We investigated the CFU-Sd8 number and concentration in the peripheral blood of bib rats to study the ... 1986, Bowen & Morgan 1987, Garrick et a1. 19931,in spite of elevated ..... Fleming MD, Romano AR, Su Ma, et a1. (19981. Nramp2 is ...
Pluripotent haemopoietic progenitor cells (CFU-Sd8) in peripheral blood of hereditarily anaemic Belgrade (bib) rats Zoran Ivanovic & Pavle Milenkovic Institute for Medical Research, Belgrade, Yugoslavia
Summary The unique anaemic syndrome of the Belgrade laboratory (bib) rat is due to an intracellular iron deficiency which is induced by a not yet defined mutation, resulting in impairment of haemopoiesis. We investigated the CFU-Sd8 number and concentration in the peripheral blood of bib rats to study the relationship between medullary and extramedullary haemopoiesis in this anaemic syndrome. The results show normal concentration of CFU-Sd8 in the peripheral blood of bib rats. This finding was unexpected in the state of severe anaemia and disturbed growth factor production in bib rats, where the mobilization of CFU-Sd8 from bone marrow to blood is expected. The results suggest that severe anaemia is not regularly accompanied by the mobilization of pluripotent progenitors from bone marrow to the blood. Keywords
CFU-Sd8i mouse assaYi progenitor cellsi Belgrade anaemic rats
The Belgrade laboratory (bib) rat is a unique experimental model in haematology, which is still not completely characterized. The main feature of bib rats is severe anaemia (Sladic-Simic et a1. 1966), originating from an intracellular iron deficiency (Edwards et a1. 1986, Bowen & Morgan 1987, Garrick et a1. 19931, in spite of elevated serum iron levels (Sladic-Simic et a1. 1969, Sladic-Simic et a1. 1972). Intracellular iron deficiency in bib rats leads to an impairment of haemopoiesis at different levels i.e. a decrease of haem synthesis (Garrick et a1. 1991), impaired synthesis of globins in erythroid lineage precursors (Marjanovic et a1. 1994), impaired 'lymphoid' regulation of erythropoiesis (BiljanoviC-Paunovic et a1.1992) and a proliferative block of pluripotent and committed progenitor cells in bone marrow (PavlovicKentera et a1. 1989, Stojanovic et a1. 1990, Correspondence to: Dr Zoran Ivanovie, Institute for Medical Research, Dr Subotica 4, PO Box 721, 11001 Beograd. Yugoslavia, Tel: 381 11 685 788. Fax: 381 11 643691 Accepted 15 April 1998
Ivanovic et a1. 1995a). In addition, haemopoiesis is affected by disturbed production of certain growth factors and their activities (Stojanovic et a1. 1990, Biljanovic-Paunovic et a1. 19921and also by regulators of pluripotent progenitor cell proliferation (Ivanovic et a1. 1995b). In earlier studies, we have characterized pluripotent progenitor cell compartments in bone marrow (Ivanovic et a1. 1995a, Ivanovic & Milenkovic 1995) and spleen (Ivanovic et a1. 1997a) of bib rats using a 'rat to mouse' colony forming unit-spleen (CFU-S) assay and more primitive stem cells (pre-CFU-S) by marrow repopulating ability (MRAI determination. Recently, we were able to measure CFU-S in rat peripheral blood (Ivanovic et a1. 1997bl, which allowed the characterization of the circulating CFU-S pool in bib rats. In this paper, our findings on CFU -Sd8 (day 8) number and incidence in the peripheral blood of bib rats are presented in order to complete earlier results related to the distribution of © Laboratory Animals Ltd. Laboratory Animals (1999) 33, 77-82
78
CFU-Sd8 in haemopoietic tissues of bib rats and to have a better insight into the relationship between medullary and extramedullary haemopoiesis in this anaemic syndrome.
