Alterations in Bone Marrow and Thymus Lymphocytes in Virus-Inoculated, Inbred, Preleukemic AKR/J Mice: Brief Communication 1,2 E~ther F. Hays, 3,
4
Dorothy R. Haskett,3 Catherine J. Kaspersky, 3 and Charles G. Craddock 3
ABSTRACT-In one study, we compared bone marrow and thy· mus lymphocytes of 5- to 6-week-old inbred AKR/J mice superinfected with Gross murine leukemia virus (AKR-V) with those of age-matched normal AKR/J mice (AKR-N). The AKR-V mice had marrow lymphopenia, a reduction of thymus weight and cellularity, and an increase in monocyte-granulocyte progenitor cells. The number of totipotent hematopoietic stem cells did not differ in the two groups of mice. In a second study, we evaluated the age-related changes of marrow and thymus lymphocytes in AKRV and AKR-N mice by comparing 5- and 8-week-old animals (a period covering the transition from adolescence to the adult state). In AKR-V mice, the normal age-related fall in marrow lymphocytes was blunted. This was due to a lesser decrease of T-Iymphocytes and null lymphocytes and an increase in immunoglobulin-bearing lymphocytes. The changes in thymus weight and cellularity between 5 and 8 weeks in the AKR-V mice reflected the actual malignant transformation in the thymus. Alterations in marrow and thymus cellularity occurring before the appearance of overt malignant lymphoma were considered in the context of virus-caused alterations of host hematopoiesis in the preleukemic (prelymphoma) state.-J Natl Cancer Inst 60: 905909,1978.
The inbred AKR mouse has a high incidence of spontaneous lymphoma in which the onset is predictably accelerated by the neonatal administration of G- M uL V (1). Using this model of accelerated lymphoma which provides the opportunity to study events occurring just prior to the onset of disease in young animals, we compared 5- to 8-week-old inbred AKR/J mice neonatally inoculated with G-MuL V (AKR- V mice) with age-matched controls of the same strain (AKR-N mice). AKR-N mice are here considered "normal," although all did develop lymphoma later. AKR- V mice develop lymphoma between 8 and 12 weeks of age (83.5±14 days), whereas in AKR-N mice the disease begins at 286±77 days (2). A morphologically similar thymic lymphoma occurs with 100% incidence in both groups. In this study, we looked at the thymuses and bone marrow of these animals with emphasis on the lymphoid population of the bone marrow. Lymphocyte populations in the thymuses and bone marrow of AKR- V mice were changed.
MATERIALS AND METHODS Inbred AKR/J mice from the breeding colony maintained in this laboratory were studied at 5, 6, and 8 weeks of age. Inbred 8-week-old C57BL/6J mice were studied for the presence of immunoglobulin-bearing cells in the bone marrow. G-MuLV (passage A) supplied by Dr. L. Gross (Veterans Hospital, Bronx, N.Y.) was VOL. 60, NO.4, APRIL, 1978
905
maintained in continuous passage by ip inoculation of 3- to 5-day-old AKR mice with cell-free filtrates of lymphomatous tissues from the virus-accelerated disease. We used as a source of virus one-tenth ml of a 20% (wt/vol) extract that was prepared by disrupting the cells and then centrifuging and filtering the supernatants with a 0.45-1L filter. This passage virus probably contained a mixture of G-MuL V passage A and the several known endogenous retroviruses from AKR mice (3, 4). We tested the passage filtrate for contaminating mouse viruses using the mouse antibody production test (Dr. Michael Collins, Microbiological Associates, Bethesda, Md.) and found the filtrate free of them. 5 We weighed and then killed the mice by lightly anesthetizing the animals with ether and bleeding them from axillary vessels. Peripheral leukocyte counts and hematocrits were determined from this blood. The spleen and thymus were removed from each animal, weighed, and fixed in Bouin's fluid; sections stained with hematoxylin and eosin were prepared. The bone marrow from both femurs of each animal was studied in the following manner. The femur was removed and cleaned of surrounding tissue. Then 5 mm of midshaft was measured and cut with a scalpel. The marrow cavity was washed through a 23-gauge needle with 3 ml McCoy's 5-A medium containing 15% FCS. The cells were separated by repeated aspiration into the syringe, and cell counts on the resulting dispersed suspension were done with a hemacytometer and expressed as cells/ 5 mm femur. In most instances, duplication of cellularABBREVIATIONS USED: G-MuLV = Gross murine leukemia virus (MuLV); FCS = fetal calf serum; CFU-s = totipotent hematopoietic stem cells; CFU-c = monocyte-granulocyte progenitor cells; HBSS = Hanks' balanced salt solution; PBS = phosphate-buffered saline.
