Stem Cells
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 Wang, X.
Right arrow Articles by Benestad, H. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by Benestad, H. B.
Stem Cells, Vol. 17, No. 5, 253-264, September 1999
© 1999 AlphaMed Press

Maturation Rate of Mouse Neutrophilic Granulocytes: Acceleration by Retardation of Proliferation, but No Detectable Influence from G-CSF or Stromal Cells

Xiuli Wanga, Hege Fjerdingstadb, Inger Strøm-Gundersena, Haakon B. Benestada

a Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway;
b Nycomed Drug Research, Oslo, Norway

Key Words. Hematopoietic cells • Bone marrow stromal cells • Proliferation • Maturation • G-CSF

Dr. Haakon B. Benestad, Department of Physiology, IMBA, UiO, P.O. Box 1103 Blindern, N-0317 Oslo, Norway.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our purpose was to examine the possible influence of stromal and humoral mediators on granulocytic maturation rates. Sorted immature murine progenitor (Lin–Sca-1+) cells were cultured in peritoneal diffusion chambers (DCs) with or without a confluent layer of irradiated bone marrow stromal cells on one of the micropore membrane walls. In other experiments, 10 µg/kg/d recombinant G-CSF (rhG-CSF) was administered continuously into DC host mice through s.c. implanted osmotic minipumps. Operationally, maturation rate was assessed as the ratio between the number of polymorphonuclear cells (PMN) and proliferative granulocytes (PG) in short-term cultures, based on the differential cell counts, and supported by flow cytometric measurement of a granulocytic differentiation marker; and by the emergence time of PMN in the DCs, obtained by extrapolation. Also, increased maturation is associated with increased cell density, as reflected by the positioning of the granulocytes during centrifugation in a discontinuous Percoll gradient. This method, as well as the conversion rate of 3H-thymidine labeled PG into the heavier non-PG maturational stages, were also used as indicators of maturation rate. After five, six, and seven days of culture in the peritoneal cavity, DC cells were harvested. Their proliferative status, based on measurement of incorporated bromodeoxyuridine, was determined, and their maturation rates were evaluated. Proliferation of immature granulocytic progenitor cells was apparently inhibited by direct contact with bone marrow stromal cells, and stimulated by G-CSF during the early stage of culturing. However, the subsequent maturation rate, which could be accelerated by increasing culture cellularity, thus decreasing PG proliferation rate, was not detectably influenced by either stromal cells or G-CSF.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The control of hematopoietic cell production is achieved by balancing the rates of differentiation (i.e., lineage commitment), proliferation, maturation (i.e., terminal differentiation), and death of parenchymal bone marrow cells. Regulation occurs at the level of the structured microenvironment (stroma), via cell-cell interactions and specific cytokines.

As a representative of late-acting, lineage-specific cytokines, G-CSF can stimulate precursor cell proliferation [1, 2] and allegedly increase the maturation process of these cells [3], thereby leading to a rapid and amplified neutrophil release to blood, when needed to kill invading microbes. Moreover, G-CSF also promotes cell survival by suppressing programed cell death (apoptosis) [4] and works as an activator of mature neutrophil function [5].

Granulocytopoiesis occurs in bone marrow in close association with stromal cells, and it has been shown that these cells not only inhibit the proliferation of primitive progenitor cells [6, 7] but also facilitate differentiation and protect the progenitors from programed cell death through, for example, integrin-mediated adhesion between hematopoietic and stromal cells [8-10]. A prevention of the maturation (terminal differentiation) of granulocytes mediated by CD34 mucin, an adhesion molecule expressed on progenitors, has also been hypothesized [11, 12].

Our present purpose was to examine possible maturational effects in a new manner; by confining granulocytopoiesis to a culture chamber in vivo, where the complicating loss of more or less mature granulocytes to the circulation (i.e., mobilization) is avoided.

