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Hematological compensation of microphthalmic mice with congenital osteopetrosis
Bone, 8, 315-317 (1987) Printed in the USA. All rights reserved
Cooyright
8756-3282187 $3.00 + .OO 0 1987 Pergamon Journals Ltd.
Hematological Compensation of Microphthalmic Mice with Congenital Osteopetrosis W. WIKTOR-JEDRZEJCZAK,
Z. POJDA,
A. AHMED,
AND M. Z. RATAJCZAK
Department of Immunology, Postgraduate MedIcal Center CKP WAM, ul. Szaserdw 728, 00-909 Warsaw, Poland, and Department Pathology and Laboratory Medlone, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
of
Address for correspondence and reprints: Dr.W. Wiktor-Jedrzejczak, Department of Immunology, Postgraduate MedIcal Center CKP WAM, ul. Szaserdw 128, 00-909 Warsaw, Poland or to Dr A. Ahmed, Department of Pathology and Laboratory Medicine, Emory University, Atlanta GA 30322, USA
Abstract
with L-glutamine (200 mm), N-2-hydroxyethyl-plperazine-N-2ethan sulfonic acid (HEPES) buffer (25 mM, Calbiochem, San Diego, CA, USA) and gentamycln (0 05 mgiml, Schering Corp., Kenilworth, NY, USA) When indicated, fetal calf serum (FCS) and/ or horse serum (HS) were used (Flow Labs)
Microphthalmic mice possess less than 10% of marrow cells. This marrow deficiency is compensated by an increase in the number of spleen cells. Additionally, 2week-old milmi mice have increased frequency of hemopoietic stem cells in the spleen and remaining marrow. In addition they have persistent stem cells in the liver. No other major hematologic abnormalities were found. Key Words: Osteopetrosis-Microphthalmic-Mice-Bone Marrow-Blood.
Hematological
measurements
Peripheral blood parameters were determined In blood drawn from the retroorbital plexus. The cell content of the organs important in hemopoiesis namely marrow, spleen, liver and thymus were determined. The cell counts of the spleen, thymus and liver, respectively, were obtained from cell suspensions from these organs. These single cell suspensions were prepared by teasing the organs separately through wire mesh and followed by repeated aspiration into a syringe (Wiktor-Jedrzejczak et al., 1986) The contents of the femoral marrow cavities were flushed out with media. The peritoneal cavity of mice was lavaged with 1 ml allquots of serum-free media and the cells obtained were enumerated (Wiktor-Jedrzejczak et al., 1986)
Introduction Microphthalmic miimi mice are homozygous for mutant mi allele at mi locus located on chromosome 6 (Wiktor-Jedrzejczak, et al., 1981 a). These mice have congenital osteopetrosis, absence of coat colour, lack incisors, and are blind (Silvers, 1979). Their osteopetrosis is a bone disorder curable by hemopoietic transplant (Walker, 1975; Loutit and Sanson, 1976; Marks and Walker, 1976). This suggest that the primary cellular lesion is located in the hemopoietic system and may imply the possible presence of other associated hematological abnormalities similary as it is the case In other murine osteopetrotic mutants (Wiktor-Jedrzejczak et al., 1982; 1986). The aim of this study is to characterize essential parameters of hemopoietic system of microphthalmic mice.
