LPF® Sodium Parenteral Isotonic Liquid Preservative-Free (Lederle Parenterals Inc.; Carolina, Puerto Rico) and stored at 4°C. Each time, the desired concentrations of MTX were freshly prepared in {alpha}-MEM. In indicated experiments, cultures were treated with a single dose of either MTX or CEF, at the desired concentration, for one week.

Statistical Analysis
The results reported are expressed as mean ± standard error (±SE). Data sets were compared using the Student's independent t-test to identify significant differences; p values <0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CSA
Figure 1A shows CSA activity and Figure 1B corresponding supernatant cell counts at daily intervals following the addition of increasing concentrations of CEF to culture. Following medium change in control cultures, CSA increased from 36.5 ± 5.3 U/ml to a peak value of 290 ± 93.6 U/ml (p < 0.05) 12 h later. The differential colony count revealed that 70% to 100% of the colonies were pure macrophage colonies. The remaining colonies were composed of granulocytes/macrophages, while no pure granulocyte colonies were detected.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 1. Daily measurement of CSA (panel A) and total supernatant cell counts (panel B) in control cultures (solid square) and cultures treated with 250 µg/ml (triangle up) or 500 µg/ml CEF (diamond) between day 0 and day 14. CEF was added once on day 0 and removed from the culture on day 7. CSA corresponds to colony number assessed by CFU-GM (Materials and Methods). Mean values (± SE) of nine individual flaskettes at each time point are given. CEF was added to culture four weeks after culture initiation. *p < .05.

 
During the week in which CEF was added to the cultures, the peak concentration of CSA was delayed by 36 h followed by a sharp decrease on day 3, at which time the value was significantly lower than control (p < 0.05). A significant dose-dependent suppression of CSA production following medium change was noted. One day after the removal of CEF from the cultures, CSA averaged 157 ± 29 U/ml (62% of control) for 250 µg/ml CEF, 79 ± 19 U/ml (33% of control) for 500 µg/ml CEF, and 237 ± 32 U/ml for control cultures. Two weeks after CEF addition, CSA production following medium change was similar to control. Because we measured CSA using a bioassay, we excluded the presence of an inhibitor by experiments in which culture supernatant and lung conditioned medium were mixed ( Table 1).


View this table:
[in this window]
[in a new window]
  		Table 1. Mixing experiment to exclude myeloid growth inhibitors
 
Figure 2A and Figure 2B demonstrate the dose-dependent effects, respectively, of MTX addition on CSA production and inhibition of myeloid cell production in LTBMC. During the week of its addition to culture, no difference in CSA production after medium change was noted between MTX-treated and control cultures. Compared to control, one week after 10 µM MTX addition, peak CSA production following medium change was delayed significantly by 12 h. The CSA concentration in cultures treated with 0.1 µM MTX was significantly higher than controls 10 and 14 days after drug addition. No significant effect on CSA production was noted when 1 nM MTX was added to cultures. In general, MTX had no important effect on CSA concentrations in the supernatant of LTBMC.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. Daily measurement of CSA (panel A) and total supernatant cell counts (panel B) in control cultures (solid square) and cultures treated with 1 nM MTX (triangle up), 0.1 µM MTX (triangle down), and 10 µM MTX (diamond) between day 0 and day 14. MTX was removed from the culture on day 7. CSA corresponds to colony number assessed by CFU-GM (Materials and Methods). Mean values (± SE) of nine individual flaskettes at each time point are given. MTX was added to culture four weeks after culture initiation. *p < .05.