Consequently, the proliferative kinetics and maturation process of murine, multipotent hematopoietic (Lin–Sca-1+) progenitor cells were investigated under stroma-contact and stroma-noncontact situations with our established diffusion chamber (DC) in vivo technique [13-15]. G-CSF was delivered by s.c. implanted osmotic minipumps. A bromodeoxyuridine (BrdUrd) incorporation method [16-18] was used to investigate the proliferative behavior of the cultured Lin–Sca-1+ cells. The relative maturation rate of granulocytes was operationally defined and determined in several ways, based on differential cell counting, Percoll density gradient centrifugation, and flux of 3H-thymidine (3H-TdR) labeled cells into the nonproliferative compartment. The findings did not support the notion that G-CSF or stroma regulates the maturation rate of granulocytes in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Dynabeads M-450 coated with sheep anti-rat IgG (Dynal; Oslo, Norway); rat gamma globulin (Pierce; Rockford, IL); fluorescein isothiocyanate (FITC)-labeled rat IgG2a kappa (Pharmingen; San Diego, CA); FITC anti-mouse Ly-6A/E (Pharmingen); Lin monoclonal cocktail: Gr-1mAb, L3T4mAb, Mac-1mAb, CD2mAb, CD45R/B220mAb, TER-119/erythroid cells (Pharmingen); antibody against BrdUrd incorporated into DNA (anti-BrdUrd, Becton Dickinson; San Jose, CA); goat anti-mouse Ig FITC (GAM-FITC, Becton Dickinson); FITC-conjugated rat anti-mouse Ly-6G (Gr-1) monoclonal antibody ([mAb], Pharmingen); BrdUrd (Boehringer Mannheim GmbH; Mannheim, Germany); propidium iodide (Calbiochem; La Jolla, CA); Pronase (Boehringer Mannheim GmbH); Tween 20 (Sigma; St. Louis, MO); pepsin (Sigma); Percoll (Pharmacia; Uppsala, Sweden); 3H-TdR, (Amersham; Buckinghamshire, UK); recombinant human G-CSF ([rhG-CSF], Chugai Pharmaceutical Co. Ltd.; Tokyo, Japan; kind gift from Dr. K.J. Mori, Faculty of Science, Niigata University, Japan); horse serum, Bio-Whittaker; Walkersville, MD); fetal bovine serum ([FBS], Hyclone Laboratories Inc.; Logan, UT); fetal calf serum ([FCS], GIBCO BRL; Paisley, Scotland); bovine serum albumin ([BSA], Sigma); minimal essential medium (MEM), Alpha Medium (GIBCO BRL); Fisher's medium (Life Technologies; Paisley, Scotland); RPMI 1640 (Bio-Whittaker); HEPES (Sigma); phosphate buffered saline ([PBS], GIBCO BRL); ampicillin (DoctacillinTM, STR; Södertälje, Sweden); sodium azide (Sigma); hydrocortisone (Sigma); hydrochloric acid ([HCl], May & Baker Ltd.; Dagenham, UK).

Mice
Adult inbred C57BL/6J/OlaHsd females, 5 to 10 weeks old, were used as donors of Lin–Sca-1+ bone marrow cells and syngeneic stromal cells. NMRI (B & K Universal AS; Sollentuna, Sweden) or ICR/OlaHsd females, five to nine weeks old, of randomly bred strains, were employed as hosts of diffusion chambers. In some experiments, NMRI mice were used as donors of bone marrow parenchymal cells and allogeneic bone marrow stromal cells. The experimental protocol was approved by the national animal experimentation committee.

Establishment of Stromal Layers in DC Cultures In Vitro
First, 30 x 106 cells /ml from femora of C57BL/6J mice were inoculated into micropore DCs (0.25 ml/DC), in the "Fisher-HEPES-antibiotics-horse serum" (FHabHS) medium [19], containing 1 µmol/l hydrocortisone as well. The DCs were incubated for 2 h to allow adherence of cells to one of the two micropore membrane walls of the DC. Then 6-ml medium was pipetted into each 35-mm diameter culture dish, which supported two DCs. At the same time, 0.25 ml medium without bone marrow cells was inoculated into control DCs. All the DCs were cultured under the same conditions (37°C with 5% carbon dioxide in air and saturated water vapor) for two weeks, and the medium in the dishes was replaced after one week.

These established stromal DCs and stroma-free DCs were irradiated with 20 Gy from a Stabilipan-x-ray machine (Siemens; Erlangen, Germany) at a dose rate of 1 Gy per minute (220 kV, 20 mA, added filtration 0.5 mm Cu) before inoculation of the parenchymal hematopoietic cells. In some experiments, host mice were exposed to 5 Gy x-ray irradiation before use.

Separation of Lin–Sca-1+ Cells
Lin–Sca-1+ cells were isolated from crushed femora, tibiae, and humeri of C57BL/6J mice according to the methods described [20]. Briefly, bone marrow cells (4 x 108/ml) were incubated with an antibody cocktail, specifically reactive with the various differentiated bone marrow cell lineages (Lin) (Gr-1; L3T4; Mac-1; CD2; CD45R/B220; TER-119/erythroid cells). Then labeled cells (Lin+) were removed by magnetic beads and Lin– cells obtained from the supernatant. Lin– cells were treated with FITC-conjugated antimouse Ly6A/E antibody for 30 min. Finally, the positive "stem cell antigen" (Sca) cells were sorted with a Coulter Epics Elite Cell Sorter (Coulter Electronics; Hialeah, FL).

Culture In Vivo
Fresh Lin–Sca-1+ cells were plated at 1,500 to 2,500/DC onto either the established and irradiated, once-washed syngeneic bone marrow stromal layers, or into the stromal cell-free DCs. The DCs were then implanted i.p.. After five-, six-, and seven-day in vivo cultures, the DCs were removed from host mice and shaken in FHabHS medium containing 0.35% Pronase at 37°C for 60 min. After washing out each DC three times, the cell suspension pooled from one group of four DCs was weighed, its particle concentration was counted electronically, and cell samples were retrieved for cytospin preparation and differential cell counting. The numbers of proliferative granulocytes (PG), nonproliferative granulocytes (NPG), polymorphonuclear cells (PMN), macrophage-like cells (MA), and lymphocytes (LY) were recorded according to morphological criteria established previously [21].