Exogenous spleen colony forming (CFU-S) assay for hemopoietic stem cells The CFU-S assay was performed as described previously (Till and McCulloch, 1961) Cell suspensions, e g marrow, spleen, liver in Medium 199. were injected intravenously into lethally irradiated k/k recipients Recipient mice were sacrificed 8 days later, their spleens were removed and placed In Tellysniczky’s fixative Subsequently, the number of colonies present on their surface was enumerated
In vitro monocyte-macrophage
Materials and Methods
colony format/on
Spleen cells were assayed for their ability to form colonies of monocytes and macrophages in the liquid culture system described earlier (Wiktor-Jedrzejczak et al 1981 b, Goud 1975b) Briefly, cells were suspended in the desired concentrations (5 x 1O4to 5 x 1OVml spleen cells) In DMEM supplemented with 20% horse serum and 20% marrow adherent layer conditioned medium (CM) that served as a source of colony stimulating actlvlty (CSA) Cells were cditured in slide chambers (Lab-Tek, Naperville, IL., USA) in 0.25 allquots at 37”C, 5% CO, In water-saturated air Cultures were carried out in octaplicate Following 4 days, cultures were termirated by discharging the media, air drying, and staining the bottom slide with Wright’s stain. Subsequently colonies of more than 30 cells were counted under 400 x magnification Both monocyte-macrophage and granulocyte colonies
Mice C57B1/6m/lm~ and k/i male and female mice were bred from breeding pairs purchased from the Jackson Laboratories, Bar Harbor, Maine. Twenty mutant animals aged between 7 and 30 days were evaluated Representative data derived from 4 animals 15 days old and their normal t/-c littermates are described Media Medium 199 (Biomed. Lublin, Poland) and Dulbecco’s modified Minimal Essential Medium (DMEM. Flow Labs, Rockville, MD, USA) were used throughout this study. They were supplemented 315
W Wiktor-Jedrzejczak
316
et al.: Hematologrcal
compensation
of microphthalmic
mice
are normally formed In those cultures. The monocyte-macrophage progenitor cell that gives rise to colonies has been termed the monoblast (Goud et al., 1975a).
suits were obtained during evaluation of the remaining 16 mutant and their respective control mice of both younger and older age in the range of 7-30 days. As shown In liable II, the hemopoietic stem cell con-
Adherent
centration as assayed using CFU-S test was significantly increased in both spleen and marrow of milmi mice. Moreover, a substantial number of CFU-S were found in the liver of milmi mice. There were no CFU-S detected in livers of ?i& mice of this age. The concentrations of macrophage colony forming units in vitro in the spleens of miimi mice were similar to the number in spleens of k/t mice. Additionally, the concentration of fibroblastoid CFU (representing cells contributing to hemopoietic microenvironment of the spleen) were similar. Moreover, the cellular composition of F-CFU-derived colonies was similar for miimi and +/+- mice (data not shown).
(stromal) colony formation by spleen cells in vitro
These cultures were set prepared as described previously (Wiktor-Jedrzejczak et al., 1981 b; Wiktor-Jedrzejczak et al., 1983b). Briefly, 5 x IO6 cells in 10 ml of DMEM supplemented with 10% HS and 10% FCS were placed in a single, sterile 2.5 cm2 disposable tissue culture flask (No. 3013, Falcon, Oxnard, CA, USA) Cultures were carried out in triplicate for 14 days at 37°C and 5% CO,. Subsequently, the supernate fluid was dscharged. Flasks were air-dried and cells were stained with Wright’s stain. Adherent colonies were visible and counted macroscopically. Colony forming units in this system are termed F-CFU (fibroblastoid colony forming units) and are assumed to be putative stem cells of at least some hemopoietic microenvironmental elements (Friedenstein, 1976).
Discussion Irradiation Recipient mice were irradiated at a dose of 120 cGys/min. The conditions of irradiation were 125 kV, 20 mA, with 0.5 mm Cu filter.
Results Compared to ?/-I littermates, there was severe cellular deficiency In the bone marrow cells in miimi mice associated with equivalent cellular deficiencies in the liver and thymus (Table I). There was a slight increase in the number of spleen cells when expressed per gram body weight. All other parameters were normal except for a slightly increased granulocyte count. Similar qualitative re-
Table I. Hematological
parameters of osteopetrotic microphthalmic miimi mice and their normal 2 /? littermatesa. Mean i- Standard
Parameter
(unit)
Body weight (gm) Hematocrit (%) White cell count x 1O-g/L Granulocyte count x 10mg/L Cells per femur x lO-6 Cells per femur x 10-6/gram body weight Cells per spleen x lO-6 Cells per spleen x lo-Ygram body weight Cells per thymus x 1O-6 Cells per thymus x 10e6/ gram body weight Cells per liver x 1O-6 Cells per liver x IO-s/gram body weight Cells per milliliter of peritoneal lavage x 1O-6 Cells per milliliter of peritoneal lavage x IO-‘Ygram body weight
-t/k 9.7 42.7 5.7 1.0 22.7
(4) ” ? 2 * t
Error
miimi (4)
0.6 0.4 1.2 0.4 0.9
6.4 44.3 6.1 1.9 2.0
5 t 2 * 5
0.3” 0.7 0.9 0.5b 0.2c
2.4 2 0.1 108.0 f 7.0
0.3 & O.lC 100.0 * 8.0
11.1 20.3 210.0 2 8.0
15 7 2 1.2c 159.0 2 lO.Ob
21.6 ? 0.7 90.0 -+ 7.0
24.8 2 0.6 70.0 2 10.0
9.2 2 03
10.9 * 1.4
3.5 2 0.2
2.9 2 0.3
0 35 2 0.01
0.45 ” 0.06
-
a Mice were evaluated at 15 days of age; half of them were male and half female b Difference significant P < 0.01. c P < 0.001, when compared to normal mice.