 
M-CSF mRNA Expression
The effects of medium change on M-CSF mRNA expression in LTBMC are shown in Figure 3. The mRNA expression of M-CSF was corrected for the percentage of stromal cell mRNA in the total extracted adherent layer mRNA. Compared to control cultures, the percent of myeloid cells in the adherent layer decreased in both CEF- and MTX-treated cultures. Thus, the proportion of stromal cell mRNA in control cultures constituted a significantly lower percentage of the total than in CEF- or MTX-treated cultures. To correct for this difference, the measured volume index for M-CSF mRNA hybridization by the PhosphorImager was normalized to 100% stromal cells. The percent of stromal cells in mobilized adherent layers, as described recently [15], averaged 36% for control cultures, 58% for CEF-treated cultures, and 89% for MTX-treated cultures one week after drug addition. After correction for stromal cell number, M-CSF hybridization was then expressed as a ratio to cytoskeletal beta-actin mRNA expression to normalize for differences in loading of total cellular RNA. Beta-actin mRNA intensity, expressed as the ratio of hybridization intensity to 18S band intensity, was constant prior to and serially following medium change in control LTBMC (range between 201 ± 49 and 222 ± 56) as well as in CEF- or MTX-treated cultures [25]. In control cultures, at the time of medium change (0 h), the ratios for the 2.3-kb M-CSF mRNA ranged from 0.269 to 1.722, and for the 4-kb M-CSF mRNA from 0.015 to 0.208. The lower hybridization intensity for the 4-kb mRNA compared with the stronger signal of the 2.3-kb transcript is shown in Figure 4. Because of marked variation in intensity of hybridization in the individual Northern blots, the ratios of the 2.3-kb and 4-kb M-CSF mRNA to beta-actin being measured were expressed as a percentage of the ratio obtained for the peak value of the corresponding 2.3-kb or 4-kb M-CSF transcripts in control cultures, which was arbitrarily set at 100%. Medium change resulted in a significant increase (p < 0.05) in expression of the 4-kb M-CSF transcript, which peaked six h after medium change, averaging 92.9 ± 4.3% compared with a value of 45.4 ± 18.3% at time 0. Expression returned toward the baseline by 24 h ( Fig. 3A). By contrast, the expression of the 2.3-kb mRNA band remained constant at all time points after medium change ( Fig. 3B). The addition of 1 µM MTX resulted in a significant suppression of both the 2.3-kb (p < 0.05) and 4-kb M-CSF (p < 0.05) transcripts. CEF addition caused significant suppression of the 2.3-kb mRNA. In contrast, the 4-kb mRNA in CEF-treated cultures did increase following medium change, but expression appeared to be delayed, peaking at nine h, rather than six h following medium change ( Fig. 3A).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. Kinetics of M-CSF mRNA expression, normalized to beta-actin mRNA expression and to 100% stromal cells (for explanation, see Results), after medium change of control cultures (solid circle) and cultures treated with 500 µg/ml CEF (triangle up) or 1 µM MTX (diamond) during the preceding week for the 4kb M-CSF mRNA (panel A) and 2.3kb M-CSF mRNA (panel B). Mean values (± SE) of four individual RNA extraction samples (pooled from three flasks) for control cultures and of two individual RNA extraction samples (pooled from three flasks) for CEF- and MTX-treated cultures are given. *p < .05

 


View larger version (44K):
[in this window]
[in a new window]
  		Figure 4. Kinetics of M-CSF mRNA expression in adherent stromal layer cells of hematopoietically active LTBMC. A representative Northern blot analysis of total cellular RNA of control and CEF- (panel A) and MTX-treated (panel B) cultures during 24 h following medium change is shown. The blots were hybridized sequentially with oligonucleotide cDNA probes for M-CSF (top line), and ß-actin (middle line). The bottom panel shows the ethidium bromide-stained Northern gel, with 28s and 18s rRNA representing the loading of RNA per line.

 
IL-6 Bioactivity
Like CSA, IL-6 bioactivity in the supernatant of LTBMC increased predictably following medium change, peaking at 12 h followed by a slow decrease, as recently reported [13]. The anti-IL-6 monoclonal antibody rat-anti-mouse (UBI) completely inhibited the proliferation of the 7TD1 cells induced by cell-free supernatant harvested from LTBMC or recombinant mouse IL-6 [13].