In density separation experiments, normal bone marrow cells (300,000/DC) were inoculated on established and irradiated (20 Gy x-rays) bone marrow stroma in DCs or in standard DCs without stromal cells. The DCs were implanted into hosts and cultured for five days. Each host mouse was injected i.p. with 10 µCi (37 kBq) 3H-TdR in 2 ml 0.9% NaCl 24 h before the removal of DCs. Three and 18 h after injection of 3H-TdR, 100 µg/kg rhG-CSF in 0.9% NaCl with 0.1% FCS was injected i.p. into the test host mice. The controls received injections of 0.9% NaCl with 0.1% FCS. The harvested DC cells (pooled from 10 DCs in each group) were positioned into four different layers during centrifugation in a discontinuous Percoll gradient (see below). Cells from each layer were enumerated (see above), and their incorporated radioactivity measured in a ß counter.

Analysis of Proliferative Status
To determine the proliferative status of the harvested cells, 0.35% Pronase containing FHabHS medium with BrdUrd was used to harvest the DC cells. To ensure optimum BrdUrd labeling of DNA, 10–4 mol/l was adopted as the highest nontoxic dose, after dose titration experiments. The BrdUrd treated cells were resuspended in 1 ml PBS, fixed in 9 ml absolute ethanol, and stored at –20°C until flow cytometric analysis could be performed. These fixed cells were processed according to the method described [17, 18], but slightly modified as follows: the samples were washed thoroughly with cold PBS, and 2 ml fresh pepsin-HCl solution (0.2% pepsin in 2N HCl) was added to each cell button. After suspension, these fixed cells were incubated for 60 min in the dark at room temperature. After washing three times, a mixture of 30 µl 0.5% Tween 20 with 0.5% BSA solution in PBS and 20 µl anti-BrdUrd mAb was added to each tube. After a 45-min incubation at room temperature and one washing, 100 µl 1:40 GAM-FITC were pipetted into the tube. This was followed by an incubation of 30 min, one washing, and the addition of 1 ml propidium iodide (2.5 µg/ml) to each tube. The samples were ready 50 min later to be analyzed with flow cytometry (FACScan, Becton Dickinson).

Analysis of Gr-1 Antigen Expression on Granulocytes
DC cells were collected after treatment with 0.35% Pronase for 60 min. Cells were labeled by Gr-1 antibody as described [22], but slightly modified. In short, 0.1 µg fluorescein-conjugated rat antimouse Gr-1/myeloid differentiation antibody was added to a 50 µl cell suspension and incubated for 45 min on ice. After washing twice with PBS containing 1% FCS and 0.1% sodium azide, the cells were resuspended in 0.5 ml PBS for fluorescence-activated cell scanning (FACS) analysis.

In Vivo Delivery of rhG-CSF
Osmotic minipumps (Model 2002, Alzet, Alza; Palo Alto, CA), loaded with rhG-CSF in Fisher's medium with HEPES (10 mmol/l, pH ~ (7.35), 0.1% BSA and ampicillin (200 µg/ml), were implanted s.c. on the back of NMRI or ICR host mice, so that 10 µg/kg rhG-CSF were delivered every day for the whole culture period of seven days. This dose has been proved effective in humans [23] and in mice [2] for stimulating myeloid proliferation in bone marrow and inducing neutrophilia in blood. Each experiment employed two groups of host mice, one with minipumps loaded with rhG-CSF, the other with minipumps delivering solvent solution only, or just control mice without minipumps implanted. Comparison between these two controls did not indicate any systematic differences concerning the relevant variables. The DCs, containing Lin–Sca-1+ cells with a stromal layer, were implanted into the peritoneal cavity, two chambers per host mouse. The DC cells were harvested and analyzed as described above.

Assessment of Maturation Rate of Granulocyte Precursors by Density Gradient Separation
A modification of the method described by Rolstad and Benestad [24] was applied to the separation of DC cells according to cell densities. Percoll was adjusted to 285 mOsM/l with 10 x PBS, and RPMI 1640 + 10% FCS to 290 mOsM/l with sterile water. Osmolarity-corrected Percoll and medium were then mixed in different proportions to create four different densities: F1 containing 37.8% v/v Percoll (corresponding to a density relative to water of 1.053); F2: 47.9% (1.065); F3: 56.3% (1.075), and F4: 62.2% (1.082). Three milliliters of each fraction were layered carefully on top of each other as depicted in Figure 1Go, with 1 ml of osmolarity-corrected medium containing 10 – 15 x 106 cells retrieved from DCs at the top, in 15-ml Falcon centrifuge tubes, and spun at 400 g for 30 min at room temperature. The DC cells in each layer were washed out of the Percoll before being used, and are hereafter termed DC cell fraction 1-4 (F1-F4), so that, for example, F2 cells are the cells just above the second density layer.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 1. Sketch of Percoll density separated cells, granulocyte density increasing with increasing maturation. The majority (>80%) of F1 cells are macrophage-like (MA) cells. Among the F4 cells, nonproliferative granulocytes (NPG) predominate. More granulocytes altogether appear in F3 than F2. See text and Figure 2Go for more details. PMN: polymorphonuclear cells; PG: proliferative granulocytes.

