Bone marrow as a potential source of hepatic oval cells.

Bone marrow stem cells develop into hematopoietic and mesenchymal lineages but have not been known to participate in production of hepatocytes, biliary cells, or oval cells during liver regeneration. Cross-sex or cross-strain bone marrow and whole liver transplantation were used to trace the origin of the repopulating liver cells. Transplanted rats were treated with 2-acetylaminofluorene, to block hepatocyte proliferation, and then hepatic injury, to induce oval cell proliferation. Markers for Y chromosome, dipeptidyl peptidase IV enzyme, and L21-6 antigen were used to identify liver cells of bone marrow origin. From these cells, a proportion of the regenerated hepatic cells were shown to be donor-derived. Thus, a stem cell associated with the bone marrow has epithelial cell lineage capability.

The effect of syngeneic marrow injection upon recovery in sub- and near-lethally irradiated mice.

Mice were given sub-lethal (200-600 cGy) or near-lethal (800 cGy) whole body irradiation and the effect of injecting syngeneic marrow on subsequent hematopoietic recovery was studied. Marrow cell injection enhanced erythropoietic recovery after sub-lethal irradiation as reflected in hematocrit values and rate of appearance of 59Fe-labeled red cells in blood. However, this enhanced erythropoiesis was only seen in the spleen, and 59Fe uptake in marrow was reduced. When the irradiation dose was kept constant and the marrow dose increased from 10(5) to 10(6) to 10(7) cells, there was a somewhat erratic increase in spleen 59Fe and a decrease in marrow 59Fe uptake. When marrow cell number was kept constant and the dose of irradiation was increased from 200 to 400 to 600 to 800 cGy, there was an exponential increase in spleen 59Fe uptake but the marrow 59Fe uptake changed from depressed after lower doses to increased after 800 cGy. Cell injection after sub-lethal irradiation did not increase or decrease granulocytopoiesis. Injection of irradiated marrow cells also reduced marrow erythropoiesis and this was evident after both sub- and near-lethal irradiation. However, injection of irradiated cells did not increase splenic erythropoiesis. Following splenectomy, the depressed marrow erythropoiesis attending injection of viable cells was virtually eliminated but no increase was seen. These data suggest that the injection of autologous or syngeneic marrow may not be effective as a means of accelerating hematopoietic recovery after irradiation unless near-lethal or lethal dose have been received.

Expression of murine CD34 by fetal liver NK cell progenitors.

Although 14.5-day murine fetal liver (FL) has few, if any, mature natural killer (NK) cells, culture of FL with recombinant human IL-2 (rhIL-2) and stroma from irradiated NK longterm bone marrow cultures (NK-LTBMC) allows proliferation and differentiation of NK cell progenitors. Using this system, NK cell progenitors were found in both CD34+ and CD34- sorted subpopulations of FL. The CD34 antigen was expressed by 14+/-1.3% of whole FL cells, while mature NK cells cultured from NK cell precursors in FL did not express the CD34 antigen. Anti-TER-119 mAb reacted with 84%+/-10.3% of the FL cells, and NK cell progenitors were enriched in the TER-119- subpopulation. After coculture with rhIL-2 and stroma, neither TER-119- nor TER-119+ cells expressed antigens associated with T cells (CD3, CD4, and CD8) or myeloid cells (Gr-1 and Mac-1). Only the TER-119 subpopulation generated NK1.1+ (77%) and B220+ (87%) cells. Within the TER-119 subpopulation, both CD34+ and CD34- cells generated cytolytic and NK1.1+ cells after culture. By a limiting dilution assay (LDA) of the Lin (i.e., negative for NK1.1, CD3, CD4, CD8, B220, Gr-1, and TER-119) CD34 positive or negative subpopulations, the calculated mean frequency of NK cell progenitors was about 1/100 for the CD34+Lin- subpopulation and about 1/(200-300) for the CD34-Lin- subpopulation. In kinetic studies, we found that NK1.1 antigen expression continued to increase with time in culture for both the CD34+Lin- and CD34-Lin- fractions. In contrast, the percentage of CD34+ cells decreased rapidly and produced CD34- cells, and the CD34- population remained CD34-. These data suggest that both CD34+ and CD34- subpopulations of FL can differentiate into NK cells when cocultured for 13 days with irradiated NK-LTBMC stroma and rhIL-2, and that CD34+ progenitors differentiate to CD34- precursors, which in turn differentiate to CD34- mature NK cells.