1 Received October 4, 1976; revised September I, 1977; accepted November 8,1977. 2 Supported by Public Health Service grant CAI3666 from the National Cancer Institute, by contract E(04-I)GENI2 between the United States Energy Research and Development Administration and the University of California, and by the Levinson Foundation for Leukemia Research. 3 Laboratory of Nuclear Medicine and Radiation Biology, Department of Medicine, School of Medicine, Center for the Health Sciences, University of California, Los Angeles, Calif. 90024. 4 Address reprint requests to Dr. Hays, Laboratory of Nuclear Medicine and Radiation Biology, University of California at Los Angeles, 900 Veteran Ave., Los Angeles, Calif. 90024. 5 These viruses were: pneumonia virus of mice (PVM), reovirus 3, Theiler's encephalomyelitis virus (GO VII), K virus, polyoma virus, ectromelia, minute virus of mice, Sendai virus, mouse adenovirus, mouse hepatitis virus, lymphocytic choriomeningitis virus, and lactic dehydrogenase elevating virus.
J NATL CANCER INST
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HAYS, HASKETT, KASPERSKY, AND CRADDOCK
Itles for each femur was good. Assays of CFU-s and CFU-c and determination of immunoglobulin-bearing cells were done on the fresh cells. Aliquots were used to make Wright's-stained cytocentrifuge preparations and 200 cell differential counts. The remainder of the marrow suspension was frozen at -70° C and used for the virus assay as outlined below. We determined the cellularity of the thymus by disrupting this organ in a known volume of tissue culture medium by pressing it through a stainless steel screen, making a unicellular suspension by repeated aspiration into a syringe, and counting with a hemacytometer. Assays for in vivo spleen colony-forming cells were done by the method of Till and McCulloch (5). The recipients were given 750 rads 60CO and received 105 bone marrow cells. Spleen colonies were counted 9 days later on spleens fixed in Bouin's fluid. Control irradiated but noninoculated AKR-N animals were used in every study, and 0-1 colony was observed in these control spleens. The radiation dose of 750 rads was chosen because many animals died before 9 days when 850 or 800 rads were used. The assay for in vitro colonies in soft agar was done as described in (6). The XC assay for MuL V was that described by Rowe et al. (7). We infected NI H/3T3 cells by treating them with DEAE-dextran for 30 minutes at 37° C and then adding a preparation of 104 marrow cells that had been frozen and thawed three times. Results were expressed as plaque-forming units per 104 marrow cells. The XC assay measures the N-ecotropic nononcogenic virus of AKR mice (3). To determine the numbers of immunoglobulin-bearing cells, bone marrow and thymus cell suspensions were washed twice with HBSS containing 5% FCS. Washed cells were resuspended in 1 ml HBSS with 5% FCS and fluorescein-conjugated goat anti-mouse IgG (heavy and light chains) or IgM (heavy chain specific) (Cappel Laboratories, Cochranville, Pa.) (final dilution, 1:20), incubated for 30 minutes at 4° C, and then washed twice with HBSS with 5% FCS. Cells were resuspended in a small volume and a drop was placed on a slide with a cover slip. We determined the number of cells with membrane fluorescence per 200 cells in each sample using a Zeiss UV microscope and by alternating phase microscopy for cell identification. T-cells and null cells as a group were determined by the subtraction of immunoglobulin-bearing cells from the total number of lymphocytes. These antisera did not stain thymus lymphocytes. The stained cells in the marrow as examined by phase microscopy represented only cells with the morphologic appearance of small lymphocytes.