 
Statistics
A two-sided, nonparametric Wilcoxon pair test was used in comparisons between the test and control DC cultures. Confidence intervals of 95% for the median values were calculated with a nonparametric method (MINITAB computer program, Minitab; State College, PA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Accelerated Granulocyte Maturation by Retardation of Proliferation Rate
After the density separation of the chamber (DC) cells, the majority of F1 (low density) cells (more than 80%) were morphologically MA (modal diameter in isotonic solution 11 µm), while the majority of F4 cells (about 80%) were NPG (modal diameter 7.3 µm). F3 contained the highest number of cells and radioactivity (from 3H-TdR injected the day before). A representative differential count is 37% PG, 58% NPG, 4% MA, and 1% LY (modal diameter 9 µm). With increased granulocyte maturity the cells were distributed to the higher-density fractions (Figs. 1 and 2GoGo), as found earlier for rat bone marrow cells [24]. DCs containing 300,000 bone marrow cells ("high cellularity DCs") or 30,000 bone marrow cells ("low cellularity DCs") were implanted i.p. into normal or 5 Gy irradiated host mice and harvested after five days. The cells were separated into the four density fractions (F1-F4). A larger portion of the 3H-TdR label and of the granulocytes appeared in F3 than in lighter fractions, when high-cellularity cultures were compared with low-cellularity cultures (Fig. 3Go). The relative expansion of the cell inocula (medians 30-, 25-, and 8-fold for 3 x 104 cells in 5 Gy irradiated hosts, 3 x 104 cells in normal hosts, and 30 x 104 cells in normal hosts, respectively) (Fig. 3Go) had been largest in the low-cellularity DCs. Accordingly, the incorporation of 3H-TdR (2.8, 1.4, and 3.9 x 104 cpm/DC, respectively) had also been most intense, on a per cell basis, in the low-cellularity cultures. Consequently, proliferation had been most accelerated and maturation—judged by the density distribution of both granulocytes and 3H activity—most retarded when the DCs had been carried by irradiated mice (Fig. 3Go).



View larger version (11K):
[in this window]
[in a new window]
  		Figure 2. More mature cells move to higher densities during Percoll gradient centrifugation. 300,000 bone marrow cells were cultured in in vivo diffusion chambers (DCs) with or without irradiated stromal cells (20 Gy x-rays) for five days. Each host mouse was injected i.p. with 10 µCi 3H-thymidine in 2 ml 0.9% NaCl 24 h before the removal of DCs. Harvested DC cells (pooled from 10 DCs in each group) were separated by density-gradient centrifugation. The distribution of granulocyte maturation stages and 3H activity to the four density fractions is shown. Two replicate and independent experiments gave similar results. NPG-PMN are metamyelocytes and band-nucleated granulocytes.

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. Maturation rate of bone marrow cells in in vivo DCs with high- or low-culture cellularity. Relative population expansion: 3 x 104 cells in 5 Gy irradiated hosts > 3 x 104 cells in normal hosts > 30 x 104 cells in normal hosts. Concerning the maturation rate, assessed as the distribution of granulocytes or 3H activity to density fractions F2 (with the less mature granulocytes) and F3 (with the more mature granulocytes), it increased with decreasing expansion of the cultured cell populations. A replicate experiment gave similar results. See Figures 1 and 2GoGo for further explanations.

 
Proliferation and Maturation of Lin–Sca-1+ Cells on Bone Marrow Stromal Cells
A good correlation was found between Gr-1+ cells measured with flow cytometry and concentration of granulocytes determined with differential counts, and between fluorescence intensity of Gr-1 expression and concentration of PMN (Fig. 4Go).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Correlation between Gr-1 antigen expression measured with flow cytometry and granulocyte concentration (PG + NPG in upper panel, PMN in lower) determined with differential counting. The cell population retrieved after in vivo culture of Lin-Sca-1+ cells with or without stroma was analyzed with both methods, the flow cytometry utilizing an FITC-labeled Gr-1 antibody. Regression lines, regression coefficients (r), and the probabilities that r != 0 are given.

 
Our DC harvesting procedure allowed virtually complete retrieval of cultured parenchymal cells from the DCs, leaving a substantial portion of the stromal cells adherent to the micropore membranes.