Latent deficiency of the hematopoietic microenvironment of aged mice as revealed in W/Wv mice given +/+ cells.

The macrocytic anemia of W/Wv mice can be cured by injection of +/+ bone marrow cells (BMC) from WBB6F1 mice. However, it has been observed that some W/Wv recipients appear to "lose" their cure with time, an effect that does not appear to be related to the age of the BMC donor. The present study was undertaken to determine the effect of recipient age on W/Wv responses to BMC injection. The effect of aging on erythroid parameters was similar in untreated W/Wv mice and +/+ controls. In both genotypes, hematocrit (HCT) and red blood cell count (RBC) decreased, and the modal red blood cell size (peak) increased between 13 and 150 weeks of age. As anticipated, mean HCT and RBC values were lower and peak values higher in W/Wv mice compared to +/+ controls at every age. However, the rate of decrease in HCT and RBC with age was the same for both genotypes, suggesting that the age effect and W gene effect were independent. Peak values increased slightly more with age for W/Wv than for +/+ controls. When female W/Wv mice in three age groups (23.5, 70, and 91.5 weeks old) were injected with 5 x 10(5) BMC from 20-week-old +/+ female donors and HCT, RBC, and peak were determined monthly, improvement was seen in most W/Wv recipients. However, in the older mice this improvement was slower and often was not sustained; 100% of the youngest recipients, 80% of the middle-aged, and only 30% of the older groups were cured after 3 months. Taken together, these data suggest a latent deficiency of the aging hematopoietic microenvironment that is revealed in W/Wv mice by the stress of continuing erythroid demand on the limited number of normal donor BMC.

Characterization of the stage in natural killer cell development in 14.5-day mouse fetal liver using adult bone marrow stroma.

Nonstimulated fetal liver (FL) from 14.5-day gestation mice had no natural killer (NK) cell activity and <3% expressed NK1.1. Even after short-term (3-4 day) culture of FL with the late-acting cytokines, interleukin (IL)-15 or IL-2, little or no NK activity was detected. However, longer-term (13 day) culture with IL-2 plus stroma derived from bone marrow (BM) of adult mice, resulted in extensive proliferation and differentiation to mature NK cells. Cell numbers began to increase after 4 days, and by day 13, they had increased 40-fold and 69% of the cells were NK1.1+ with high NK activity and 5%-10% were NK1.1- B220+. With stroma, but no IL-2, equivalent proliferation occurred, but differentiated cells were predominantly NK1.1- B220+, not NK cells. Culture for 13 days without stroma, but with either IL-2, IL-15, FLTK3-ligand (L) or stroma-conditioned medium, resulted in less than fivefold expansion, and minimal NK activity. Culture with combinations of FLTK3-L or ckit-L plus IL-15 or IL-2 increased both cell number and NK activity, but the increase in cell number was less than that seen with stroma plus IL-2. By limiting dilution assay on stroma plus IL-2, the precursor frequency was 1/(2660+/-292) whole FL cells and the absolute number, but not the frequency, increased during culture on stroma without IL-2. The NK cell progenitors were found in sorted NK1.1- and Sca-1+ c-kit+ lineage- subpopulations at a frequency of 1/(156+/-52.5). Together, these data suggest that the NK lineage cells in FL are primarily in early stages of development. They are highly proliferative, respond to early acting cytokines and express stem cell markers.

16,16-Dimethyl prostaglandin E2 and/or syngeneic bone marrow transplantation increase mouse survival after supra-lethal total body irradiation.