However, the concentration of IL-6 varied in different culture sets. Therefore, we normalized the IL-6 concentrations for these studies in which either MTX or CEF was added to cultures ( Fig. 5). Of each culture set, the IL-6 concentrations of untreated cultures at day 7 were arbitrarily used as 100%. At day 7, the absolute IL-6 concentrations in control cultures ranged between 38 ± 7 mU/ml and 476 ± 89 mU/ml per set. During the week of drug addition, there was no difference between IL-6 concentrations in treated cultures and control cultures (data not shown). IL-6 peaked 12 h after medium change, averaging between 179 ± 9 mU/ml and 3,234 ± 318 mU/ml per set. A significant suppression of IL-6 concentrations was seen after treating cultures with 500 µg/ml CEF ( Fig. 5A). No suppression of IL-6 production occurred when 250 µg/ml were added ( Fig. 5A). MTX treatment at concentrations between 1 µM to 10 µM did not significantly affect IL-6 bioactivity in the supernatant of LTBMC ( Fig. 5B).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. IL-6 concentration in the supernatant of control cultures (solid circle) and cultures treated with 250 µg/ml CEF (triangle up) and 500 µg/ml CEF (triangle down) (panel A), and cultures treated with 1 nM (solid circle), 0.1 µM (triangle down), and 10 µM (diamond) MTX (panel B). The supernatants for these IL-6 measurements were from the same cultures grown in glass flaskettes in which the CSA was measured and summarized in Figure 1 for CEF and Figure 2 for MTX. The absolute IL-6 concentrations are normalized to the day 7 control cultures as 100% of each of the three culture sets, averaging 476 ± 89 mU/ml, 241 ± 43 mU/ml, and 38 ± 7 mU/ml, respectively. *p < .05

 
IL-6 mRNA Expression
We then examined IL-6 mRNA expression in the adherent stromal cells prior to and following medium change. Expressed as a ratio to cytoskeletal beta-actin mRNA expression, Figure 6 shows a significant increase in IL-6 mRNA expression averaging 82.2 ± 8.0 six h following medium change compared with 0.3 ± 0.2 at time 0 (immediately prior to medium change). One week after the addition of 500 µg/ml CEF to LTBMC, IL-6 mRNA expression was significantly reduced, averaging 42.2 ± 9.0 (p = 0.019, compared with control value) six h after medium change. A representative Northern blot analysis of total cellular RNA of control and CEF-treated cultures is shown in Figure 7A. MTX at a concentration of 1 µM produced a significant inhibition of IL-6 mRNA, averaging 43.1 ± 14.6 (p = 0.034, compared with control value) six h after medium change ( Fig. 6). A representative Northern blot analysis of total cellular RNA of control and MTX-treated cultures is presented in Figure 7B. Myeloid cells in the supernatant did not express mRNA of IL-6 as assessed by Northern hybridization ( Fig. 7B).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 6. Kinetic of IL-6 mRNA expression normalized to ß-actin mRNA expression after medium change of control cultures (solid circle) and cultures treated with 500 µg/ml CEF (triangle up) or 1 µM MTX (diamond) during the preceding week. Mean values (± SE) of six individual RNA extraction samples (each pooled from three flasks) for control cultures and of three individual RNA extraction samples (each pooled from three flasks) for CEF- and MTX-treated cultures are given. *p < .05

 


View larger version (41K):
[in this window]
[in a new window]
  		Figure 7. Kinetics of mRNA expression in adherent stromal layer cells of hematopoietically active LTBMC. Representative Northern blot analysis of total cellular RNA of control and CEF-treated (panel A) and of control and MTX-treated (panel B) cultures during 24 h following medium change is shown. In panel B, the 11 left lines contain mRNA harvested from adherent stromal layer cells and the five right lines contain mRNA extracted from supernatant myeloid cells. The blots were hybridized sequentially with oligonucleotide cDNA probes for IL-6 (top line) and beta-actin (middle line). The bottom panel shows the ethidium bromide-stained Northern gel, with 28s and 18s rRNA representing the loading of RNA per line.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Both MTX and CEF are known to produce significant neutropenia and to suppress marrow myeloid cell production [26, 27]. MTX competitively inhibits the dihydrofolate reductase function that consequently decreases the cellular pool of tetrahydrofolate, a carrier of one-carbon groups, which serves as a cofactor in the de novo synthesis of purine nucleotides and thymidylate [28]. MTX is the most widely used antifolate in the treatment of a variety of cancers and is a second-line agent to treat severe psoriasis, psoriasis arthritis, and rheumatoid arthritis.