RESULTS Comparison of 5- to 6-Week-Old AKR-N and AKR-V Mice Thirty AKR- V mice (11 males, 19 females) were compared with 29 AKR-N mice (15 males, 14 females). The animals were 5- to 6-week-old mice. When new-
J
NATL CANCER INST
born, 22 AKR-N mice were inoculated with 0.1 ml PBS. The values for these animals were not significantly different than those from noninoculated AKR-N mice (Student's t-test). Therefore, PBS-inoculated and noninoculated animals were both included in the AKR-N group. At the time of killing, the animals from both groups were in vigorous health (table 1). The studies of bone marrow revealed interesting differences in the two groups. Differential counts of representative marrow showed a significant decrease in lymphocytes in the AKR- V mice, whereas values for promyelocytes and erythroid precursors were similar in both groups. In terms of absolute numbers, the decrease in marrow lymphocytes was highly significant. This is of particular interest in view of the red uction in thymus lymphocytes to be described. CFU-s and CFU-c were also evaluated. The numbers of CFU-s were not significantly different, whereas those of the CFU-c of the bone marrow from AKR-V mice were significantly increased. This discovery confirmed our previous findings (6). These findings were not, as noted above, reflected in any change in marrow promyelocytes. Virus concentration as measured by the XC assay was increased (P
4
Dorothy R. Haskett,3 Catherine J. Kaspersky, 3 and Charles G. Craddock 3
ABSTRACT-In one study, we compared bone marrow and thy· mus lymphocytes of 5- to 6-week-old inbred AKR/J mice superinfected with Gross murine leukemia virus (AKR-V) with those of age-matched normal AKR/J mice (AKR-N). The AKR-V mice had marrow lymphopenia, a reduction of thymus weight and cellularity, and an increase in monocyte-granulocyte progenitor cells. The number of totipotent hematopoietic stem cells did not differ in the two groups of mice. In a second study, we evaluated the age-related changes of marrow and thymus lymphocytes in AKRV and AKR-N mice by comparing 5- and 8-week-old animals (a period covering the transition from adolescence to the adult state). In AKR-V mice, the normal age-related fall in marrow lymphocytes was blunted. This was due to a lesser decrease of T-Iymphocytes and null lymphocytes and an increase in immunoglobulin-bearing lymphocytes. The changes in thymus weight and cellularity between 5 and 8 weeks in the AKR-V mice reflected the actual malignant transformation in the thymus. Alterations in marrow and thymus cellularity occurring before the appearance of overt malignant lymphoma were considered in the context of virus-caused alterations of host hematopoiesis in the preleukemic (prelymphoma) state.-J Natl Cancer Inst 60: 905909,1978.
The inbred AKR mouse has a high incidence of spontaneous lymphoma in which the onset is predictably accelerated by the neonatal administration of G- M uL V (1). Using this model of accelerated lymphoma which provides the opportunity to study events occurring just prior to the onset of disease in young animals, we compared 5- to 8-week-old inbred AKR/J mice neonatally inoculated with G-MuL V (AKR- V mice) with age-matched controls of the same strain (AKR-N mice). AKR-N mice are here considered "normal," although all did develop lymphoma later. AKR- V mice develop lymphoma between 8 and 12 weeks of age (83.5±14 days), whereas in AKR-N mice the disease begins at 286±77 days (2). A morphologically similar thymic lymphoma occurs with 100% incidence in both groups. In this study, we looked at the thymuses and bone marrow of these animals with emphasis on the lymphoid population of the bone marrow. Lymphocyte populations in the thymuses and bone marrow of AKR- V mice were changed.
MATERIALS AND METHODS Inbred AKR/J mice from the breeding colony maintained in this laboratory were studied at 5, 6, and 8 weeks of age. Inbred 8-week-old C57BL/6J mice were studied for the presence of immunoglobulin-bearing cells in the bone marrow. G-MuLV (passage A) supplied by Dr. L. Gross (Veterans Hospital, Bronx, N.Y.) was VOL. 60, NO.4, APRIL, 1978
905
maintained in continuous passage by ip inoculation of 3- to 5-day-old AKR mice with cell-free filtrates of lymphomatous tissues from the virus-accelerated disease. We used as a source of virus one-tenth ml of a 20% (wt/vol) extract that was prepared by disrupting the cells and then centrifuging and filtering the supernatants with a 0.45-1L filter. This passage virus probably contained a mixture of G-MuL V passage A and the several known endogenous retroviruses from AKR mice (3, 4). We tested the passage filtrate for contaminating mouse viruses using the mouse antibody production test (Dr. Michael Collins, Microbiological Associates, Bethesda, Md.) and found the filtrate free of them. 5 We weighed and then killed the mice by lightly anesthetizing the animals with ether and bleeding them from axillary vessels. Peripheral leukocyte counts and hematocrits were determined from this blood. The spleen and thymus were removed from each animal, weighed, and fixed in Bouin's fluid; sections stained with hematoxylin and eosin were prepared. The bone marrow from both femurs of each animal was studied in the following manner. The femur was removed and cleaned of surrounding tissue. Then 5 mm of midshaft was measured and cut with a scalpel. The marrow cavity was washed through a 23-gauge needle with 3 ml McCoy's 5-A medium containing 15% FCS. The cells were separated by repeated aspiration into the syringe, and cell counts on the resulting dispersed suspension were done with a hemacytometer and expressed as cells/ 5 mm femur. In most instances, duplication of cellularABBREVIATIONS USED: G-MuLV = Gross murine leukemia virus (MuLV); FCS = fetal calf serum; CFU-s = totipotent hematopoietic stem cells; CFU-c = monocyte-granulocyte progenitor cells; HBSS = Hanks' balanced salt solution; PBS = phosphate-buffered saline.