The proliferative state of the irradiated (20 Gy x-ray) bone marrow stromal cells was also checked. DCs with stromal cells only, but without inoculated stem cells, were harvested and analyzed. Only about 1.5% (mean value from three experiments) of bone marrow stromal cells were apparently in S-phase of the cell cycle after five to seven days' in vivo culture.

To evaluate the proliferative kinetics of the cultured Lin–Sca-1+ cells, the number of cells that incorporated BrdUrd in stroma-contact and stroma-noncontact DCs was recorded on days 5, 6, and 7. Throughout the period of observation, 14% to 27% of S-phase cells (medians) were recorded for stroma-contact and stroma-noncontact groups of DCs. However, proliferating granulocytes appeared earlier in stroma-noncontact than in stroma-contact cultures (Table 1Go versus Fig. 5Go).


View this table:
[in this window]
[in a new window]
  		Table 1. Numbers of DNA-synthesizing cells and granulocytes (Gr-1+) harvested per Lin–Sca-1+ cell inoculated in diffusion chamber culture with or without a bone marrow stromal layer (SL)
 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Maturation rate of Lin–Sca-1+ cells cultured with or without contact with irradiated bone marrow stroma in vivo. At each time point, the number of PG and PMN were determined, based on differential counting. Medians and their 95% confidence intervals from 10 replicate experiments for both groups are given.

 
Concerning granulocyte maturation rate, no detectable differences were found between the stroma-contact and stroma-noncontact cultures, as assessed with the PMN:PG ratios (Fig. 5Go). The appearance of PMN in stroma-contact cultures was nonsignificantly delayed, 4.4 days versus 3.3 days for stroma-noncontact cultures (Fig. 6Go), the retardation possibly being caused by the delayed wave of proliferating PG in the stromal DCs. Finally, the density centrifugation experiments did not reveal any maturational effect of the stroma on the later stages of granulocyte development (Fig. 7Go).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 6. Granulocytopoiesis from sorted Lin–Sca-1+ cells cultured under in vivo conditions. Absolute values (with their 95% confidence intervals) of all granulocytes (PG + NPG) retrieved after five, six, and seven days' culturing are given (left-hand panels). Normalized PMN values (values for stroma-contact group on day 5 = 1.0 in each experiment) are also presented (right-hand panels). Extended linear regression lines (with their 95% confidence belts) gave the times of appearance of PMN in the cultures. n = 11 five to seven-day experiments for stromanoncontact and stroma-contact groups. n = 6 for stroma-contact with or without G-CSF groups.

 


View larger version (12K):
[in this window]
[in a new window]
  		Figure 7. Maturation of bone marrow cells cultured in vivo with or without stromal cells. 300,000 bone marrow cells were cultured in each DC on micropore membrane or on a layer of irradiated (20 Gy x-rays) bone marrow stromal cells for five days. Harvested cells, which had been labeled with 3H-thymidine one day before retrieval, were subjected to Percoll density gradient centrifugation. See legend to Figure 2Go. Two replicate experiments gave similar results.

 
Proliferation and Maturation of Lin–Sca-1+ Cells in Stroma-Contact Cultures Given Supplementary rhG-CSF
Continuously delivered rhG-CSF, from s.c. osmotic minipumps, gave rise to a higher percentage of Gr-1+ cells in the bone marrow of test than control, solvent infused mice (91% versus 82%). The proliferative activity in DCs between days 5 and 7 was high and was increased slightly on day 5 by the rhG-CSF infusion ( Table 2Go, Fig. 8Go). PMN appeared in the DC cultures at almost the same time in test as in control—4.0 days with G-CSF treatment versus 4.4 days for the control (Fig. 6Go). The rhG-CSF had no detectable influence on the maturation rate of granulocytes as judged by the occurrence of Gr-1+ cells and the PMN:PG ratios either (Table 2Go, Fig. 9Go). Likewise, the density centrifugation approach could not substantiate any significant maturational effect (Fig. 10Go).


View this table:
[in this window]
[in a new window]
  		Table 2. Numbers of DNA-synthesizing cells and granulocytes (Gr-1+) harvested per Lin–Sca-1+ cell inoculated in diffusion chamber culture with a bone marrow stromal layer, with or without G-CSF infusion to chamber hosts
 


View larger version (28K):
[in this window]
[in a new window]
  		Figure 8. BrdUrd incorporation profiles of five-day cultures. Freshly sorted Lin–Sca-1+ cells were cultured on bone marrow stromal cells in DCs in the peritoneal cavity of host mice. An osmotic minipump loaded with rhG-CSF was implanted s.c. into each host, so that 10 µg/kg rhG-CSF (A) or solvent (B) was delivered every day. DC cells were labeled with BrdUrd in vitro before cell retrieval from the chambers, and the cells which incorporated BrdUrd (enclosed in rectangles), were considered S-phase cells. Five independent, replicate experiments gave similar results.