Penicillins and cephalosporins exert their inhibitory effect on the bacterial cell viability by specific covalent binding to high molecular weight penicillin-binding proteins in the outer side of the plasma membrane [29]. In eukaryotic cells, betalactam antibiotics selectively inhibit the replicative DNA polymerase {alpha} (=DNA polymerase II), {delta}, and {epsilon} whereas the reparative DNA polymerase ß (=DNA polymerase I) and DNA polymerase {gamma}, which is responsible for mitochondrial DNA replication, are not affected [30-32]. With the use of higher doses of betalactam antibiotics and longer applications, more direct, non-allergic toxicities have been observed clinically. Agranulocytosis has been reported in 5%-15% of patients treated with high-dose (>200 g) parenteral betalactam antibiotics for 20 days or longer [27]. In the literature, five cases with CEF-induced neutropenia have been reported [33, 34].

Like MTX-induced myeloid suppression, CEF-induced myeloid suppression is dose-dependent and predictable [15, 27]. When either of these drugs was added to LTBMC, a dose-dependent suppression of myelopoiesis occurred, followed by a recovery of myeloid cell production to control levels [14, 15]. CEF appears to cause a greater direct toxic effect on stromal cells than does MTX [16].

We confirmed previous results that medium change resulted in a highly significant increase in CSA production which peaked at 24 h [11, 14]. Four days after medium change, only minimal levels of CSA could be detected in LTBMC supernatant. The CFU-GM assay was employed to measure CSA in the supernatant of the LTBMC. The differential colony count revealed that 70% to 100% of the colonies were pure macrophage colonies. Therefore, we assumed that predominant CSA in the LTBMC supernatant was caused by M-CSF, and to a lesser extent, by GM-CSF activity. The presence of these cytokines in LTBMC has been shown in numerous previous reports [4, 35].

One week after CEF addition, a dose-dependent suppression of CSA concentration in the supernatant was observed. By contrast, the addition of MTX minimally affected supernatant CSA concentrations. The results obtained from mixing experiments indicated that reduced CSA response to medium change following CEF addition was not due to the presence of an inhibitor.