Materials and methods Animals The anaemic homozygous (mutant) rats (bib) and normal genotype and phenotype (+1+) litter mate rats, both sexes, from the New Belgrade colony (Military Medical Academy, Belgrade, Yugoslavia) were used for the determination of blood CFU-Sd8 when 8 weeks old. The bib rat colony was obtained by mating male and female heterozygous rats in a randomly bred closed system, which has been maintained since 1985 without outcrossing. Anaemia in bib rats is inherited as an autosomal recessive mutation (which occurred in Wistar rats at the Institute for Nuclear Sciences Vinca, Yugoslavia and as demonstrated in inbred crossing mating systems (Sladic-Simic et a1. 1966). The breeding system used in the current colony produced around 30% anaemic offspring with an estimated 1% of inbreeding per generation. CBA/H mice (Military Medical Academy, Belgrade), weighing 24-30 g at 10-12 weeks, both sexes, were irradiated and used as the recipients of the rat peripheral blood mononuclear cells (PBMe). The mice of this strain have a very stable haematological status, and have been used for CFU-S assays in our laboratory since 1978. The mice were placed in compartmentalized perspex box with 12 individual compartments (9x3x2.5 em each) (Lord 1993L and then irradiated. After irradiation, the mice were transferred to cages (24 mice per cage), and immediately supplied with fresh water and food. The mice were injected with PBMC 1-3 h after irradiation, and separated into various cages (7-10 mice per cage for each experimental group). All animals used had conventional microbiological status, and were kept in open systems, 3-4 rats per cage, or 7-10 mice per cage. Environmental temperature (20-22°e) and relative humidity (57%) were automatically regulated by an integral climatic system,
Ivanovic & Milenkovic
without special ventilation and filtration regimes, and under natural lighting. The animals were fed a standard industrially fabricated diet for laboratory rodents ('brickets') (Mixture M2, minimal content: 19% proteins, 7% cellulose, 8% ashes-Veterinarski Zavod Subotica, Yugoslavia), and given water from the public water system ad libitum. Separation of peripheral blood mononuclear cells The blood samples were obtained by heart puncture from rats following ether euthanasia. The detailed separation of PBMC has been previously described (Ivanovic et a1. 1997b): the blood was collected in sterile tubes with preservative-free sterile heparin (40 U/mIL and mixed in the proportion 10: 1.5 with 6% 0-(2-hydroxyethylJ-amylopectin-hydrolysate and isotonic sodium chloride (HES)-450 kDa solution (Plasmasteril) Fresenius} Bad Homburg) as an erythrocyte sedimenting agent. The blood was then centrifuged and the 'buffy coat' harvested. The 'buffy coat' was placed on Lymphoprep (Nycomed, Oslo), and after centrifugation, the PBMC at the interface were removed and washed twice in cell medium optimized for murine progenitor cells (Dulbecco's Modification of Eagle's Medium (DMEMI). The PBMC count was made using a haemocytometer and was then adjusted to 2, 4 or 8 x 106 cells per 0.2 ml. 'Rat to mouse' CFU-S assay The 'rat to mouse' assay performed in these experiments has been previously described for determining CFU-Sd8 in bone marrOw (Ivanovic et a1. 1995a, Ivanovic & Milenkovic 1995L spleen (Ivanovic et a1. 1997a) and peripheral blood of rats (Ivanovic et a1. 1997b). In short, the mice were irradiated with 9Gy of X-rays (dose rate 0.959 Gy/min for 10.69 min, RT 305; Philips, at the Institute of Radiology, Military Medical Academy, Belgrade). The applied conditions of irradiation resulted in the complete depletion of CFU-S in mice, i.e. endogenous colonies were not detected. The rat PBMC suspension (0.2 ml) was injected into the tail veins of the irradiated mice. The mice were killed 8 days
CFU-Sd8 in blood of Belgrade anaemic rats
later, the spleens removed, fixed in Telleyesnitzky solution (87% of 70% ethanolj 43.5% glacial acetic acid; 8.7% formaldehyde) and the colonies defined as macroscopically visible nodules> 1 mm were counted (Ivanovic. et al. 1997b). Calculating CFU-Sd8 numbers The number of CFU-Sd8 per ml of blood was calculated by multiplying the CFU-Sd8/106 injected PBMC by the number of PBMC per ml of donor rat blood. The number of PBMC per ml of blood was calculated on the basis of white blood cell (WBC) numbers, and the percentage of mononuclear cells calculated from blood smear differential cell counts (stained by May-Griinwald-Giemsa stain). The total number of circulating CFU-Sd8 was calculated on the basis of the previous determination of volemia in bib and normal rats by Sladic-Simic et al. (1972) using a radioactive tracer dilution method. Determination of peripheral blood parameters Red blood cell (RBC) counts were made using a haemocytometer. Blood haemoglobin concentration was determined by the cyanmethaemoglobin technique. Peripheral blood smears were stained with May-GriinwaldGiemsa stain. Packed cell volume (PCVl was determined using a microhaematocrit method. Statistical analysis The differences between variables were evaluated using the Student's t-test.