We evaluated the effects of 16,16-dimethyl prostaglandin E2 (dm-PGE2), with and without syngeneic bone marrow transplantation (BMT) on the survival and hematopoietic recovery of mice given 14-20 Gy total body irradiation (TBI). Survival of mice given combined dm-PGE2 and BMT was improved significantly over that of mice given either treatment alone. The 30-day survival after 14, 15, 16 or 18 Gy TBI for combined treatment was 97, 90, 20 or 10 percent, respectively. The corresponding 30-day survival rates for mice given BMT alone were 69, 60, 7 or 0 percent, respectively. For dm-PGE2 alone, 30-day survival was 63, 20, 10 or 0 percent, respectively. Deaths in both dm-PGE2 treated groups generally occurred after day 10 whereas deaths in the BMT group occurred before day 10. All irradiated controls were dead on or before day 10; after larger doses, deaths clustered around day 5. After 20 Gy TBI, all mice in all groups were dead by day 7. Studies of white blood cell recovery 1-9 days after 14 Gy TBI showed improvement with BMT, whereas dm-PGE2 did not enhance recovery. Nucleated cells per humerus, spleen weight, and spleen iron uptake (erythropoiesis) were also improved by BMT but not dm-PGE2.

Short-term myeloid reconstitution following TBI is not adversely affected by doses of FK506 that abrogate lethal GVHD.

Studies were undertaken to determine whether the doses of FK506 that are effective for acute GVHD prophylaxis following lethal irradiation and bone marrow transplantation (BMT) would also suppress myeloid cell reconstitution. FK506 (3 mg/kg/day) abrogated acute lethal graft versus host disease (GVHD) in lethally irradiated C57BL/10SnJ (H-2b) recipient mice given histoincompatible BM plus spleen cells from B10.BR (H-2k) donors and this dose was used in all of the studies. Endogenous and exogenous myeloid repopulation was studied in mice given daily injections of either FK506, an equivalent amount of carrier solvent or no treatment throughout the interval between total body irradiation (TBI) and the day of assay. Repopulation was studied after 400 or 500 cGy TBI (endogenous) and after 950 cGy TBI plus injection with syngeneic BM (exogenous). No consistent adverse effects of FK506 were seen during either exogenous or endogenous recovery. Parameters studied included hematocrit (Hct), WBC count, cells per humerus, spleen weight, splenic colony-forming units, % spleen or BM 59Fe uptake and colony forming cells per humerus. Similarly, when lethally irradiated secondary recipients were reconstituted with BM from FK506 treated primary recipients (lethal irradiation plus exogenous BM), no consistent effects were observed. These data suggest that FK506 given to prevent GVHD would not compromise the myeloid recovery that is critical for survival in the interval of time following shortly after BMT.

A minimal conditioning approach to achieve stable multilineage mouse plus rat chimerism.

Transplantation of untreated rat bone marrow into lethally irradiated (950 cGy) mouse recipients results in durable xenogeneic (rat-->mouse) chimerism and confers donor-specific transplantation tolerance for subsequent xenografts. The purpose of the present study was to characterize the minimal dose of total body irradiation (TBI) which would allow engraftment of rat bone marrow in mouse recipients. We report here that durable and stable lymphohaematopoietic cross-species chimerism can be achieved using a less than totally ablative radiation-based conditioning approach. The percentage of B10 mouse recipients which engrafted with rat bone marrow cells correlated with the dose of TBI. Engraftment of rat bone marrow stem cells occurred in all animals receiving 750 cGy prior to bone marrow transplantation, while no engraftment was detected at doses less than 650 cGy. Although most of the recipients were repopulated with mixed mouse and rat multilineage chimerism, some exhibited a predominance of rat cells. Although mixed xenogeneic rat/mouse chimeras prepared by lethal TBI produced only mouse derived RBC (red blood cells), chimeras prepared by sublethal conditioning produced both rat and mouse RBC. Only animals with detectable chimerism exhibited specific functional transplantation tolerance to donor xenoantigens, as assessed in vitro by mixed lymphocyte reaction assay. This model may offer an in vivo approach to study the role of species-specific growth factors in stem cell biology as well as the mechanisms for the induction of tolerance across species barriers.

Trisomy of chromosome 6.15 is not necessary for proliferation of AKR(Rb6.15)1Ald lymphoma cells.