The multifunctional cytokine IL-6 is produced by bone marrow stromal cells and stimulates growth and differentiation of B and T lymphocytes; it is involved in the regulation of inflammatory responses and influences many bone and bone marrow functions [36-39]. In recent years, a great deal of attention has focused on the critical role played by IL-6 in marrow stromal and hematopoietic function, and in the pathophysiology of multiple myeloma [40, 41] and osteoporosis [42]. IL-6 is a survival factor for human CFU-GM [43], and anti-IL-6 antibodies suppressed the generation of CFU-GM and myeloid cell production in murine long-term bone marrow cultures [44]. In our LTBMC grown on glass flaskettes, the IL-6 concentrations at the time of medium change (= 0 hour) were about twice as high as the values reported by Hudak et al. [44]. They employed an ELISA to measure IL-6 in the supernatant of LTBMC established from CBA/J mice using 10% horse serum and 10% FBS. We determined the specificity of our bioassay by demonstrating a dose-dependent inhibition of supernatant IL-6 bioactivity by the simultaneous addition to the assay of an anti-IL-6 monoclonal antibody rat-anti-mouse [13]. Following medium change, we found a highly significant eightfold increase in IL-6 bioactivity in hematopoietically active LTBMC. As with CSA, IL-6 production appeared to be suppressed by CEF but not by MTX. The high concentrations of IL-6 in LTBMC and the earlier reported observation that CEF damaged the stromal histology [16] and suppressed IL-6 but not MTX bioactivity suggest that IL-6 contributes a regulatory role in the stromal integrity and hematopoietic activity of the marrow stroma [13]. To our knowledge, the kinetics of M-CSF and IL-6 mRNA expression in a hematopoietically active murine LTBMC in response to medium change and myelotoxic agents have not been reported. We demonstrated the presence of the 2.3-kb and 4.0-kb M-CSF and of the 1.6-kb IL-6 transcript in murine LTBMC. Following medium change, the 2.3-kb mRNA remained constant over a 24-h period. The 4.0-kb mRNA expression increased significantly, peaking six h after medium change. The cycling nature of the 4.0-kb mRNA following medium change suggests that it may be the main regulator of medium-change-induced increases in M-CSF production. The purified murine M-CSF is a 40- to 90-Kd homodimeric glycoprotein synthesized primarily by fibroblasts [45], bone marrow stromal cells, endothelial cells, monocytes and macrophages, uterine epithelium, and others [46, 47]. The cDNA clones for the 4-kb and 2.3-kb mRNA transcripts contain essentially the same coding sequence and result in one protein [48]. It is assumed that in the murine genomic sequence, as in the human counterpart, the two 3' untranslated sequences reflect an alternative splicing choice of exons [46]. The 2-kb 3’ untranslated sequence found in the 4-kb murine clone contains three AU sequences within 58 bp [48]. The 2.3-kb clone does not contain the AU sequence. This motif may at least partly regulate the mRNA degradation and lead to a faster turnover of the 4-kb transcript. Our results support the idea of a differential turnover of the two transcripts.

MTX treatment resulted in significant inhibition of expression of both the 2.3-kb and 4.0-kb M-CSF mRNA and IL-6 mRNA expression following medium change. Despite reduction in message, MTX had minimal if any effects on supernatant CSA concentrations and IL-6 bioactivity at various time points following medium change. It is possible that MTX-induced suppression of mRNA expression is not sufficient to significantly inhibit posttranslational synthesis of CSA or IL-6 bioactivity. If, however, CSA or IL-6 production is compromised by MTX treatment, the only explanation for unaffected supernatant CSA concentrations or IL-6 bioactivity is its reduced utilization. This may occur as a consequence of reduced numbers of myeloid elements or altered stromal cells leading to lower CSA or IL-6 requirements. The phenomenon of growth factor utilization in hematopoietically active cultures compared with irradiated cultures has been demonstrated for GM-CSA and CSA [9, 11].

CEF (500 mg/ml) treatment caused a delay in expression of the 4.0-kb M-CSF transcript, but the peak level was not affected. The expression of IL-6 mRNA was significantly reduced, approximately 50%. These data suggest that impaired CSA or IL-6 production following CEF treatment must reflect impaired protein synthesis rather than suppression at the transcription level. This finding is in line with observations that penicillin significantly suppressed protein synthesis in primary rat hepatocyte cultures in a dose-dependent manner [49, 50]. Penicillin at a concentration of 1.4 mM (about 500 mg/ml) inhibited the synthesis of both cellular and medium proteins by 50% [50].

