In vivo sister chromatid exchange analysis of a mouse plasmacytoma cell line NP-38

In vivo sister chromatid exchange (SCE) frequencies have been compared between the mouse plasmacytoma NP-38 and normal bone marrow cells of the host BALB/c mouse. NP-38 cells, transplanted subcutaneously showed a two-fold increase in SCEs (4.35-5.76/cell) compared with the bone marrow cells of the host (1.65-2.14/cell). Such an increase in SCE rates was also observed in NP-38 cells metastasized in spleen, bone marrow, liver, or mesentery, upon inoculation of NP-38 cells by intravenous injection. Even in such tumor-bearing mice, the SCE rates of the bone marrow cells were equivalent to the SCE level found in uninfected mice. These results indicate that the high SCE incidence in NP-38 cells is an inherent characteristic of this tumor cell line.     

A phenotypic and functional analysis of long-lived B and T lymphocytes

The lymphocyte composition of spleen, lymph nodes, bone marrow, and thymus of mice submitted to hydroxyurea treatments for four consecutive days was studied. The treatment selects for small lymphocyte populations that represent between 4 and 20% of control numbers in the various organs. Spleen and bone marrow contain the same B cell population with a low IgM, high IgD, low I-E phenotype, which respond to LPS at control clonal frequencies. The T cell compartment is equally depleted, and the lymphocytes remaining contain frequencies of clonable cells in response to mitogens and IL-2 that are comparable to those detected in normal spleen cells. Overall, the results suggest that only a minor fraction of all lymphocytes in a normal young adult mouse have life spans longer than 4 days.     

Booster irradiation to the spleen following total body irradiation. A new immunosuppressive approach for allogeneic bone marrow transplantation

Graft rejection presents a major obstacle for transplantation of T cell-depleted bone marrow in HLA-mismatched patients. In a primate model, after conditioning exactly as for leukemia patients, it was shown that over 99% of the residual host clonable T cells are concentrated in the spleen on day 5 after completion of cytoreduction. We have now corroborated these findings in a mouse model. After 9-Gy total body irradiation (TBI), the total number of Thy-1.2+ cells in the spleen reaches a peak between days 3 and 4 after TBI. The T cell population is composed of both L3T4 (helper) and Lyt-2 (suppressor) T cells, the former being the major subpopulation. Specific booster irradiation to the spleen (5 Gy twice) on days 2 and 4 after TBI greatly enhances production of donor-type chimera after transplantation of T cell-depleted allogeneic bone marrow. Similar enhancement can be achieved by splenectomy on day 3 or 4 after TBI but not if splenectomy is performed 1 day before TBI or 1 day after TBI, strengthening the hypothesis that, after lethal TBI in mice, the remaining host T cells migrate from the periphery to the spleen. These results suggest that a delayed booster irradiation to the spleen may be beneficial as an additional immunosuppressive agent in the conditioning of leukemia patients, in order to reduce the incidence of bone marrow allograft rejection.     

小鼠体内脾脏,骨髓及精原细胞姐妹染色单体交换的比较研究

本文对几种化学诱变剂诱发小鼠体内脾脏、骨髓和精原细胞的SCE进行了比较研究,同时分析了几类常见化合物在小鼠脾脏细胞中诱发SCE的活力。结果显示诱变剂在脾脏细胞中诱发SCE比骨髓和精原细胞敏感。几类化合物都能显著地诱发小鼠脾脏SCE的增加,与对照相比差异显著(P<0.05)或极显著(P<0.01),说明利用小鼠脾脏细胞检测环境诱变物是相当灵敏的。     

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.     

Haematopoietic stem cell content of murine bone marrow, spleen, and blood. Limiting dilution analysis of diffusion chamber cultures

Suspensions of mouse bone marrow cells, spleen cells, and blood leucocytes were cultured in diffusion chambers in dilution series in order to establish the minimum concentrations of haematopoietic stem cells (HSC). The observed frequencies of empty chambers after seven days of culture conformed to the expected frequencies of a null response in a Poisson distribution. The proportions of empty chambers could therefore be used to estimate the concentrations of HSC in the cell suspensions. The following numbers of HSC per 10⁵ cells were found (with 95% confidence limits): Bone marrow: 50 (44–56). Spleen: 3.5 (2.8–4.3). Blood leucocytes: 1.4 (1.2–1.8). The mean (± standard error) HSC-content per femur, spleen, and milliliter blood when pooling cells from three to six donor mice was 8240 ± 600, 7660 ± 490, and 56 ± 6.5 respectively. For comparison, the HSC concentrations were also determined with the spleen colony technique; the ratio between the HSC-concentrations of bone marrow, spleen, and blood determined with the diffusion chamber technique was similar to that determined with the spleen colony technique.     