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Table 1 Mean ± SE values indices in bib and +1+ rats
of
peripheral
blood
Group of rats
Hb (g/dl)
RBC(x10'2/1)
PCV (S)
Control (+1+) n=24 Anaemic (bib) n=9
14.2±O.9
5.7± 1.2
43± 1.7
3.4±O.2*
13±4.9*
2.7±O.6*
*P < 0.001 (t-test). n = no. of animals, PCV= packed cell volume, RBC= red blood cells, Hb = haemoglobin
The calculations for body weight, volume of blood and the total number of circulating CFU-Sd8 are presented in Table 3. The mean body weight of bib rats used in these experiments was 3.4 times lower than in normal (+1+) rats, as previously described IIvanovic et al. 1995a, Ivanovic et al. 1995b, Ivanovic & Milenkovic 1995), and the estimated volume of blood of the bib rats was 2.66 times lower than for normal rats.
Discussion The results demonstrate that the number of CFU-Sd8 per ml of blood and the incidence of these cells per 107 PBMC for bib and normal homozygous dominant 1+/+1 rats were similar. The number of total circulating CFU-Sd8: body weight ratio in bib rats was higher than in +1+ rats, but this was not statistically significant. In severe anaemia, elevated erythropoietin (Epo) levels (Pavlovic.-Kentera et al. 1989, Biljanovic-Paunovic et al. 1992) and disturbed growth factor production (Stojanovic. et al. 1990, Biljanovic-Paunovic. et al. 1992, Ivanovic et al. 1995b), may lead to a massive
Results The level of anaemia present in the bib rats used in these experiments is similar to previously published data showing extremely low levels of Hb and low PCV values. (Table 1). The number of CFU-Sd8 per 107 PBMC is lower in bib rats than in +1+ rats, however there is no statistically significant difference (Table 2). When these CFU-Sd8 numbers were expressed per ml of blood, almost identical values (1O.7±4.3 : 1O.3±2.2) were obtained for bib and normal rats.
Table 2 Mean ± SE for CFU-Sd8 numbers eral blood of bib and +1+ rats
Group of rats
No. of donor rats
Controls
24 9
in periph-
CFU-Sd8j1 07 PBMN cells
CFU-Sd8/ml of blood
8.1 ±1.1
17.1 ±3.4
10.3±1.7
10.8± 1.8
11.4±6.6
10.7±4.3
WBC (x109il)
(+i+) Anaemic (bib) WBC=white
blood cells
Ivanovic & Milenkovic
80
Table 3
Mean ± SE for body weight,
estimated
blood
volume,
and CFU-Sd8 in
bib and +/+ rat circulation
Group of rats
Body weight (g)
Blood vol (ml)t
Total no. of circulating CFU-Sd8
CFU-Sd8 per gram of body weight
Controls
193.0±22.0
11.6± 1.32
119.9±31.7
O.62±O.13
(+!+) n=24 Anaemic
4.3±0.7*
56.51: 9.4
46.3±20.9*
0.82±0.33
(bib) n=9 *P < 0.001 (t-test), n = no. of animals. Results from three independent experiments. t The volume of blood calculated according to the determination of Siadic-Simic et a/. 1972
mobilization of progenitor cells from bone
of CFU-Sd8 from bone marrow to the spleen,
marrow to blood. A decreased number of CFU-Sd8 in bone marrow of bib rats (Iva-
but these data neither support this hypothesis nor the phenomenon of CFU-Sd8 mobi-
novie et al. 1995a, Ivanovie & Milenkovie 1995) with an increased number of these cells in the spleen (Ivanovie et al. 1997a, Ivanovie 1997), suggests a potential intensive 'transfer'
lization from bone marrow to the blood in certain anaemic mouse models, as previously reported (Rencricca et ai. 1970, Gidali & Feher 19851.