Based on our results, the following conclusions are warranted. CSA and IL-6 concentration in the supernatant of LTBMC, as well as the expression of the 4-kb M-CSF and IL-6 mRNA in the adherent stromal layer cells, increase in concentration following medium change. mRNA expression peaks six h after medium change, cytokine concentrations peak after 12 h and significant increases in myeloid cell numbers in the supernatant are seen after 48 to 72 h. This time frame indicates that in response to medium change, increased mRNA expression leads to increased synthesis of cytokines, which in turn stimulates myeloid cell proliferation. Agents which cause myeloid suppression have different effects on cytokine production. CEF causes more acute damage to stromal histology [16] and a greater suppression of CSA and IL-6 bioactivity production by stromal cells than does MTX. In the earlier report, we showed that the stromal layer of untreated cultures consisted of about 49% macrophages, 22% fibroblastoid cells, 25% fat-containing cells, and 4% endothelial cells. Cultures treated with methotrexate showed a transient absolute increase of macrophages and fat-containing cells, whereas CEF-treated cultures had a transient decrease of fat-containing and endothelial cells [16]. MTX-induced suppression of myelopoiesis occurs primarily as a consequence of direct interference in cell division, whereas CEF effects may well be toxic to both rapidly dividing hematopoietic elements and to resting stromal cells. This would explain the early alterations in stromal cell morphology and function in CEF-treated cultures. A clearer understanding of these effects should allow more rational approaches to minimizing the adverse effects of therapy with these agents. It is possible that the administration of myeloid growth factors will show greater responsiveness and be more rational in the treatment of neutropenia induced by drugs that affect stromal function and suppress cytokine production.


   Acknowledgments
 
The authors would like to thank Virginia Fitzhugh for her excellent technical assistance.

This work was supported by Grants AG 07473 and AG 09458 from the National Institutes of Health, by the Arkansas EPSCoR program funded by the National Science Foundation, Arkansas Science and Technology Authority and the University of Arkansas for Medical Sciences and by funds from the Department of Veterans Affairs. S.P.H. was partly supported by grants from the Swiss Cancer League and the Bernese Cancer League, Switzerland.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dexter TM. Stromal cell associated haemopoiesis. J Cell Physiol (suppl) 1982;1:87-94.

  2. Allen TD, Dexter TM, Simmons PJ. Marrow biology and stem cells. In: Dexter TM, Garland JM, Testa NG, eds. Colony Stimulating Factors. Molecular and Cellular Biology. Immunology Series Vol. 49. New York: Marcel Dekker, 1990:1-38.

  3. Dorshkind K. Regulation of hematopoiesis by bone marrow stromal cells and their products. Annu Rev Immunol 1990;8:111-137.[Medline]

  4. Kittler ELW, McGrath H, Temeles D et al. Biologic significance of constitutive and subliminal growth factor production by bone marrow stroma. Blood 1992;79:3168-3178.[Abstract/Free Full Text]

  5. Sutherland HJ, Hogge DE, Eaves CJ. Growth factor regulation of the maintenance and differentiation of human long-term culture-initiating cells (LTC-IC). Leukemia 1993;7(suppl 2):S122-S125.

  6. Shadduck RK, Waheed A, Greenberger JS et al. Production of colony stimulating factor in long-term bone marrow cultures. J Cell Physiol 1983;114:88-92.[Medline]

  7. Eliason JF, Thorens B, Kindler V et al. The roles of granulocyte-macrophage colony-stimulating factor and interleukin 3 in stromal cell-mediated hemopoiesis in vivo. Exp Hematol 1988;16:307-312.[Medline]

  8. Lipschitz DA, Udupa KB, Taylor JM et al. Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures. Blood 1987;69:1211-1217.[Abstract/Free Full Text]

  9. Gualtieri RJ, Shudduck RK, Baker DG et al. Hematopoietic regulatory factors produced in long-term murine bone marrow cultures and the effect of in vitro irradiation. Blood 1984;64:516-525.[Abstract/Free Full Text]

  10. McGrath HE, Wade PM, Kister VK et al. Lithium stimulation of HPP-CFC and stromal growth factor production in murine Dexter culture. J Cell Physiol 1992;151:276-286.[Medline]

  11. Udupa KB, Lipschitz DA. An evaluation of the role of microenvironmental factors in the limitation of myelopoiesis in murine long-term bone marrow culture. Exp Hematol 1988;16:112-116.[Medline]

  12. Eaves CJ, Cashman JD, Kay RJ et al. Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within adherent layer. Blood 1991;78:110-117.[Abstract/Free Full Text]