These studies failed to detect any specific hematological abnormalities in milmi mice other than those that could be interpreted as secondary consequences of osteopetrosis. The predominant abnormality was a lo-fold decrease in marrow cellularity, which was compensated for by increased stem cell concentration in the marrow itself and also by increased extramedullary hemopoiesis in the spleen and additional extramedullary hemopoiesis in the liver. The nature of the slight increase in granulocyte count is not clear at present, In contrast to thymic deficiency in other osteopetrotic mutants, e.g. the grey lethal mouse (Wiktor-Jedrzejczak et al., 1983a) or the op rat (Milhaud and Labat, 1978) no quantitative thymic cell deficiency could be found in microphthalmic mice. Therefore, the hematological profile of osteopetrotic milmi mice is more similar to estrone-induced osteopetrosis (Morse et al., 1974) and not to other murine congenital osteopetroses (WiktorJedrzejczak et al., 1986; Wiktor-Jedrzejczak et al., 1982) where other characteristic defects are observed such as monocyte and macrophage deficiencies (oplop and ocioc mice, Wiktor-Jedrzejczak et al., 1982, 1986) lymphocyte deficiency (ocioc mice, Wiktor-Jedrrejczak et al., 1986) Table II. Colony forming units (CFU) in hemopoietic
organs of
microphthalmic mi/m/ mice and their normal -+i* littermates.a Mean 2 Standard CFU assayed CFU-spleen per 1O5 marrow cells CFU-spleen per 1O6 spleen cells CFU-spleen per 1O6 liver cells CFU-macrophage per 1O4 spleen cells CFU-fibroblastoid per lo7 spleen cells
*I?
Error
m//m
212?31 65.6 ‘- 7.4 none
46.0 & 4.5b 111.2 2 7.lb 27.1 t 4.6b
29.1 ? 5.4
26.6 ? 6.2
39.0 ‘- 2.8
35.3 2 1.9
a Mice were evaluated at 15 days of age four donor mice per group, 10 recipient mice for each donor in in vivo assays, 8 simultaneous cultures for each donor for CFU-macrophage assay, and triplicate cultures for CFU-fibroblastoid assay per each donor mouse. b Dtfferences significant P < 0.001 when compared to normal mice
W Wiktor-Jedrzejczak
et al.: Hematological
compensation
of microphthalmic
and increased number of cells of monocyte-macrophage lineage (g//g/ mice, Wiktor-Jedrzejczak et al., 1981).
Research was supported ical Section, Polish Academy of Sciences.