Table 4
Published
observations
in bib rats and suggested
mechanisms
Observation
Reference
CFU-Sd8 of bib rats have an iron deficiency-induced proliferative block
Ivanovic et al. 1995a, 1997a, 'b' defect is expressed in CFU-Sd8 Ivanovic 1997 population in bib rats, resulting in an intracellular iron deficiency Ivanovic et al. 1997a Iron imported by haemin in cells with
Treatment of bib rats with haemin abrogated the proliferative block of CFU-Sd8
Suggested mechanisms
'b' defect could partially correct the intracellular iron deficiency
Garrick et al. 1978 Bypassing of the 'b' defect with exogenous haemin results in a partially corrected globin synthesis in reticulocytes of bib rats Maximal saturation
of transferrin
in bib rats
Siadit-Simit
1972
Increased extracellular concentration
iron
may not enhance
the transferrin-dependent
uptake
of iron in bib rat cells Non-transferrin-bound rat reticulocytes
iron uptake by bib
Farchich & Morgan 1992
The barrier present in membrane transport of non-transferrin-bound iron in 'b' affected cells is of a
is decreased, but can be
partially corrected by an elevation of extracellular iron concentration
quantitative nature. Transferrinindependent uptake of iron could be partially achieved by a sufficient elevation of extracellular
Increased ferritin
mRNA in the splenic tissue
of bib rats Decreased activity of the haemopoietic stem cell proliferation inhibitor, tetrapeptyde AcSDKP (Frindel & Monpezat splenic tissue of bib rats
1989), in
Savkovic et a/. 1996
iron concentration Elevated iron concentration
in splenic
tissue of bib rats Our unpublished
data
Active proliferation stem cells
of haemopoietic
CFU-Sd8in blood of Belgrade anaemic rats
Two possible explanations could be proposed for the normal levels of circulating CFU-Sd8 in bib rats: Firstly, the severe chronic life-long anaemia may result in an intensive 'transfer' of CFU-Sd8 from bone marrow to the spleen, via blood, in the first few weeks of life, leading to an altered bib rat 'steady state'. This results in a depletion of CFU-S in bone marrow (Ivanovic et al. 1995a, Ivanovic & Milenkovic 1995), an increase of CFU-Sd8 pool in the spleen (Ivanovic et aI. 1997a), and a normal level of CFU-Sd8 in peripheral blood as an adaptation to hypoxia and other related phenomena (e.g. growth factor production). Secondly, the increased spleen content of CFU-Sd8 may be due to intensive proliferation of the local CFU-Sd8 population and their progenitors. Molecular evidence for increased haemopoietic proliferation in bib rat spleen (Savkovic et al. 19961, together with several other observations (Table 4) indirectly support this hypothesis. Thus, it seems that the increased concentration of iron and haemin, due to RBC disintegration occurring predominantly in the spleen, may be the critical factor enabling the proliferation of haemopoietic progenitors in the spleen of bib rats. The results presented here, demonstrate normal CFU-Sd8 concentration in the peripheral blood of bib rats, contributing to the validity of the conclusions related to CFUSd8 proliferation in the bone marrow of these animals (Ivanovic et aI. 1995a, Ivanovic & Milenkovic 1995, Ivanovic et al. 1997a, Ivanovic 1997). It is also evident that severe anaemia is not always accompanied by CFUSd8 mobilization from bone marrow to blood. Acknowledgments The excellent technical assistance of Mrs M. Dokic is fully appreciated, as well as the language corrections made by Ms Liliana Costa. This work was supported by a grant from the Ministry of Science and Technology of Serbia. Note: During the processing of this paper, 'b' mutation has been identified like a single polymorphism resulting in a glycine-to-arginine substitution at codon 185 Nramp 2 (Fleming et al. 1998). This mutation is responsible for impairment of the Nramp 2, which probably has a marked physiological role in cellular iron metabolism.
81
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