  13. Hauser SP, Kajkenova O, Lipschitz DA. The pivotal role of interleukin-6 in hematopoietically active murine long-term bone marrow cultures. STEM CELLS 1997;15:125-132.[Abstract/Free Full Text]

  14. Williams LH, Udupa KB, Lipschitz DA. Long-term bone marrow culture as a model for host toxicity: the effect of methotrexate on hematopoiesis and adherent layer function. Exp Hematol 1988;16:80-87.[Medline]

  15. Hauser SP, Udupa KB, Lipschitz DA. Effects of ceftazidime, a betalactam antibiotic, on murine haemopoiesis in vitro. Br J Haematol 1994;86:733-739.[Medline]

  16. Hauser SP, Udupa KB, Lipschitz DA. Murine marrow stromal response to myelotoxic agents in vitro. Br J Haematol 1996;95:596-604.[Medline]

  17. Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of hematopoietic stem cells in vitro. J Cell Physiol 1977;91:335-344.[Medline]

  18. Hauser SP, Waldron JA, Udupa KB et al. Morphological characterization of stromal cell types in hematopoietically active murine long-term bone marrow cultures. J Histochem Cytochem 1995;43:371-379.[Abstract]

  19. Worton RG, McCulloch EA, Till JE. Physical separation of hemopoietic stem cells from cells forming colonies in cultures. J Cell Physiol 1969;74:171-182.[Medline]

  20. Sheridan JW, Metcalf D. A low molecular weight factor in lung-conditioned medium stimulating granulocyte and monocyte colony formation in vitro. J Cell Physiol 1973;81:11-23.[Medline]

  21. Van Snick J, Cayphas S, Vnik A et al. Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci USA 1986;83:9679-9683.[Abstract/Free Full Text]

  22. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Meth 1986;89:271-277.[Medline]

  23. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159.[Medline]

  24. Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci USA 1984;81:1991-1995.[Abstract/Free Full Text]

  25. Hauser SP, Udupa KB, Lipschitz DA. Kinetics of interleukin-6 mRNA expression in murine long-term bone marrow cultures and effects of myelotoxic drugs. Blood 1993;82 (suppl 1):499a.

  26. Chu E, Takimoto CH. Antimetabolites. Antifolates. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: Principles and Practice of Oncology. 4th ed. Philadelphia, PA: JB Lippincott, 1994:358-374.

  27. Neftel KA, Hauser SP, Müller MR. Inhibition of granulopoiesis in vivo and in vitro by beta-lactam antibiotics. J Infect Dis 1985;152:90-98.[Medline]

  28. Sweeny DJ, Diasio RB. Toxicity of antimetabolites. In: Powis G, Hacker MG, eds. The Toxicity of Anticancer Drugs. New York: Pergamon Press, 1990:63-81.

  29. Waxman DJ, Strominger JL. ß-Lactam antibiotics: Biochemical modes of action. In: Morin RB, Gorman M, eds. The Chemistry and Biology of ß-Lactam Antibiotics. New York: Academic Press, 1982:209-285.

  30. Huynh UD, Neftel KA, Spadari S et al. Betalactam antibiotics interfere with eukaryotic DNA-replication by inhibiting DNA-polymerase alpha. Nucleic Acids Res 1987;15:10495-10506.[Abstract/Free Full Text]

  31. Neftel KA, Hübscher U. Effects of ß-lactam antibiotics on proliferating eukaryotic cells. Minireview. Antimicrob Agent Chemother 1987;31:1657-1661.[Free Full Text]

  32. Neftel KA, Hafkemeyer P, Cottagnoud P et al. Did evolutionary forerunners of betalactam-antibiotics bind to nucleic acid replication enzymes? In: Kleinkauf H, Döhren H, eds. 50 Years of Penicillin Application, History and Trends. Symposium, TU Berlin. Prag: Public Ltd, 1993:394-403.

  33. Eron LJ, Park CH, Hixon DL et al. Ceftazidime in patients with pseudomonas infections. J Antimicrob Chemother 1983;12(suppl A):161-169.