Acknowledgment:
in part by the Med-
References Fnedenstein A J Precursor cells for mechanocytes. Inf. Rev. Cyfol 47.327, 1976 Goud T J L M Schotte C and Van Furth R ldentlflcation and characterlzatlon of the monoblast in mononuclear phagocyte colonies grown In vitro. J Exp. Med. 142-l 180, 1975a Goud T J L M and Van Furth R.: Proliferative characteristics of monoblasts grown in vitro J. Exp. Med. 142:1200, 1975b Loutit J F and Sanson J M.: Osteopetrosis oi microphthalmlc mice-a defect of the hemopoletic stem cell? &/c/f. T!ss Res. 20251, 1976 Marks S C and Walker D G.: Mammalian osteopetrosis-a model for studying cellular and humoral factors In bone resorption. In The 8/ochem&y and Physfology of the Bone. E. Bourne. Ed Academic Press, New York, 1976, Vol. 4 p. 227 Milhaud G and Labat M L ,Thymus and osteopetrosls C/in Orthopaed Rel Res 135:260, 1978 Morse B S Giuliani D Soremekun M , DiFlno S and Giuiiani E R Adaptation of hemopoletlc tissue resultmg from estrone-Induced osteosclerosis in mice Cell Trssue Kmet. 7-I 13, 1974 Silvers W K The coat colors of mice A model for mammalian gene action and interachon Springer Verlag, New York, 1979. p 268
mice
317
Toil J E and McCulloch E.A A direct measurement of the radlatlon sensetlvity of normal mouse bone marrow cells Radiat Res 14 213. 1961 Walker D G Control of bone resorption by hematopoietlc tissue. The Induction and reversal of congenital osteopetrosls in mice through use of bone marrow and splenic transplants J Exp. Med. 142 651, 1975 Wiktor-Jedrzefczak W , Skelly RR., and Ahmed A Hematopoietlc stem cell differenttallon and iis role In osteopetrosis In immunologrc Defects UI Laboratory An/ma/s M E Gershwin and E Merchant eds Plenum Press, New York, 1981a, p 51 Wiktor-Jedrzelczak W , Ahmed A and Szczyllk C Conditions of adherent cell growth from murlne bone marrow In liquid cultures, and partial characterization of function Exp. Hem&o/ 9 835. 1981 b Wiktor-Jedrzelczak W Ahmed A Szczylik C and Skelly R R Hematologtcai charactenzahon of congenital osteopetrosis in op/op mouse J Exp Med. 156 1516. 1982 Wiktor-Jedrzelczak W Grzybowskl J Ahmed A, and Kaczmarek L 0s~ teopetrosis associated with premature thymtc tnvolutlon In grey-lethal mice In vitro studies of thymlc mlcroenvlronment C//n. txp fmmunol. 52 465, 1983a Wiktor-Jedrzejczak W., Ptasznlk A., Ahmed A and Szczylik C Adherent ceil growth from murine bone marrow in Isquid cultures Inbred strain vartablllty and etfects of mutations affectmg hemopoiesis Exp. Hematol. 11 63, 1983b Wlktor-Jedrzejczak W , Szczylik C Ratalczak M Z and Ahmed A Congenital murine osteopetrosis InherIted with the osteosclero6c (oc) gene Hematologlcal characterization Exp Hematol. 14 819. 1986
Recetved. May 7, 1986 Revised April 14, 1987 Accepted May 26, 1987
Cooyright
8756-3282187 $3.00 + .OO 0 1987 Pergamon Journals Ltd.
Hematological Compensation of Microphthalmic Mice with Congenital Osteopetrosis W. WIKTOR-JEDRZEJCZAK,
Z. POJDA,
A. AHMED,
AND M. Z. RATAJCZAK
Department of Immunology, Postgraduate MedIcal Center CKP WAM, ul. Szaserdw 728, 00-909 Warsaw, Poland, and Department Pathology and Laboratory Medlone, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
of
Address for correspondence and reprints: Dr.W. Wiktor-Jedrzejczak, Department of Immunology, Postgraduate MedIcal Center CKP WAM, ul. Szaserdw 128, 00-909 Warsaw, Poland or to Dr A. Ahmed, Department of Pathology and Laboratory Medicine, Emory University, Atlanta GA 30322, USA
Abstract
with L-glutamine (200 mm), N-2-hydroxyethyl-plperazine-N-2ethan sulfonic acid (HEPES) buffer (25 mM, Calbiochem, San Diego, CA, USA) and gentamycln (0 05 mgiml, Schering Corp., Kenilworth, NY, USA) When indicated, fetal calf serum (FCS) and/ or horse serum (HS) were used (Flow Labs)
Microphthalmic mice possess less than 10% of marrow cells. This marrow deficiency is compensated by an increase in the number of spleen cells. Additionally, 2week-old milmi mice have increased frequency of hemopoietic stem cells in the spleen and remaining marrow. In addition they have persistent stem cells in the liver. No other major hematologic abnormalities were found. Key Words: Osteopetrosis-Microphthalmic-Mice-Bone Marrow-Blood.
Hematological
measurements
Peripheral blood parameters were determined In blood drawn from the retroorbital plexus. The cell content of the organs important in hemopoiesis namely marrow, spleen, liver and thymus were determined. The cell counts of the spleen, thymus and liver, respectively, were obtained from cell suspensions from these organs. These single cell suspensions were prepared by teasing the organs separately through wire mesh and followed by repeated aspiration into a syringe (Wiktor-Jedrzejczak et al., 1986) The contents of the femoral marrow cavities were flushed out with media. The peritoneal cavity of mice was lavaged with 1 ml allquots of serum-free media and the cells obtained were enumerated (Wiktor-Jedrzejczak et al., 1986)
Introduction Microphthalmic miimi mice are homozygous for mutant mi allele at mi locus located on chromosome 6 (Wiktor-Jedrzejczak, et al., 1981 a). These mice have congenital osteopetrosis, absence of coat colour, lack incisors, and are blind (Silvers, 1979). Their osteopetrosis is a bone disorder curable by hemopoietic transplant (Walker, 1975; Loutit and Sanson, 1976; Marks and Walker, 1976). This suggest that the primary cellular lesion is located in the hemopoietic system and may imply the possible presence of other associated hematological abnormalities similary as it is the case In other murine osteopetrotic mutants (Wiktor-Jedrzejczak et al., 1982; 1986). The aim of this study is to characterize essential parameters of hemopoietic system of microphthalmic mice.
Exogenous spleen colony forming (CFU-S) assay for hemopoietic stem cells The CFU-S assay was performed as described previously (Till and McCulloch, 1961) Cell suspensions, e g marrow, spleen, liver in Medium 199. were injected intravenously into lethally irradiated k/k recipients Recipient mice were sacrificed 8 days later, their spleens were removed and placed In Tellysniczky’s fixative Subsequently, the number of colonies present on their surface was enumerated
In vitro monocyte-macrophage
Materials and Methods
colony format/on
Spleen cells were assayed for their ability to form colonies of monocytes and macrophages in the liquid culture system described earlier (Wiktor-Jedrzejczak et al 1981 b, Goud 1975b) Briefly, cells were suspended in the desired concentrations (5 x 1O4to 5 x 1OVml spleen cells) In DMEM supplemented with 20% horse serum and 20% marrow adherent layer conditioned medium (CM) that served as a source of colony stimulating actlvlty (CSA) Cells were cditured in slide chambers (Lab-Tek, Naperville, IL., USA) in 0.25 allquots at 37”C, 5% CO, In water-saturated air Cultures were carried out in octaplicate Following 4 days, cultures were termirated by discharging the media, air drying, and staining the bottom slide with Wright’s stain. Subsequently colonies of more than 30 cells were counted under 400 x magnification Both monocyte-macrophage and granulocyte colonies
Mice C57B1/6m/lm~ and k/i male and female mice were bred from breeding pairs purchased from the Jackson Laboratories, Bar Harbor, Maine. Twenty mutant animals aged between 7 and 30 days were evaluated Representative data derived from 4 animals 15 days old and their normal t/-c littermates are described Media Medium 199 (Biomed. Lublin, Poland) and Dulbecco’s modified Minimal Essential Medium (DMEM. Flow Labs, Rockville, MD, USA) were used throughout this study. They were supplemented 315
W Wiktor-Jedrzejczak
316
et al.: Hematologrcal
compensation
of microphthalmic
mice
are normally formed In those cultures. The monocyte-macrophage progenitor cell that gives rise to colonies has been termed the monoblast (Goud et al., 1975a).
suits were obtained during evaluation of the remaining 16 mutant and their respective control mice of both younger and older age in the range of 7-30 days. As shown In liable II, the hemopoietic stem cell con-
Adherent
centration as assayed using CFU-S test was significantly increased in both spleen and marrow of milmi mice. Moreover, a substantial number of CFU-S were found in the liver of milmi mice. There were no CFU-S detected in livers of ?i& mice of this age. The concentrations of macrophage colony forming units in vitro in the spleens of miimi mice were similar to the number in spleens of k/t mice. Additionally, the concentration of fibroblastoid CFU (representing cells contributing to hemopoietic microenvironment of the spleen) were similar. Moreover, the cellular composition of F-CFU-derived colonies was similar for miimi and +/+- mice (data not shown).
(stromal) colony formation by spleen cells in vitro
These cultures were set prepared as described previously (Wiktor-Jedrzejczak et al., 1981 b; Wiktor-Jedrzejczak et al., 1983b). Briefly, 5 x IO6 cells in 10 ml of DMEM supplemented with 10% HS and 10% FCS were placed in a single, sterile 2.5 cm2 disposable tissue culture flask (No. 3013, Falcon, Oxnard, CA, USA) Cultures were carried out in triplicate for 14 days at 37°C and 5% CO,. Subsequently, the supernate fluid was dscharged. Flasks were air-dried and cells were stained with Wright’s stain. Adherent colonies were visible and counted macroscopically. Colony forming units in this system are termed F-CFU (fibroblastoid colony forming units) and are assumed to be putative stem cells of at least some hemopoietic microenvironmental elements (Friedenstein, 1976).
Discussion Irradiation Recipient mice were irradiated at a dose of 120 cGys/min. The conditions of irradiation were 125 kV, 20 mA, with 0.5 mm Cu filter.
Results Compared to ?/-I littermates, there was severe cellular deficiency In the bone marrow cells in miimi mice associated with equivalent cellular deficiencies in the liver and thymus (Table I). There was a slight increase in the number of spleen cells when expressed per gram body weight. All other parameters were normal except for a slightly increased granulocyte count. Similar qualitative re-
Table I. Hematological
parameters of osteopetrotic microphthalmic miimi mice and their normal 2 /? littermatesa. Mean i- Standard
Parameter
(unit)
Body weight (gm) Hematocrit (%) White cell count x 1O-g/L Granulocyte count x 10mg/L Cells per femur x lO-6 Cells per femur x 10-6/gram body weight Cells per spleen x lO-6 Cells per spleen x lo-Ygram body weight Cells per thymus x 1O-6 Cells per thymus x 10e6/ gram body weight Cells per liver x 1O-6 Cells per liver x IO-s/gram body weight Cells per milliliter of peritoneal lavage x 1O-6 Cells per milliliter of peritoneal lavage x IO-‘Ygram body weight
-t/k 9.7 42.7 5.7 1.0 22.7
(4) ” ? 2 * t
Error
miimi (4)
0.6 0.4 1.2 0.4 0.9
6.4 44.3 6.1 1.9 2.0
5 t 2 * 5
0.3” 0.7 0.9 0.5b 0.2c
2.4 2 0.1 108.0 f 7.0
0.3 & O.lC 100.0 * 8.0
11.1 20.3 210.0 2 8.0
15 7 2 1.2c 159.0 2 lO.Ob
21.6 ? 0.7 90.0 -+ 7.0
24.8 2 0.6 70.0 2 10.0
9.2 2 03
10.9 * 1.4
3.5 2 0.2
2.9 2 0.3
0 35 2 0.01
0.45 ” 0.06
-
a Mice were evaluated at 15 days of age; half of them were male and half female b Difference significant P < 0.01. c P < 0.001, when compared to normal mice.
These studies failed to detect any specific hematological abnormalities in milmi mice other than those that could be interpreted as secondary consequences of osteopetrosis. The predominant abnormality was a lo-fold decrease in marrow cellularity, which was compensated for by increased stem cell concentration in the marrow itself and also by increased extramedullary hemopoiesis in the spleen and additional extramedullary hemopoiesis in the liver. The nature of the slight increase in granulocyte count is not clear at present, In contrast to thymic deficiency in other osteopetrotic mutants, e.g. the grey lethal mouse (Wiktor-Jedrzejczak et al., 1983a) or the op rat (Milhaud and Labat, 1978) no quantitative thymic cell deficiency could be found in microphthalmic mice. Therefore, the hematological profile of osteopetrotic milmi mice is more similar to estrone-induced osteopetrosis (Morse et al., 1974) and not to other murine congenital osteopetroses (WiktorJedrzejczak et al., 1986; Wiktor-Jedrzejczak et al., 1982) where other characteristic defects are observed such as monocyte and macrophage deficiencies (oplop and ocioc mice, Wiktor-Jedrzejczak et al., 1982, 1986) lymphocyte deficiency (ocioc mice, Wiktor-Jedrrejczak et al., 1986) Table II. Colony forming units (CFU) in hemopoietic
organs of
microphthalmic mi/m/ mice and their normal -+i* littermates.a Mean 2 Standard CFU assayed CFU-spleen per 1O5 marrow cells CFU-spleen per 1O6 spleen cells CFU-spleen per 1O6 liver cells CFU-macrophage per 1O4 spleen cells CFU-fibroblastoid per lo7 spleen cells
*I?
Error
m//m
212?31 65.6 ‘- 7.4 none
46.0 & 4.5b 111.2 2 7.lb 27.1 t 4.6b
29.1 ? 5.4
26.6 ? 6.2
39.0 ‘- 2.8
35.3 2 1.9
a Mice were evaluated at 15 days of age four donor mice per group, 10 recipient mice for each donor in in vivo assays, 8 simultaneous cultures for each donor for CFU-macrophage assay, and triplicate cultures for CFU-fibroblastoid assay per each donor mouse. b Dtfferences significant P < 0.001 when compared to normal mice
W Wiktor-Jedrzejczak
et al.: Hematological
compensation
of microphthalmic
and increased number of cells of monocyte-macrophage lineage (g//g/ mice, Wiktor-Jedrzejczak et al., 1981).
Research was supported ical Section, Polish Academy of Sciences.
Acknowledgment:
in part by the Med-
References Fnedenstein A J Precursor cells for mechanocytes. Inf. Rev. Cyfol 47.327, 1976 Goud T J L M Schotte C and Van Furth R ldentlflcation and characterlzatlon of the monoblast in mononuclear phagocyte colonies grown In vitro. J Exp. Med. 142-l 180, 1975a Goud T J L M and Van Furth R.: Proliferative characteristics of monoblasts grown in vitro J. Exp. Med. 142:1200, 1975b Loutit J F and Sanson J M.: Osteopetrosis oi microphthalmlc mice-a defect of the hemopoletic stem cell? &/c/f. T!ss Res. 20251, 1976 Marks S C and Walker D G.: Mammalian osteopetrosis-a model for studying cellular and humoral factors In bone resorption. In The 8/ochem&y and Physfology of the Bone. E. Bourne. Ed Academic Press, New York, 1976, Vol. 4 p. 227 Milhaud G and Labat M L ,Thymus and osteopetrosls C/in Orthopaed Rel Res 135:260, 1978 Morse B S Giuliani D Soremekun M , DiFlno S and Giuiiani E R Adaptation of hemopoletlc tissue resultmg from estrone-Induced osteosclerosis in mice Cell Trssue Kmet. 7-I 13, 1974 Silvers W K The coat colors of mice A model for mammalian gene action and interachon Springer Verlag, New York, 1979. p 268
mice
317
Toil J E and McCulloch E.A A direct measurement of the radlatlon sensetlvity of normal mouse bone marrow cells Radiat Res 14 213. 1961 Walker D G Control of bone resorption by hematopoietlc tissue. The Induction and reversal of congenital osteopetrosls in mice through use of bone marrow and splenic transplants J Exp. Med. 142 651, 1975 Wiktor-Jedrzefczak W , Skelly RR., and Ahmed A Hematopoietlc stem cell differenttallon and iis role In osteopetrosis In immunologrc Defects UI Laboratory An/ma/s M E Gershwin and E Merchant eds Plenum Press, New York, 1981a, p 51 Wiktor-Jedrzelczak W , Ahmed A and Szczyllk C Conditions of adherent cell growth from murlne bone marrow In liquid cultures, and partial characterization of function Exp. Hem&o/ 9 835. 1981 b Wiktor-Jedrzelczak W Ahmed A Szczylik C and Skelly R R Hematologtcai charactenzahon of congenital osteopetrosis in op/op mouse J Exp Med. 156 1516. 1982 Wiktor-Jedrzelczak W Grzybowskl J Ahmed A, and Kaczmarek L 0s~ teopetrosis associated with premature thymtc tnvolutlon In grey-lethal mice In vitro studies of thymlc mlcroenvlronment C//n. txp fmmunol. 52 465, 1983a Wiktor-Jedrzejczak W., Ptasznlk A., Ahmed A and Szczylik C Adherent ceil growth from murine bone marrow in Isquid cultures Inbred strain vartablllty and etfects of mutations affectmg hemopoiesis Exp. Hematol. 11 63, 1983b Wlktor-Jedrzejczak W , Szczylik C Ratalczak M Z and Ahmed A Congenital murine osteopetrosis InherIted with the osteosclero6c (oc) gene Hematologlcal characterization Exp Hematol. 14 819. 1986
Recetved. May 7, 1986 Revised April 14, 1987 Accepted May 26, 1987