By utilizing a unique AKR(Rb6.15)Ald (AKRb) mouse line we have been able to determine the incidence and frequency of cells with and without abnormalities of chromosome number (specifically trisomy 6.15) in spontaneous and transplanted lymphoma. The transplanted lymphoma cells were easily distinguished from the normal cells of AKR/J recipients by the presence in diploid cells of 2 metacentric and 36 acrocentric chromosomes. The majority of the cells from 17 normal control AKRb mice were diploid and trisomy 6.15 (3 metacentric and 36 acrocentric chromosomes) was not seen. In 73% of the 59 AKRb mice with spontaneous lymphoma, the majority of cells were diploid. In 54% of the mice none of the cells had trisomy 6.15, and in 22% of these mice a majority (greater than 50%) of cells had trisomy 6.15. In order to determine which of the cells from AKRb mice with spontaneous lymphoma were malignant, they were transplanted into normal young AKR/J recipient mice, and when lymphoma developed in recipients, their cells were analyzed. The majority of first passage lymphoma cells from 30 recipient mice were donor-type and the majority of these donor cells were diploid. Ten (17%) of these recipients had a majority of cells with trisomy 6.15. In order to further determine whether or not trisomic lymphoma cells had a different proliferative rate than diploid lymphoma cells, eight first passage recipient mice were used to start sequential passage lines. In two diploid lines and four trisomic lines, the same relative frequency of the trisomy was maintained in the sequential passages. However, in two trisomic lines, the frequency of lymphoma cells with three metacentric chromosomes diminished after passage. These studies suggest that aneuploidy is not necessary for proliferation of lymphoma and that diploid cells are a major part of the malignant cell population.

Hematopoiesis and aging III: Anemia and a blunted erythropoietic response to hemorrhage in aged mice.

Whether the hematocrit normally declines in the aged or whether such a decline represents inapparent disease in addition to aging is a matter of dispute. Female B6D2f1 mice were studied at ages 3, 13, or 27-28 months, and there was no difference in hematocrit between the younger groups. The hematocrit of 45 aged mice was slightly lower than that of 66 younger mice; mean 43% vs 49% (p less than .001). However, rather unexpectedly, the total red cell mass was not decreased in the aged; rather, the plasma volume was expanded. Survival of mature red blood cells did not differ significantly between young and aged mice. Mice were bled 0.4 ml from the orbital sinus for 4 days, reducing the hematocrit of all groups to a nadir of 20-25%. Recovery of hematocrit began more slowly in aged than in young mice. That this reflected a difference in erythropoiesis rather than a difference in plasma volume equilibration was suggested by studies with 59Fe. 59Fe was given following the second bleed, and 1 day later RBC 59Fe was more than twice as high in young mice than in groups of aged mice. Aged mice that did not appear healthy had been excluded. Aged mice were divided into a group with significant amounts of gray hair and/or patches of hair loss and two groups with normal-appearing hair; the latter was subdivided into those weighing less (25-26 g) or more (30-34 g) than most aged mice. Neither hair condition nor weight influenced hematocrit or response to bleeding. These results suggest, but do not prove, that a mild "dilutional" anemia and a blunted erythropoietic response to hemorrhage may be an expected part of the murine aging process.

Marrow mass and distribution in murine skeletons cleaned by beetles as compared to cut up carcasses and a further simplification of the latter technique.

Distribution of 59Fe into various bone groups of the complete murine skeleton was studied using two methods of dividing up the bones: 1) our previously reported technique of simply cutting up a skinned, eviscerated carcass and 2) separating bones from skeletons cleaned of overlying tissue by beetles, Dermestes species. The total percentage of injected 59Fe recovered in the sum of all skeletal parts, the percentage of total skeletal 59Fe found in each bone group, and the overall accuracy of determining these values were quite similar for the two techniques. The only statistically significant difference shown was a modest decrease in the percentage of total skeletal iron found in ribs plus sternum plus cervical and thoracic spine in beetle-cleaned as compared to cut up groups and we would not consider this to be of biological significance. Cutting up carcasses is the simpler of the two techniques but there are circumstances in which beetle digestion would be advantageous. In addition, we collected data on the reproducibility and precision of determining the percentage of 59Fe injected which is found in a "pulled off" foreleg plus scapula and of the distribution of 59Fe within three cut up pieces from the leg and within the scapula. These data can be used as a measure of overall changes in marrow mass and/or distribution, or at least they can be used as a screening procedure to detect such. This simple procedure adds potentially useful values for fully interpreting hematopoietic changes in the mouse.

Purified murine hematopoietic stem cells function longer on nonirradiated W41/Wv than on +/+ irradiated stroma.