  34. Rouveix B, Lassoued K, Vittecoq D et al. Neutropenia due to ß lactamine antibodies. Br Med J 1983;287:1832-1834.

  35. Abboud SL, Pinzani M. Peptide growth factors stimulate macrophage colony-stimulating factor in murine stromal cells. Blood 1991;78:103-109.[Abstract/Free Full Text]

  36. Van Snick J, Cayphas S, Szikora J-P et al. cDNA cloning of murine interleukin-HP1: homology with human interleukin 6. Eur J Immunol 1988;18:193-197.[Medline]

  37. Chiu C-P, Moulds C, Coffman RL et al. Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells. Proc Natl Acad Sci USA 1988;85:7099-7103.[Abstract/Free Full Text]

  38. Suda T, Yamaguchi Y, Suda J et al. Effect of interleukin 6 (IL-6) on the differentiation and proliferation of murine and human hemopoietic progenitors. Exp Hematol 1988;16:891-895.[Medline]

  39. Kishimoto T. The biology of interleukin-6. Blood 1989;74:1-10.[Free Full Text]

  40. Caligaris-Cappio F, Bergui L, Gregoretti MG et al. Role of bone marrow stromal cells in the growth of human multiple myeloma. Blood 1991;77:2688-2693.[Abstract/Free Full Text]

  41. Kinashi T, Springer TA. Adhesion molecules in hematopoietic cells. Blood Cells 1994;20:25-44.[Medline]

  42. Manolagas SC, Jilka RL. Bone marrow, cytokines, and bone remodeling. Emerging insight into the pathophysiology of osteoporosis. N Engl J Med 1995;332:305-311.[Free Full Text]

  43. Kerst JM, Slaper-Cortenbach ICM, Van der Schoot CE et al. Interleukin-6 is a survival factor for committed myeloid progenitor cells. Exp Hematol 1993;21:1550-1557.[Medline]

  44. Hudak S, Thompson-Snipes L, Rocco C et al. Anti-IL-6 antibodies suppress myeloid cell production and the generation of CFU-C in long-term bone marrow cultures. Exp Hematol 1992;20:412-417.[Medline]

  45. Das SK, Stanley ER. Structure-function studies of a colony-stimulating factor (CSF-1). J Biol Chem 1982;257:13679-13684.[Abstract/Free Full Text]

  46. Kawasaki ES, Ladner MB. Molecular biology of macrophage colony-stimulating factor. In: Dexter TM, Garland JM, Testa NG, eds. Colony-Stimulating Factors. Molecular and Cellular Biology. New York, Basel: Marcel Dekker, 1990:155-176.

  47. Roth P, Stanley ER. The biology of CSF-1 and its receptor. Curr Top Microbiol Immunol 1992;181:141-167.[Medline]

  48. Ladner MB, Martin GA, Noble JA et al. cDNA cloning and expression of murine macrophage colony-stimulating factor from L929 cells. Proc Natl Acad Sci USA 1988;85:6706-6710.[Abstract/Free Full Text]

  49. Schwarze PE, Seglen PO. Effects of antibiotics on protein synthesis and degradation in primary cultures of rat hepatocytes. In Vitro (Rockville) 1981;17:71-76.[Medline]

  50. Vonen B, Morland J. Penicillin toxicity in isolated rat hepatocytes revealed by decreased incorporation of valine into proteins. Acta Pharmacol Toxicol 1982;51:81-86.[Medline]

accepted for publication April 28, 1998.



This article has been cited by other articles:


Home page
 The Annals of PharmacotherapyHome page
C. B Whitman, J. M Joseph, and L. O Sjoholm
Cephalosporin-Induced Leukopenia Following Rechallenge with Cefoxitin
Ann. Pharmacother., September 1, 2008; 42(9): 1327 - 1332.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hauser, S. P.
Right arrow Articles by Lipschitz, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hauser, S. P.
Right arrow Articles by Lipschitz, D. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS