Hematologic changes during spleen colony development in nonirradiated mice

The mechanism of spleen colony formation in nonirradiated mice was investigated. When the spleen cells of C57BL mice immunized against CBA- T6T6 mice were injected into the nonirradiated BT6F1 (C57BL X CBA-T6T6) hybrid mice, the number of hematopoietic stem cells (CFU-S of C57BL mouse origin that settled in the spleen of the BT6F1 mice continued to decrease in the first 9 days and then started to increase, with a doubling time of about 36 hr. Colonies were detected on the surface of the spleen 16-22 days after the cell injection. The slower appearance of spleen colonies in nonirradiated mice (compared with 6-10 days in the irridiated animals) appears to be due to retarded start of differentiation and to the prolonged doubling time of CFU-S in non- irradiated mice.     

Effect of cyclosporine A and methylprednisolone on the graft-versus-leukemia effects across major histocompatibility barriers in mice following allogeneic bone marrow transplantation

The effect of post-transplant immunosuppressive agents used in anti-GVHD prophylaxis on leukemic relapse was tested using a murine model of originally spontaneous, subsequently transplantable and non-immunogenic B cell leukemia (BCL1). (BALB/c x C57BL/6)F1 mice inoculated with 10(7) BCL1 cells were conditioned by total lymphoid irradiation (TLI) (1600 cGy), cyclophosphamide (200 mg/kg) or total body irradiation (TBI) 750 cGy and reconstituted with C57BL/6 (C57) bone marrow cells (30 x 10(6] or 10 x 10(6) bone marrow cells with additional 2 x 10(6) donor-type spleen cells, respectively. Mice were treated by cyclosporine A (CSA) 20 mg/kg i.p., or methylprednisolone (MP) 10 mg/kg i.p. for 10 days each and one group of controls received no post-transplant therapy. Stable chimerism was documented in all recipients with greater than or equal to 90% donor-type C57 cells in the peripheral blood. Eighty-nine percent of the mice treated by CSA following conditioning with TLI developed leukemia within 70 days, whereas none of the MP-treated mice and none of untreated chimeras showed any evidence of leukemia for more than 150 days. Adoptive transfer experiments using 10(5) spleen cells obtained from recipients conditioned with TBI were done to monitor residual leukemic cells following different post-transplant treatments. Eight-five percent of recipients of spleen cells obtained from mice treated with CSA developed leukemia in contrast with 33% and 25% when spleen cells were obtained from mice treated with MP or untreated controls, respectively (p = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of hematopoietic spleen colonies in nonirradiated genetically normal mice.

The question as to whether prior irradiation or injection of cytotoxic drugs is essential for the development of spleen colonies was examined in genetically normal mice. Mixtures of lymph node and bone marrow cells from C57BL mice were injected into (C57BL X CBA-T6T6) F1 hybrid mice without pretreatment. Hematopoietic nodules were observed in the spleens of F1 hybrid mice killed 18 days after injection. The average number of nodules increased linearly with increased numbers of injected bone marrow cells. Hematopoietic stem cells (CFU-S) and dividing cells in the nodules were shown to be of C57BL origin. Histologic examination showed that erythroid cell colonies predominated over granulocytic cell colonies. These results suggest that any kind of treatment that causes the depletion of CFU-S in the spleen of hosts would provide a suitable environment for the production of colonies by transplanted CFU-S.     

Mobilization of hematopoietic primitive and committed progenitor cells into blood in mice by anti-vascular adhesion molecule-1 antibody alone or in combination with granulocyte colony-stimulating factor

OBJECTIVE: One of the mechanisms for mobilization of hematopoietic stem cells and progenitor cells is alternation of adhesion molecules. We investigated the mobilization of hematopoietic progenitor cells in blood by administration of anti-vascular cell adhesion molecule (VCAM)-1 antibody (Ab) in mice. MATERIALS AND METHODS: Twelve- to 14-week old C57BL/6J mice were injected intravenously with anti-VCAM-1 Ab and anti-very late antigen (VLA)-4 Ab at a dose of 5 mg/kg for 2 days. RESULTS: The number of colony-forming cells (CFCs) in blood was increased 11.4-fold after anti-VCAM-1 Ab treatment, but the number of CFCs was not increased after treatment with anti-VLA-4 Ab. The number of colony-forming unit spleen (CFU-S) also was increased 21.6-fold in the peripheral blood by administration of anti-VCAM-1 Ab. The number of CFCs and CFU-S in the bone marrow of mice treated with anti-VCAM-1 Ab was decreased and that in the spleen also was decreased. On administration of recombinant human granulocyte colony-stimulating factor (125 microg/kg twice daily) with anti-VCAM-1 Ab, the numbers of CFCs and CFU-S were increased 141.8-fold and 439-fold, respectively. CONCLUSIONS: These observations demonstrated that administration of anti-VCAM-1 Ab induced mobilization of hematopoietic progenitor cells into blood from bone marrow and spleen and that granulocyte colony-stimulating factor has synergistic effects on anti-VCAM-1 Ab-induced mobilization.     