Mouse bone marrow (BM) was separated into low-density, lineage-negative, wheat germ agglutinin-positive (WGA+), Rhodamine-123 bright (Rhbright) or dim (Rhdim) cells to obtain populations that were highly enriched for committed progenitors (Rhbright cells) or for more primitive stem cells (Rhdim). When 2,500 Rhbright or Rhdim cells were seeded onto 6-week-old irradiated (20 Gy) long-term BM cultures (LTBMC), the nonadherent cell production from Rhbright cells was transient and ended after 5 weeks. Production from Rhdim cells did not begin until week 3, peaked at week 5, and ended at week 8, when the irradiated stroma seemed to fail. Termination of cell production from Rhdim cells did not occur in nonirradiated LTBMC from W41/Wv mice. During peak nonadherent cell production, 25% to 30% of the cells in the nonirradiated LTBMC from W41/Wv mice had donor cell markers. Two approaches were tested to try to enhance the proportion or number of donor cells. Addition of Origen-HGF at the time of seeding Rhdim cells caused a nonspecific increase in both host and donor cell production, but a specific increase in production of donor cells was obtained by seeding the cultures at 2 weeks rather than 6 weeks. Limiting dilution of Rhdim cells gave the same frequency of wells producing cells on both irradiated +/+ and nonirradiated W41/Wv or W/Wv cultures.

Alterations of nuclear size profiles in AKR/J mice with spontaneous or transplanted leukemia.

Electronic sizing with a Coulter Counter was used to measure the frequency and number of cells with a large nuclear volume in tissues from AKR/J mice with spontaneous, long passaged, or first passage leukemia. The mean percentage of cells with a large nuclear volume was 1% in the blood, 4% in the spleen, 10% in the bone marrow, and 5% in the thymus of nonleukemic AKR/J mice. The percentage of cells with a large nucleus increased to 14-83% in all four tissues from AKR/J mice with spontaneous leukemia. In mice with a long passaged leukemia, a progressive increase in the percentage of cells with a large nucleus in the spleen and blood was associated with an increase in the number of cells/tissue with a large nucleus. However, in the bone marrow, an increase in percentage of cells with a large nuclear volume appeared to be the result of a decrease in the number of cells/humerus with a small nuclear volume. In the thymus, there was only a slight increase in the percentage of cells with a large nucleus. In mice with a first passage leukemia, there was a progressive increase, until death of the animals (21 +/- 1.9 days), in thymus weight and in the percentage of cells with a large nucleus. Also, the number of cells with a large nucleus increased in the spleen and bone marrow until day 12. Thereafter, there was a decrease in the total number of cells and number of cells with a large nucleus.

Constitutive systemic expression of IL-1Ra or soluble TNF receptor by genetically modified hematopoietic cells suppresses LPS induction of IL-6 and IL-10.

We have been developing both local and systemic gene therapy approaches to treat inflammatory and autoimmune diseases. To determine if systemic, constitutive expression of biologically active anti-inflammatory agents is therapeutic and/or has associated toxicity, mouse hematopoietic stem cells were infected with retroviral vectors carrying the genes for human IL-1 receptor antagonist (IL-1Ra), human soluble TNF receptor p75 (sTNFR), or the beta-galactosidase (lacZ) gene, and transplanted into lethally irradiated recipients. The serum levels of human IL-1Ra and human sTNFR in the long-term reconstituted mice, 2-7 months after transplantation, were 596 and 158 ng/ml respectively. The long-term expression of human IL-1Ra had minimal effects on the PBMC profile whereas human sTNFR expression increased the percentage of B220 and Mac.1 stained cells and decreased slightly the specific T cell subsets. The ability of these proteins to protect the transplanted mice from endotoxin treatment was determined by measuring serum interleukin-6 (IL-6) and interleukin-10 (IL-10) responses after LPS injection at 1.5, 3, 4.5 and 24 h after treatment. The IL-1Ra group showed diminished IL-10 levels and less mortality after injection of LPS. These results demonstrate that constitutive, systemic expression of IL-1Ra and sTNFR is able to confer partial protective effects following treatment with endotoxin. These results further demonstrate that gene transfer methods which result in systemic, long-term expression of immunodulatory proteins could be applied to the treatment of inflammatory diseases.

Natural killer cell precursors in the CD44neg/dim T-cell receptor population of mouse bone marrow.

Natural killer (NK) cells develop from the nonadherent cell component of NK long-term bone marrow (BM) cultures (NK-LTBMC). Because these nonadherent cells are depleted of mature NK cells and T cells, but appear to enriched for NK precursors, they were used as a starting population to begin to define the NK precursors that function in NK-LTBMC. As the stromal cell component of NK-LTBMC has been shown to support interleukin (IL)-2-induced, CD44 dependent, NK cell development from nonadherent NK precursors, NK-LTBMC stroma was used in a limiting dilution assay (LDA) to quantitate the precursors. NK-LTBMC in 96-well plates were irradiated (20 Gy) to kill hematopoietic cells (including the NK precursors), seeded with limiting dilutions of the cells to be quantitated, cultured with 500 U/mL IL-2 for 13 days and assayed for development of NK activity by adding 51Cr-labeled YAC-1 cells to the wells and evaluating the release of 51Cr after 4 hours. Flow cytometric analysis, sorting, and quantitation of the nonadherent cell component of NK-LTBMC showed that NK precursors were concentrated in the CD44neg/dim subset that comprised 10% of the "lymphoid" gated cells. When the CD44neg/dim subset was sorted from BM of mice treated with 5-fluorouracil (5-FU) day before (-1FUBM), there were about 30% T cells, but no NK-1.1+ cells. When the T cells were removed by sorting and the CD44neg/dim, alphabeta, gammadelta T-cell receptorneg (TCR-) subpopulation was seeded onto irradiated stroma with IL-2, they proliferated, developed NK activity, became NK-1.1+ and CD44bright and remained alphabeta, gammadelta TCR-. The frequency of NK precursors in this population as estimated from the LDA was about 1/500.

The effect of DFMO induced uptake of [3H] putrescine on human glioma cells.

Polyamine synthesis inhibitors, such as a-difluoromethylornithine (DFMO), inhibit tumor cell growth in vitro and in vivo. However, upon cessation of treatment, tumor growth resumes. We hypothesized that incorporation of radioactive polyamines might kill the growth-arrested cells. This hypothesis was previously tested in rat 9L brain tumor cells in which DFMO increased both the uptake and the retention of [3H] putrescine. In these rat cells, DFMO-induced retention of high-specific-activity [3H] putrescine for 20 days resulted in several logs killing. In the present studies all of the 5 different human glioma cell lines tested with DFMO treatment also showed enhanced uptake of exogenous [3H] putrescine, reduced cell counts and enhanced killing of colony forming cells (CSF). Extending the time of DFMO treatment of cells that had taken up high-specific-activity (80 Ci/mmol) [3H] putrescine further increased the killing. A 10-day extension resulted in a 10,000-fold reduction in cumulative cell growth. A 5-day extension resulted in a 2-3 log decrease in numbers of surviving CFC. These data further support the hypothesis and suggest that DFMO-induced cell cycle arrest enhances cellular retention of [3H] putrescine, increasing the effective internal radiation dose enough to cause proliferative death. In a clinical setting, the short (approximately 1 microm) path-length of the tritium beta particle should limit effects to the tumor cells and spare adjacent normal cells. These results support the concept that treatment with the combination of polyamine inhibitors and radioactive polyamines might be a useful adjunct to current therapies for glioblastoma multiforme.

Durable mixed allogeneic chimerism and tolerance by a nonlethal radiation-based cytoreductive approach.

For over 40 years, the association between hemopoietic chimerism and donor-specific tolerance for allografts has been recognized. However, toxicity associated with lethal conditioning has prevented the clinical application of bone marrow (BM) chimerism to induce tolerance. We previously demonstrated that engraftment could be achieved with less than total recipient myeloablation (700 cGy) and that the incidence of engraftment correlated with the dose of total body irradiation (TBI). Administration of cyclophosphamide (CyP) on Day +2 reduced the minimum TBI dose sufficient to permit engraftment to 500 cGy. In the current study, addition of antilymphocyte globulin (ALG) to the TBI/CyP-based conditioning approach reduced the radiation required for engraftment to < or = 300 cGy. B10 (H-2b) mice conditioned with ALG on day -3, 300 cGy of TBI with transplantation of B10.BR (H-2k) or BALB/c (H-2d) BM on day 0, and CyP on day +2 exhibited evidence of donor chimerism (49.6 +/- 3.7% and 38.2 +/- 2.4%, respectively) in 97% of recipients. ALG eliminated CD4+ and CD8+ cells and decreased NK1.1+ cells in the peripheral circulation at the time of transplantation. Moreover, T and NK cells in the host BM were significantly decreased compared with cells of recipients conditioned with TBI alone. CyP delayed repopulation of host thymocytes, providing time for the establishment of donor chimerism before production of mature T cells. Chimeric animals exhibited stable multilineage chimerism and donor-specific tolerance to skin grafts and in in vitro assays. This model may provide a clinically acceptable approach for the induction of donor-specific transplantation tolerance.