1 Received October 4, 1976; revised September I, 1977; accepted November 8,1977. 2 Supported by Public Health Service grant CAI3666 from the National Cancer Institute, by contract E(04-I)GENI2 between the United States Energy Research and Development Administration and the University of California, and by the Levinson Foundation for Leukemia Research. 3 Laboratory of Nuclear Medicine and Radiation Biology, Department of Medicine, School of Medicine, Center for the Health Sciences, University of California, Los Angeles, Calif. 90024. 4 Address reprint requests to Dr. Hays, Laboratory of Nuclear Medicine and Radiation Biology, University of California at Los Angeles, 900 Veteran Ave., Los Angeles, Calif. 90024. 5 These viruses were: pneumonia virus of mice (PVM), reovirus 3, Theiler's encephalomyelitis virus (GO VII), K virus, polyoma virus, ectromelia, minute virus of mice, Sendai virus, mouse adenovirus, mouse hepatitis virus, lymphocytic choriomeningitis virus, and lactic dehydrogenase elevating virus.
J NATL CANCER INST
906
HAYS, HASKETT, KASPERSKY, AND CRADDOCK
Itles for each femur was good. Assays of CFU-s and CFU-c and determination of immunoglobulin-bearing cells were done on the fresh cells. Aliquots were used to make Wright's-stained cytocentrifuge preparations and 200 cell differential counts. The remainder of the marrow suspension was frozen at -70° C and used for the virus assay as outlined below. We determined the cellularity of the thymus by disrupting this organ in a known volume of tissue culture medium by pressing it through a stainless steel screen, making a unicellular suspension by repeated aspiration into a syringe, and counting with a hemacytometer. Assays for in vivo spleen colony-forming cells were done by the method of Till and McCulloch (5). The recipients were given 750 rads 60CO and received 105 bone marrow cells. Spleen colonies were counted 9 days later on spleens fixed in Bouin's fluid. Control irradiated but noninoculated AKR-N animals were used in every study, and 0-1 colony was observed in these control spleens. The radiation dose of 750 rads was chosen because many animals died before 9 days when 850 or 800 rads were used. The assay for in vitro colonies in soft agar was done as described in (6). The XC assay for MuL V was that described by Rowe et al. (7). We infected NI H/3T3 cells by treating them with DEAE-dextran for 30 minutes at 37° C and then adding a preparation of 104 marrow cells that had been frozen and thawed three times. Results were expressed as plaque-forming units per 104 marrow cells. The XC assay measures the N-ecotropic nononcogenic virus of AKR mice (3). To determine the numbers of immunoglobulin-bearing cells, bone marrow and thymus cell suspensions were washed twice with HBSS containing 5% FCS. Washed cells were resuspended in 1 ml HBSS with 5% FCS and fluorescein-conjugated goat anti-mouse IgG (heavy and light chains) or IgM (heavy chain specific) (Cappel Laboratories, Cochranville, Pa.) (final dilution, 1:20), incubated for 30 minutes at 4° C, and then washed twice with HBSS with 5% FCS. Cells were resuspended in a small volume and a drop was placed on a slide with a cover slip. We determined the number of cells with membrane fluorescence per 200 cells in each sample using a Zeiss UV microscope and by alternating phase microscopy for cell identification. T-cells and null cells as a group were determined by the subtraction of immunoglobulin-bearing cells from the total number of lymphocytes. These antisera did not stain thymus lymphocytes. The stained cells in the marrow as examined by phase microscopy represented only cells with the morphologic appearance of small lymphocytes.
RESULTS Comparison of 5- to 6-Week-Old AKR-N and AKR-V Mice Thirty AKR- V mice (11 males, 19 females) were compared with 29 AKR-N mice (15 males, 14 females). The animals were 5- to 6-week-old mice. When new-
J
NATL CANCER INST
born, 22 AKR-N mice were inoculated with 0.1 ml PBS. The values for these animals were not significantly different than those from noninoculated AKR-N mice (Student's t-test). Therefore, PBS-inoculated and noninoculated animals were both included in the AKR-N group. At the time of killing, the animals from both groups were in vigorous health (table 1). The studies of bone marrow revealed interesting differences in the two groups. Differential counts of representative marrow showed a significant decrease in lymphocytes in the AKR- V mice, whereas values for promyelocytes and erythroid precursors were similar in both groups. In terms of absolute numbers, the decrease in marrow lymphocytes was highly significant. This is of particular interest in view of the red uction in thymus lymphocytes to be described. CFU-s and CFU-c were also evaluated. The numbers of CFU-s were not significantly different, whereas those of the CFU-c of the bone marrow from AKR-V mice were significantly increased. This discovery confirmed our previous findings (6). These findings were not, as noted above, reflected in any change in marrow promyelocytes. Virus concentration as measured by the XC assay was increased (P