 


View larger version (17K):
[in this window]
[in a new window]
  		Figure 9. Maturation rate of Lin–Sca-1+ cells cultured on bone marrow stromal cells in vivo, with or without continuous delivery of G-CSF. Purified Lin–Sca-1+ cells were plated at 1,500-2,500/DC onto the irradiated stromal layer in DC and cultured for five, six, and seven days, rhG-CSF or solvent being administered from osmotic minipumps. See legends to Figures 5 and 8GoGo. Cells from four DCs were pooled in each group. Medians and their 95% confidence intervals for six replicate experiments are depicted.

 


View larger version (15K):
[in this window]
[in a new window]
  		Figure 10. Maturation of bone marrow cells on stromal cells cultured in vivo with or without exposure to rhG-CSF, as assessed with density gradient separation. See legends to Figures 2 and 7GoGo. The test host mice were injected i.p. with 100 µg/kg rhG-CSF in 300 µl 0.9% NaCl with 0.1% FCS 3 and 18 h after injection of 3H-TdR and 6 h before DC harvest on day 5. Controls received solvent injections. Representative data from one of two independent experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Long-term stroma-based culturing [25, 26] has shown that sustained hematopoiesis depends on close proximity between hematopoietic cells and marrow stromal cells and extracellular matrix molecules. Inter alia, integrin-mediated signaling can affect motility, cell division, differentiation, and programed death of hematopoietic cells [9, 10]. But it is not known whether or how maturation of progenitor and precursor cells is influenced by the adhesion between parenchymal and stromal cells.

In the present study, two main ways—each with two subdivisions—were adopted to measure maturation rate: A) density gradient centrifugation, with scoring of both granulocyte distribution in the gradient, and the flux of 3H-TdR-prelabeled cells into the denser fractions of the gradient, where the more mature granulocytes are found, and B) recording the appearance of PMN in in vivo, cell-tight DC cultures, initiated from sorted stem cells. Moreover, in this system, provided a variable rate of apoptosis does not confound the process, PMN production after a certain time is positively correlated to the number of cell divisions and the flux rate from the proliferative to the end stage of granulocyte maturation. In the same vein, PG numbers are likewise dependent on the cell divisions, but inversely related to the loss by further maturation. Consequently, we also chose the PMN:PG ratio as an indicator of maturation rate. An even increase of the PMN:PG ratios from day 5 to day 7, with no detectable differences between test and control values, would strongly suggest both similar maturation and death (apoptosis) rates in the tests and controls.

Our present studies suggested that stroma-contact culturing retarded the initiation of development from purified Lin–Sca-1+ cells toward PMN, perhaps for one day, compared with stroma-noncontact cells. But when it started, the maturation of granulocytes proceeded at approximately the same rate with and without stromal contact.

G -CSF, initially identified as a differentiation-inducing factor for a leukemia cell line [27], is certainly a granulopoietin with an important stimulatory role for baseline granulocytopoiesis, as demonstrated with gene knock-out mice [28, 29]. Its granulocyte differentiation function (via a STAT-3 signal pathway) can apparently occur at lower concentrations of G-CSF than needed to accelerate proliferation [30]. G-CSF can increase the production of mature granulocytes [23, 31] and effect mobilization to blood of granulocytes and clonogenic hematopoietic progenitors [1, 31, 32]. Such findings suggest that G-CSF might also accelerate—or at least affect—maturation of granulocytes. However, the present experiments could not support this hypothesis. When the high proliferation rate of granulocyte precursors on a stromal cell layer in peritoneal DC was marginally increased by a continuous s.c. infusion of rhG-CSF, we could not find any changed maturation rate. This was in contrast to the acceleration of maturation rate that occurred when proliferation rate was slowed down by utilizing high-cellularity DC cultures in nonstimulated host mice. These latter results are in line with our previously published results from perturbations of proliferation rate of mouse granulocyte precursors [13]. A possible mechanism is that marked proliferation inhibition could result in one (or more) skipped cell divisions, and the cells enter the nonproliferative compartment instead. This would increase the maturation rate.

The discrepancies between the results of Lord et al. [1, 3] and ours are not easily explained. In their experiments, after labeling with 3H-TdR, labeled neutrophils appeared in the circulation of control mice after 24 h and peaked (about 15% labeling index) after 72 to 96 h. In G-CSF-treated mice, label appeared in blood after about 12 h and peaked (about 25% labeling index) after 24 h. This earlier appearance could be due to acceleration of maturation, release to blood (mobilization), or both. In our experience, it may not be easy to distinguish between segmented and band-nucleated rodent neutrophils in liquid emulsion radioautographs. Moreover, it has been shown that G-CSF can certainly mobilize neutrophils from the bone marrow [3, 33], whereas an effect on maturation rate per se has not been unequivocally demonstrated by others. Lord et al. used a higher dose of rhG-CSF (250 versus 10 µg/kg x d), given as two daily injections, whereas we provided a constant infusion rate with osmotic minipumps. Their dose was chosen to give a maximal blood leukocytosis, but they reported that 10 µg/kg x d gave a similar degree of marrow stimulation as the higher dose. The high G-CSF dose would decrease marrow cellularity by about 50% after 70 h, due to a redistribution of hematopoiesis between the bone marrow and the spleen [34]. They studied granulocytopoiesis in situ, whereas granulocyte formation in our experiments took place in a confined, ectopic, and artificial microenvironment. It may also be necessary to take into account that we have used different strains of mice, which may react differently to G-CSF.

One further finding by Lord et al. does indeed suggest that G-CSF accelerates maturation through the myelocyte stage—that is, the lack of increase in the proportion of myelocytes, morphologically defined, in the marrow—despite their more rapid proliferation after G-CSF treatment. However, this might also be explained by an increased rate of apoptosis, since there are instances where enhanced cell cycling is associated with enhanced apoptosis [35, 36]. In any case, more experiments need to be done to clarify this matter.

Our results are also in line with the observation that G-CSF receptor signals are not required for terminal differentiation, that is, maturation, since no accumulation of immature granulocytic cells was observed in the bone marrow of G-CSF receptor-deficient mice [37]. It would appear that a rather constant maturation rate of granulocytes is maintained, possibly at a genetically fixed level, under most physiological circumstances [38], since actual maturation programs are not dependent on the particular hematopoietic regulator used as the proliferative stimulus [39]. In combination with possibilities to regulate, for example, proliferative rate, mobilization to blood, and apoptosis, a fixed maturation rate would still provide enough flexibility to allow granulocytopoietic adaptation to the challenges of ordinary inflammatory states.


   Acknowledgments
 
We thank Arne Bøyum and Dagfinn Løvhaug for critically reading and Anne Berit Morrow for reviewing the manuscript. This research was supported by grants from Anders Jahre's Foundation for the Promotion of Science, The Norwegian Cancer Society, and the Norwegian Research Council.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lord BI, Molineux G, Pojda Z et al. Myeloid cell kinetics in mice treated with recombinant interleukin-3, granulocyte colony-stimulating factor (CSF), or granulocyte-macrophage CSF in vivo. Blood 1991;77:2154-2159.[Abstract/Free Full Text]

  2. Pojda Z, Molineux G, Dexter TM. Hemopoietic effects of short-term in vivo treatment of mice with various doses of rhG-CSF. Exp Hematol 1990;18:27-31.[Medline]

  3. Lord BI. Myeloid cell kinetics in response to haemopoietic growth factors. Baillieres Clin Haematol 1992;5:533-550.[Medline]

  4. Williams GT, Smith CA, Spooncer E et al. Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 1990;343:76-79.[Medline]

  5. Nicola NA. Granulocyte colony-stimulating factor. Immunol Ser 1990;49:77-109.[Medline]

  6. Verfaillie CM. Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis. Blood 1992;79:2821-2826.[Abstract/Free Full Text]

  7. Hurley RW, McCarthy JB, Verfaillie CM. Direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation. J Clin Invest 1995;96:511-519.

  8. Foley KP, McArthur GA, Queva C et al. Targeted disruption of the MYC antagonist MAD1 inhibits cell cycle exit during granulocyte differentiation. EMBO J 1998;17:774-785.[Medline]

  9. Howe A, Aplin AE, Alahari SK et al. Integrin signaling and cell growth control. Curr Opin Cell Biol 1998;10:220-231.[Medline]

  10. Giancotti FG. Integrin signaling: specificity and control of cell survival and cell cycle progression. Curr Opin Cell Biol 1997;9:691-700.[Medline]

  11. Cheng J, Baumhueter S, Cacalano G et al. Hematopoietic defects in mice lacking the sialomucin CD34. Blood 1996;87:479-490.[Abstract/Free Full Text]

  12. Fackler MJ, Krause DS, Smith OM et al. Full-length but not truncated CD34 inhibits hematopoietic cell differentiation of M1 cells. Blood 1995;85:3040-3047.[Abstract/Free Full Text]

  13. Benestad HB, Rytömaa T. Regulation of maturation rate of mouse granulocytes. Cell Tissue Kinet 1977;10:461-468.[Medline]

  14. Benestad HB, Reikvam A. Diffusion chamber culturing of haematopoietic cells: methodological investigation and improvement of the technique. Exp Hematol 1975;3:249-260.[Medline]

  15. Benestad HB, Toogood EO. Diffusion chamber (DC) culturing of haemopoietic cells: a reliable assay system for regulators of proliferation? Exp Hematol 1982;10:161-171.[Medline]

  16. Dolbeare F, Gratzner H, Pallavicini MG et al. Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci USA 1983;80:5573-5577.[Abstract/Free Full Text]

  17. van Erp PEJ, Brons PPT, Boezeman JBM et al. A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation. Cytometry 1988;9:627-630.[Medline]

  18. Kirkhus B, Fraas Clausen OP. Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry. Cytometry 1990;11:253-260.[Medline]

  19. Breivik H. Haematopoietic stem cell content of murine bone marrow, spleen, and blood. Limiting dilution analysis of diffusion chamber cultures. J Cell Physiol 1971;78:73-78.[Medline]

  20. Spangrude GJ, Scollay R. A simplified method for enrichment of mouse hematopoietic stem cells. Exp Hematol 1990;18:920-926.[Medline]

  21. Benestad HB. Formation of granulocytes and macrophages in diffusion chambers of mouse blood leucocytes. Scand J Haematol 1970;7:279-288.[Medline]

  22. Tapley P, Shevde NK, Schweitzer PA et al. Increased G-CSF responsiveness of bone marrow cells from hematopoietic cell phosphatase deficient viable motheaten mice. Exp Hematol 1997;25:122-131.[Medline]

  23. Campbell LJ, Maher DW, Tay DLM et al. Marrow proliferation and the appearance of giant neutrophils in response to recombinant human granulocyte colony stimulating factor (rhG-CSF). Br J Haematol 1992;80:298-304.[Medline]

  24. Rolstad B, Benestad HB. Spontaneous alloreactivity of natural killer (NK) and lymphokine-activated killer (LAK) cells from athymic rats against normal haemic cells. NK cells stimulate syngeneic but inhibit allogeneic haemopoiesis. Immunology 1991;74:86-93.[Medline]

  25. Klein G. The extracellular matrix of the hematopoietic microenvironment. Experientia 1995;51:914-926.[Medline]

  26. Dexter TM, Coutinho LH, Spooncer E et al. Stromal cells in haemopoiesis. Ciba Found Symp 1990;148:76-86.[Medline]

  27. Nicola NA, Metcalf D, Matsumoto M et al. Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor. J Biol Chem 1983;258:9017-9023.[Abstract/Free Full Text]

  28. Lieschke GJ, Grail D, Hodgson G et al. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood 1994;84:1737-1746.[Abstract/Free Full Text]

  29. Liu F, Wu HY, Wesselschmidt R et al. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice. Immunity 1996;5:491-501.[Medline]

  30. Ward AC, Hermans MHA, van Aesch YM et al. Tyrosine-dependent and -independent mechanisms of STAT3 activation by the human granulocyte colony-stimulating factor (G-CSF) receptor are differentially utilized depending on G-CSF concentration. Blood 1999;93;113-124.[Abstract/Free Full Text]

  31. Bungart B, Loeffler M, Goris H et al. Differential effects of recombinant human colony stimulating factor (rhG-CSF) on stem cells in marrow, spleen and peripheral blood in mice. Br J Haematol 1990;76:174-179.[Medline]

  32. Lord BI, Woolford LB, Wood LM et al. Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein-1a. Blood 1995;85:3412-3415.[Abstract/Free Full Text]

  33. Price TH, Chatta GS, Dale DC. Effect of recombinant granulocyte colony-stimulating factor on neutrophil kinetics in normal young and elderly humans. Blood 1996;88:335-340.[Abstract/Free Full Text]

  34. Molineux G, Pojda Z, Dexter TM. A comparison of hematopoiesis in normal and splenectomized mice treated with granulocyte colony-stimulating factor. Blood 1990;75:563-569.[Abstract/Free Full Text]

  35. Long MW, Dixit VM. Thrombospondin functions as a cytoadhesion molecule for human hematopoietic progenitor cells. Blood 1990;75:2311-2318.[Abstract/Free Full Text]

  36. Traycoff CM, Orazi A, Ladd AC et al. Proliferation-induced decline of primitive hematopoietic progenitor cell activity is coupled with an increase in apoptosis of ex vivo expanded CD34+ cells. Exp Hematol 1998;26:53-62.[Medline]

  37. Jacob J, Haug JS, Raptis S et al. Specific signals generated by the cytoplasmic domain of the granulocyte colonystimulating factor (G-CSF) receptor are not required for G-CSF-dependent granulocytic differentiation. Blood 1998;92:353-361.[Abstract/Free Full Text]

  38. Ogawa M. Hemopoietic stem cells: stochastic differentiation and humoral control of proliferation. Environ Health Perspect 1989;80:199-207.[Medline]

  39. Metcalf D. Lineage commitment and maturation in hematopoietic cells: the case for extrinsic regulation. Blood 1998;92:345-347.[Free Full Text]

accepted for publication July 31, 1999.



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
J. R. Allport, Y.-C. Lim, J. M. Shipley, R. M. Senior, S. D. Shapiro, N. Matsuyoshi, D. Vestweber, and F. W. Luscinskas
Neutrophils from MMP-9- or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro
J. Leukoc. Biol., May 1, 2002; 71(5): 821 - 828.
[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 Wang, X.
Right arrow Articles by Benestad, H. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by Benestad, H. B.

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