Successful engraftment of blood derived normal hemopoietic stem cells in chronic myelogenous leukemia

The ability of blood-derived stem cells to restore hemopoietic function was investigated in a patient with chronic myelogenous leukemia with bone marrow cells containing the Philadelphia chromosome marker (Ph1+). After treatment with high dose cyclophosphamide, 26.3 X 10(9) blood mononuclear leukocytes, among them 26.2 X 10(5) granulocyte/macrophage progenitor cells (CFUC), were harvested by means of 5 successive leukaphereses when the bone marrow cells had converted to Ph1--. When the patient entered the aggressive phase (blast crisis), myeloablative treatment with busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg) was given, followed by transfusion of the cryopreserved blood leukocytes. Restoration of marrow and blood cellularity was completed about 20 days after this autologous blood stem cell transplantation (ABSCT). Marrow CTUC recovery was complete 2 weeks after ABSCT, and all karyotypes of the patient's marrow cells were free of the marker chromosome. The patient died of toxicity but with normal bone marrow cellularity. This report confirms the therapeutic usefulness of autologous blood-derived stem cells harvested in remission in restoring hemopoietic function after myeloablative treatment.     

Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.     

三种品系小鼠对伴刀豆球蛋白A所致急性肝损伤的耐受性比较研究

目的：应用不同剂量伴刀豆球蛋白 A（ConA）诱导三种不同品系小鼠急性肝损伤,比较肝酶变化、死亡率和肝组织病理学变化。方法分别用6、12、20和30 mg·kg-1 ConA 处理 C57BL/6J、BABL/c 和 KM 小鼠,8 h 后对比观察急性肝损伤动物血清酶学及肝组织病理学变化情况。结果在6 mg·kg-1 ConA 的作用下,三个品系小鼠均被成功制造出急性肝损伤模型;在12 mg·kg-1 ConA 作用下,KM 小鼠血清 ALT 和AST 分别为（1006.8±12.1） U/L 和（1391.8±13.4）U/L,显著高于 BABL/c 小鼠[（75.7±0.5）U/L 和（284.7±23.4） U/L）和 C57BL/6J 小鼠（104.4±32.2）U/L 和（492.2±12.3）U/L,P 均〈0.05];同样,KM 小鼠肝指数和脾指数分别为（5.4±0.3）和（0.7±0.5）,均显著高于 BABL/c 小鼠[（5.2±0.4）和（0.6±0.3）和 C57BL/6J 小鼠（5.0±0.6）和（0.6±0.2）,P 均〈0.05];在20 mg·kg-1 ConA 作用下,三组小鼠血清 AST 和 ALT 水平无统计学差异,但 BABL/c 小鼠（4/10）和 C57BL/6J 小鼠（5/10）死亡率显著高于 KM 小鼠（1/10）;在30 mg·kg-1 ConA 作用下,三个品系小鼠死亡率均较高（KM 小鼠为30%,BABL/c 和C57BL/6J 小鼠均为100%）。结论不同品系的小鼠对 ConA 的耐受性不同,KM 小鼠对 ConA 的耐受性明显优于BABL/c 小鼠和 C57BL/6J 小鼠,且呈剂量依赖性。     

High persistence rate of hepatitis B virus in a hydrodynamic injection-based transfection model in C3H/He N mice

AIM: To optimize the viral persistence rate in a hydrodynamic injection(HI) based hepatitis B virus(HBV) transfection mouse model.METHODS:(1) 5-6-wk-old male C3H/He N and C57BL/6 mice were hydrodynamically injected with 10 μg endotoxin-free p AAV/HBV1.2 plasmid DNA via the tail vein. Hepatitis B surface antigen(HBs Ag), hepatitis B e antigen(HBe Ag) and HBV DNA, both in the serum and liver, were detected at different time points post HI by ELISA, immunohistochemical staining or quantitative polymerase chain reaction(PCR);(2) male C3H/He N and C57BL/6 mice, either hydrodynamically injected mice at 10 wk post HI or na?ve mice, were all immunized subcutaneously with 5 μg HBs Ag formulated in complete Freund’s adjuvant three times at a 2-wk interval. Two weeks after the final immunization, splenocytes were isolated for T cell function analysis by ELISPOT assay; and(3) five weeks post HI, C3H/He N mice were intragastrically administered 0.1 mg/kg entecavir once a day for 14 d, or were intraperitoneally injected with 1 mg/kg interferon(IFN)-α twice a week for 2 wk, or were treated with PBS as controls. The sera were collected and assayed for HBV DNA on days 0, 7 and 14 after drug treatment. RESULTS:(1) Approximately 90%(22/25) of the injected C3H/He N mice were still HBs Ag-positive at 46 wk post HI, whereas HBs Ag in C57BL/6 mice were completely cleared at 24 wk. Serum levels of HBe Ag in C3H/He N mice were higher than those in C57BL/6 mice from 4 wk to 46 wk. HBV DNA levels in the hydrodynamically injected C3H/He N mice were higher than those in the C57BL/6 mice, both in the serum(from 4 wk to 46 wk) and in the liver(detected at 8 wk and 46 wk post HI). Histology showed that hepatitis B core antigen and HBs Ag were expressed longer in the liver of C3H/He N mice than in C57BL/6;(2) HBs Ag specific T cell responses after HBs Ag vaccination in hydrodynamically injected C3H/He N and C57BL/6 mice, or naive control mice were detected by ELISPOT assay. After stimulation with HBs Ag, the frequencies of IFN-γ producing splenocytes in the hydrodynamically injected C3H/He N mice were significantly lower than those in hydrodynamically injected C57BL/6 mice, control C3H/He N and control C57BL/6 mice, which were 0, 17 ± 7, 18 ± 10, and 41 ± 10 SFCs/106 splenocytes, respectively, and the mean spot sizes showed the same pattern. Even just stimulated with PMA and ionomysin, T-cell responses elicited in the vaccinated control C3H/He N were much higher than those in hydrodynamically injected C3H/He N mice; and(3) For drug treatment experiments on the hydrodynamically injected C3H/He N mice, serum HBV DNA levels in the entecavir treatment group declined(131.2 folds, P < 0.01) on day 7 after treatment and kept going down. In the group of IFN-α treatment, serum HBV DNA levels declined to a lowest point(6.42 folds, P < 0.05) on 7 d after treatment and then rebounded.CONCLUSION: We have developed a novel HI-based HBV transfection model using C3H/He N mice, which had a higher HBV persistence rate than the classic C57BL/6 mouse model.     

Influence of dose and route of inoculation and of mouse strain on the production of interleukin 2 in mice infected with Mycobacterium lepraemurium

In order to evaluate the influence of route and dose of inoculation on interleukin 2 (IL2) production, C57BL/6 mice were infected either intravenously (I.V.) or subcutaneously (S.C.) with 10(5) or 10(8) Mycobacterium lepraemurium. The role of genetic factors on the production of IL2 during M. lepraemurium infection, was investigated in 7 inbred mouse strains (C57BL/6, DBA/2, F1 (C57BL/6 X DBA/2), DBA/1, BALB/c, CBA and A/J) after I.V. infection with 10(7) M. lepraemurium. At different times after M. lepraemurium inoculation, the number of AFB within the spleens of infected mice was counted and the ability of Con A-activated spleen cells to produce IL2 was studied. In S.C. inoculated C57BL/6 mice the increase in footpad thickness was measured during the progression of infection. After one month of infection heavily infected C57BL/6 mice (10(8) bacilli) showed an early and strong deficiency of IL2 production, regardless of the route of inoculation, whereas mice infected with a lower dose (10(5) bacilli) did not. In S.C. infected mice the decrease of IL2 production was observed when the footpad enlargement reached to the plateau phase. The data obtained from the numeration of AFB within the spleens of infected mice allowed to rank the infected mouse strains into 2 separated groups according to the pattern of the Bcg gene expression. An IL2 deficiency was only observed in C57BL/6, DBA/1, (C57BL/6 X DBA/2)F1 and DBA/2 infected mouse strains. No evident correlation could be shown between splenic IL2 activity upon Con A stimulation and the number of AFB recovered from the spleens of these 7 inbred mouse strains.     

Lysis of leukemia cells by spleen cells of normal mice.

Spleen cells from 2- to 3-month-old normal mice of some strains having a low incidence of spontaneous leukemia were found to lyse cells of the spontaneous AKR leukemia K36 in the 51Cr release assay. Incubation of 51Cr-labeled ADR K36 cells with spleen cells from normal C57BL/6, C57L, C57BL/10, and RF mice resulted in the release of significantly more 51Cr than that released in the presence of medium alone. In contrast, 51Cr released from AKR K36 cells after incubation with spleen cells from mice of the high leukemic strains AKR and C58 was less than that released spontaneously. The results of competitive inhibition tests when C57BL/6 spleen cells were incubated simultaneously with 51Cr-labeled AKR K36 target cells and varying numbers of nonlabeled cells demonstrated that the cytotoxic activity of normal C57BL/6 spleen cells was directed against an antigen(s) associated with several leukemias, but that was undetectable on normal thymocytes. Pretreatment of C57BL/6 spleen cells with carbonyl iron and a magnet, which removed phagocytic macrophages, did not decrease the cytotoxic acitivity for AKR K36 cells.     

On the role of the spleen in Friend virus (F-MuLV-P) erythroleukemia

About 30% of Friend virus (F-MuLV-P)-infected C57BL/6 mice became leukemic more than ten weeks after virus infection. This late leukemia development could not be essentially influenced by drug treatment, such as injection of hydroxyurea (2 X 500 mg/kg), actinomycin D (3 X 120 micrograms/kg), phenylhydrazine (3 X 60 mg/kg), or 30 micrograms endotoxin or by bone marrow transplantation following lethal irradiation. Endotoxin (30 micrograms) given prior to virus caused the loss of resistance to F-MuLV-P, but it had only a slight effect if applied one or three months after virus infection. Leukemia development has never been observed in C57BL/6 mice after splenectomy. In DBA/2 mice, highly susceptible to F-MuLV-P, leukemia development was markedly impaired if the mice were splenectomized. These results clearly indicate that the spleen plays a crucial role in the mechanism of susceptibility or resistance to the Friend virus.     

The effect of splenectomy on resistance of mice to Entamoeba histolytica infection

The role of the spleen in amoebic infection was examined in mice, using strains selected as being either genetically-susceptible (C57BL/6) or genetically-resistant (A/J) to amoebiasis. Splenectomized and sham-operated animals were inoculated intracaecally with 2.5 X 10(5) polyxenic trophozoites of E. histolytica at 6, 12 and 15 days post-splenectomy. The animals were killed 6 or 12 days after infection and the parasite burden was evaluated. Removal of the spleen in both susceptible and resistant mouse strains rendered these hosts extremely resistant to amoebic infection by this criterion. Gross examination of the caeca of non-splenectomized, genetically-susceptible mice showed numerous ulcers over the mucosal surface when compared to the splenectomized group which had superficial lesions or none. These observations suggest that the spleen plays a suppressive role in early anti-amoebic resistance.     

Transfer of experimental autoimmune insulitis by spleen cells in mice

Summary We have tested whether experimental insulitis induced by multiple subdiabetogenic injections of streptozotocin can be transferred by lymphocytes to normal recipients. C57BL/6J mice were treated on 5 consecutive days with 40 mg streptozotocin /kg body weight. 5×10⁷ nucleated spleen cells from 20 animals which had developed hyperglycaemia with concomitant insulitis three weeks after the first streptozotocin-injection, were transferred into congenic thymusless C57BL/6J-nu/nu mice. The cell transfer led to lymphocytic infiltrations of pancreatic islets in 75% of the recipients. Hyperglycaemia was not observed. It is concluded that lowdose streptozotocin treatment induces cellular immune reactions against pancreatic islets.     

Reduction of lethal graft-versus-host disease: transplantation of cultured murine bone marrow across minor histocompatibility differences

The murine bone marrow culture technique was used to prepare donor marrow for bone marrow transplantation across minor histocompatibility complex differences. Previous studies have shown that theta-positive cells are rapidly lost from such cultures and that transplantation of cultured marrow across major histocompatibility complex differences results in a delay in the development of lethal graft-v-host disease (GVHD). In this study, a total of 1 to 2 X 10(7) nonadherent cells (740 to 1560 CFUs [colony-forming units]) from three-day-old cultures were used as a source of donor marrow. Three strain combinations were evaluated; LP/J into C57BL/6; BIO.BR into CBA/J; and C57BL/6 into LP/J. Donor mice were immunized with recipient spleen cells prior to culture in order to increase the graft-v-host response. For LP/J marrow into C57BL/6 mice, 5 X 10(7) donor spleen cells transplanted along with the marrow were needed to induce lethal GVHD. However, lethal GVHD was seen without the addition of spleen cells for BIO.BR into CBA/J and C57BL/6 into LP/J strain combinations. Most animals receiving fresh marrow were dead of GVHD five weeks after transplantation. With the use of cultured marrow the three-month survival was 80%, 51%, and 93%, respectively, for LP/J into C57BL/6, BIO.BR into CBA/J, and C57BL/6 into LP/J strain combinations. Long-term donor engraftment in all recipient animals receiving cultured marrow was confirmed by analyzing hemoglobin polymorphisms between the strain combinations. These results demonstrate that in contrast to transplantation across major histocompatibility complex differences, the use of cultured cells for bone marrow transplantation across minor histocompatibility complex differences allows for engraftment while reducing the risk of lethal GVHD.     

Extensive proliferation of subsequently injected marrow cells in parental-to-F1 hematopoietic chimeras that restored normal stem cell concentration after initial transplantation

We investigated the fate of donor stem cells that were injected into hosts with a normal concentration of spleen colony-forming unit (CFU-S). Radiation chimeras were used as hosts. When CFU-S concentration in the marrow and spleen recovered to preirradiation levels after the initial bone marrow transplantation, the subsequent transplantation was done without reirradiation. Giant granules of beige C57B1/6 (bg) mice were used as a marker and proliferation and differentiation of the stem cells of the subsequent donor origin were evaluated by measuring the proportion of neutrophils with giant granules. No beige-type neutrophils were detectable at week 24 after transplantation of 5 X 10(7) marrow cells from bg mice to intact (WB X C57B1/6)F1 (F1) mice, which were used as control recipients. In contrast, transplantation of 5 X 10(7) marrow cells to radiation chimeras resulted in the appearance of neutrophils of second-donor origin. The proportion of beige-type neutrophils was 12% at week 24 after transplantation of bg marrow cells to F1-to-F1 (syngenic) or C57B1/6-+/+-to-bg (B6-to-bg) (congenic) chimeras; the proportion of beige-type neutrophils was 43% when bg marrow cells were transplanted to B6-to-F1 semiallogenic chimeras; the proportion of normal-type neutrophils was 82% when F1 marrow cells were injected to bg-to-F1 semiallogenic chimeras. Thus, the interaction of the host hematopoietic microenvironment with the stem cells of the initial donor as well as with the stem cells of the second donor seems to influence the proliferation and differentiation of the latter stem cells.     

In vivo and in vitro growth of the TK-1 strain of Coxiella burnetii]

All A/J, BALB/c and C57BL/6 murine strains were resistant against the intraperitoneal infection with TK-1 strain, but an enhancement of susceptibility of mice were revealed by the administration of cyclophosphamide (CPA) in BALB/c and A/J strains. CPA-treated BALB/c mice allowed an increase of TK-1 strain up to 1.7 x 10(6) coxiellar particles/mg spleen. But athymic nude mice of BALB/c strain showed only a slight increase of coxiellar particles in spleen. The resident peritoneal macrophages from BALB/c and A/J, but C57BL/6, showed proliferation of the TK-1 strain in the large infected cell population, and a part of the infected macrophages allowed TK-1 strain to survive. On the other hand, the elicited peritoneal macrophages from resistant C57BL/6 showed the largest infected cell population, number of intracellular coxiellar particles, and following decrease of TK-1 strain in later stage of infection. These in vitro infectivity of TK-1 strain seemed to relate to the in vivo infectivity in mice, and indicated existence of macrophage subpopulation, in which destruction or proliferation of TK-1 strain occurred.     

Regeneration of hemopoietic precursor cells in spleen organ cultures from irradiated mice: influence of genotype of cells injected and of the spleen microenvironment

The regeneration of hemopoietic precursor cells (colony-forming cells, CFC) was monitored in spleen organ cultures from lethally irradiated mice injected with 10(7) normal syngeneic or allogeneic bone marrow cells. The important role of the microenvironment in supporting hemopoiesis was confirmed by the failure of mutant S1/S1d spleens to support CFC regeneration in organ cultures. However, the extent and quality of the CFC regeneration was clearly dependent on the genetic properties of the injected cells. Evidence for this was obtained from the regeneration patterns of various CFC types in organ cultured spleens derived from different mouse donor-recipient strain combinations (CBA/CBA, CBA/C57BL, CBA/BALB/c, C57BL/C57BL, C57BL/CBA, C57BL/BALB/c) that maintained the differences in the bone marrow frequency of various CFC types characteristic of the donor strain.     

Hemopoietic effects of short-term in vivo treatment of mice with various doses of rhG-CSF.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was injected s.c., twice daily, into normal adult male BDF1 mice for a period of 4-5 days in doses ranging from 10 to 2500 micrograms/kg/day. The number of peripheral blood cells and their morphology was examined daily from the beginning of the experiment for 6 days. On the 5th day of treatment parameters such as spleen weight and cellularity, bone marrow morphology and cellularity, content of granulocyte-macrophage colony-forming cells (GM-CFC) or multipotential stem cells (CFU-S) in spleen and marrow, and also histology of lung and liver were examined. We observed a dose-dependent increase in the number of blood neutrophils, and an increase in weight, cellularity, and numbers of GM-CFC and CFU-S in the spleen. In the bone marrow, cellularity decreased to 40% of normal. Numbers of GM-CFC and CFU-S in marrow were also decreased, and examination of marrow morphology revealed an inhibition of erythropoiesis.     

Cell-mediated cytotoxicity of sensitized spleen cells against target liver cells--in vivo and in vitro study with a mouse model of experimental autoimmune hepatitis

Spleen cells obtained from C57BL/6 (B6) mice with an experimental autoimmune hepatitis were transferred to normal C57BL/6 recipient mice. Most prominent liver damages occurred in the recipient mice injected with sensitized nylon wool column-adherent spleen cells from the donor mice. Production of such liver damage was blocked by treatment of the sensitized adherent spleen cells with anti-Thy 1,2 monoclonal antibody and complement before injection. Based on these in vivo results, a microcytotoxicity assay was performed using isolated C57BL/6 hepatocytes as target cells and sensitized spleen cells obtained from hepatitis donor mice as effector cells. The fraction of sensitized nylon wool-adherent spleen cells demonstrated a high cytotoxic activity against isolated syngeneic hepatocytes, although the other fractions and spleen cells of control animals showed no such effect. The cytotoxic activity of sensitized-adherent spleen cells against target hepatocytes was significantly reduced after treatment with anti-Thy 1,2 antibody and complement, but it increased after depletion of B cells and Fc receptor-bearing T-cells. Although these sensitized nylon wool-adherent spleen cells showed high cytotoxic activities against syngeneic hepatocytes, their cytotoxicity against allogeneic hepatocytes was lower. They exerted no cytotoxic activity against syngeneic renal glomerular cells and EL-4 thymoma cells. These results suggest that sensitized T-cells in the nylon wool column-adherent fraction play the role of cytotoxic killer cells against target liver cells in vitro.     

Effects of purified bacterially synthesized murine multi-CSF (IL-3) on hematopoiesis in normal adult mice

Normal adult C57BL, BALB/c, and C3H/HeJ mice were injected intraperitoneally three times daily for up to 6 days with 102,000 U (200 ng) per injection of purified, bacterially synthesized, Multipotential colony-stimulating factor (CSF) (Interleukin-3) (rMulti- CSF) and compared with control mice injected with serum/saline with or without added endotoxin (1 ng/mL). Mice injected with rMulti-CSF exhibited tenfold rises in blood eosinophil and twofold to threefold rises in neutrophil and monocyte levels. The spleens from mice injected with rMulti-CSF showed a 50% increase in weight, elevated levels of maturing granulocytes, eosinophils, nucleated erythroid cells and megakaryocytes, and up to 100-fold rises in mast cells. Progenitor cell frequencies in the spleen were elevated sixfold to 18-fold. No significant changes were observed in the marrow. Sixfold to 15-fold rises were observed in peritoneal cell populations of mice injected with rMulti-CSF with evidence of increased peritoneal macrophage phagocytic activity. Livers of C57BL mice, but not of the other strains, exhibited increased numbers of infiltrating hematopoietic cells whereas rises in mast cell numbers were observed in the mesenteric lymph node, skin, and gut in BALB/c and C3H/HeJ mice. Endotoxin was excluded as being responsible for the observed changes except possibly those involving peritoneal macrophage phagocytic activity. The results indicate that the injection of normal mice with rMulti-CSF significantly stimulates the same types of hematopoietic populations as are stimulated in vitro by Multi-CSF and indicate that this and other CSFs should be useful in stimulating hematopoietic repopulation and functional activity in vivo.