The origin of antibody-forming cells detected in the bone marrow after thymus-independent antigen-stimulation and abnormality in migration of B cells of X-linked immunodeficient CBA/N mice

The anti-TNP IgM plaque-forming cells (PFC) were generated in the spleen and bone marrow of non-immunodeficient normal mice after intraperitoneal administration of TNP-LPS. Irradiation of normal mice while shielding bone marrow completely abrogated the generation of bone marrow PFC, indicating that they are derived from extramedullary sites. The bone marrow PFC, response to TNP-LPS was low in X-linked immunodeficient CBA/N strain mice, while the spleen response was comparable to that seen in the normal mice. To further study the basis of the deficient bone marrow PFC response in CBA/N mice, spleen cells were adoptively transferred to irradiated syngeneic mice stimulated with TNP-LPS. While spleen cells from normal mice generated high numbers of PFC in recipient bone marrow and spleen, those from CBA/N strain mice could not generate bone marrow PFC. This result was obtained regardless of whether normal or CBA/N recipients were used. These results indicate that TNP-LPS administration normally results in the migration of B lymphocytes from the periphery into the bone marrow and that B cells from immunodeficient CBA/N strain mice bear an inherent defect in this migratory function. This migratory defect was shown to be X-linked, as are the other previously reported B cell defects in this inbred mouse strain. The possible relationship between this migratory defect and the maturational defects of B cell lineage as reported previously in CBA/N strain mice is discussed.     

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

Mechanism of loss of natural killer activity in P815 ascites tumor bearing DBA/2 mice

P815 tumor cells (10(7] were administered intraperitoneally to DBA/2 mice. As the ascites tumor grew in the syngeneic host, a decline leading to a total loss of host spleen natural killer (NK) activity could be demonstrated. Removal of T and B cells or macrophages from the tumor-bearing (TB) mouse spleen cells did not raise the level of NK activity. Spleen cells from TB mice did not inhibit the NK activity of normal spleen cells. Comparable target (YAC cells) binding capacity could be demonstrated in spleen cells derived from normal or TB mice, but interferon failed to significantly stimulate the NK activity of TB mouse spleen cells. In adoptive transfer experiments, transfer of spleen or bone marrow cells from TB mice resulted in the development of significant levels of spleen NK activity in lethally X-irradiated recipient DBA/2 mice. These results indicate that the impairment of NK cell differentiation pathway rather than active suppression at the level of effector cells may be the mechanism of loss of NK activity in P815 TB DBA/2 mice.     

In vivo studies on the regeneration kinetics of enriched populations of haemopoietic spleen colony-forming cells from normal bone marrow

Functional properties of mouse haemopoietic spleen colony-forming cells, enriched 40- to 80-fold, from normal bone marrow were studied. It was found that: (1) the number of partially purified CFU-s (colony forming unit-spleen) required to rescue lethally irradiated mice was similar to the number of normal unfractionated bone marrow CFU-s giving the same level of protection; (2) the homing of partially purified CFU-s was similar to that of CFU-s from unfractionated bone marrow; (3) the regeneration of CFU-s in spleen was similar for enriched and unfractionated cell populations between 4 and 11 days after transplantation. In contrast, the rate of regeneration of CFU-s in femur was slower with enriched progenitor cells than with unfractionated bone marrow. The growth rate in femur, however, could be restored to normal by injecting freshly isolated syngeneic thymocytes with the enriched CFU-s population. The results indicate that the partially purified CFU-s are by themselves functionally normal and show that the rate of CFU-s repopulation in bone marrow can be affected by cell types other than spleen colony-forming cells.     

Sister-chromatid exchange studies on direct- and indirect-acting clastogens in mouse primary cell cultures

An in vitro sister-chromatid exchange (SCE) assay using mouse primary bone marrow and spleen cells was conducted with both direct- and indirect-acting genotoxic agents. 2,4,7-Trinitrofluorenone, a direct-acting genotoxic agent, induced a significant dose-related increase in SCEs. In both bone marrow and spleen cells, 2.0 micrograms/ml caused an approx. 3-fold increase in SCE level over control values. Cyclophosphamide, an indirect-acting genotoxicant which requires metabolic activation for its clastogenicity, induced a significant increase in SCEs in the presence of S9 from liver of rats pretreated with Aroclor-1254. A dose of 2 micrograms/ml resulted in a 2-fold increase in bone marrow and a greater than 5-fold increase in spleen cells. Benzo[a]pyrene, another indirect-acting genotoxicant, also induced significant dose-related SCE responses in both cell types. It seems that primary bone marrow and spleen cell culture systems can detect both direct- and indirect-acting genotoxicants and may be useful for routine and/or comparative cytogenetic studies.     

An in vitro assay for T lymphocyte progenitors (CFU-preT)

Thy-1.2 negative progenitors give rise to Thy-1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen-stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy-1 positive, peanut agglutinin-positive, and active in a pre-T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU-preT.     

Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice

The genotoxicity of an acetone extract of locally collected airborne particles was evaluated both in vitro and in vivo using the sister-chromatid exchange (SCE) assay in mice. At the highest concentration (5.36 mg/5 ml culture), the extract caused approximately a 3-fold increase in SCEs over controls in mouse bone marrow and spleen primary cells in vitro. However, the same airborne particle extract did not induce a significant increase in the SCE level over controls in vivo in mouse bone marrow and spleen cells when administered intraperitoneally or through oral gavage. This indicates that bone marrow and spleen primary cell cultures can be used in in vitro genotoxicity studies of complex mixtures, and that the genotoxicity of airborne particles detected in the in vitro system cannot always be detected in vivo with the same cell types. In addition, the same acetone extract of airborne particles caused dose-related his+ revertants in the strain TA98 of Salmonella typhimurium, both with and without S9 activation. The significant finding of this study is that the in vitro genotoxicity results of airborne particle extract may not be very meaningful in an in vivo situation.     

PROPERTIES OF HAEMOPOIETIC STEM CELLS IN PHENYLHYDRAZINE TREATED MICE

Recovery of erythropoiesis was fast in Balb/c mice irradiated 700 R 5 days after initiation of phenylhydrazine treatment and took place predominantly in the spleen, which showed numerous large frequently confluent endogenous colonies. Post irradiation phenylhydrazine induced anaemia did not accelerate recovery of erythropoiesis; it did, however, produce a slight but significant rise in endogenous colony formation.
Radiosensitivity of spleen CFU-S from phenylhydrazine treated mice was similar to that of CFU-S in normal mouse spleen.
Spleen CFU-S in mice 5 days after initiation of phenylhydrazine treatment were sensitive to the lethal action of Hydroxyurea, while bone marrow CFU-S were not.
The self-renewal capacity of CFU-S in the endogenously repopulated spleen of phenylhydrazine pretreated 700 R X-irradiated mice was low when compared to that of spleen exogenously repopulated by cells from normal mouse bone marrow, normal and phenylhydrazine treated mouse spleen. CFU circulating in blood of phenylhydrazine treated mice had a low self-renewal capacity.
The marked strain differences in self-renewal capacity of spleen CFU-S, and of the capacity of spleen CFU-S to increase by proliferation are discussed.     

Effect of fibroblasts from monolayer cultures of hematopoietic and lymphoid tissue on the immune response in vitro]

Stromal fibroblasts from the monolayer cultures of human bone marrow, guinea pig bone marrow, spleen, thymus and peripheral blood suppressed the response of the plagueforming cells against sheep erythrocytes in the suspension cultures of mouse spleen cells. Combined cultivation of 20 X 10(6) fibroblasts from all the mentioned sources led to complete suppression of the immune response. This suppression was less in mice immunized three days before the spleen cell explantation into the suspension cultures and was absent entirely in case the pre-immunization of spleen cell donors was accomplished nine days before the explantation.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.     

In vivo sister chromatid exchange in cells of various organs of the mouse

In vivo sister chromatid exchange (SCE) in mouse cells derived from various organs was studied by infusing BrdU from the tail vein. It was found that at BrdU concentrations ranging from 2.2–13.5 g/g/h, the SCE frequency in bone marrow cells seemed to stay at a constant level (1.5–2/cell/two cell cycles) whereas it started to rise as the BrdU dose exceeded this dose range. When BrdU within this dose range was infused continuously from the tail vein for appropriate hours to label chromosomes in various organs, the average SCE frequencies per cell were found to be 1.64 in bone marrow cells, 1.82 in spermatogonia, 1.99 in splenic cells, 2.89 in intestinal cells and 3.69 in cells from adjuvant stimulated lymph nodes. It is suggested that the spontaneous level of the in vivo SCE frequency might be about 1.5–2/cell/two cell cycles in the mouse. In cells derived from intestine and adjuvant stimulated lymph node, some unknown factors might work as a inducer of SCEs resulting in a significant increase in the SCE frequency in these organs.