Generation of natural killer cells from long-term cultures of mouse bone marrow.

The features of a mouse long-term bone marrow culture (LTBMC) system that produces natural killer (NK) cell activity are described. Over a 4-week period in the NK-LTBMC, cellularity dropped from approximately 2.5 x 10(7) to 8 x 10(5) cells/25-cm2 flask. About 3 x 10(5) of these cells were loosely adherent. The cultures at this time contained about one-third the spleen colony forming units, one-tenth the granulocyte macrophage colony forming units and about one-third the transplantable NK progenitor activity of fresh bone marrow (BM), and no detectable NK cell activity. In the 4-week NK-LTBMC, IL-2-responsive precursor cells appeared to be selectively maintained and gave an 8-fold higher activity after culture with human recombinant IL-2 (rIL-2) than did fresh BM. The addition of 50-5,000 IU/ml of rIL-2 resulted, after a minimal 3-day lag, in progressively increased cellularity for as long as 13 days. The percentage and staining intensity of NK-1.1+ cells increased with time after addition of rIL-2. CD3 epsilon + cells were occasionally seen and B220+ cells were present in low numbers at day 7 and slowly increased through day 13. The stroma was necessary for IL-2-dependent development of NK activity.

Prolonged systemic expression of human IL-1 receptor antagonist (hIL-1ra) in mice reconstituted with hematopoietic cells transduced with a retrovirus carrying the hIL-1ra cDNA.

This study was designed to test the feasibility and safety of long-term expression of high levels of secreted human interleukin-1 receptor antagonist (hIL-1ra) protein in mice by retroviral transduction of hematopoietic stem cells. The retroviral vector, CRIP-MFG-hIL-1ra (MFG-IRAP), carrying the hIL-1ra gene was used to infect mouse bone marrow (BM) which was subsequently injected into lethally irradiated mice. All of the mice survived and greater than 98% of the white blood cells (WBC) of these mice were of donor type from 2-13 months after transplantation. All of the mice had hIL-1ra protein in their sera (40-1200 ng of hIL-1ra/ml) at all assay periods for at least 15 months after transplantation. Bone marrow from seven of seven primary recipients produced at least one secondary recipient with sustained, high serum levels of hIL-1ra, indicating that hematopoietic stem cells had been successfully transduced. Although the hIL-1ra was biologically active when assayed in vitro, the mice appeared to be well and their WBC counts and hematocrit (HCT) were not significantly different from those of lethally-irradiated mice given BM cells infected with the same vector carrying the lacZ gene. There was also no evidence of alterations of white cell subpopulations. These results demonstrate that systemic production of biologically active hIL-1ra can be obtained by retrovirus-mediated gene transfer to hematopoietic stem cells and that this level of expression and secretion into the serum is compatible with normal BM engraftment, hematopoietic recovery and survival of the lethally irradiated recipient mice. These hIL-1ra-expressing mice represent a model to examine the functions of IL-1 and hIL-1ra and to determine the ability of hIL-1ra to reduce susceptibility to chronic diseases such as rheumatoid arthritis as well as effects of aging such as bone degeneration. The data further suggest that transduction and transplantation of hematopoietic stem cells is a potential method for delivery of hIL-1ra and other secreted therapeutic gene products for systemic diseases.

Mixed allogeneic chimerism induced by a sublethal approach prevents autoimmune diabetes and reverses insulitis in nonobese diabetic (NOD) mice.

Evidence in experimental models suggests that many autoimmune diseases can be prevented by transplantation of bone marrow from disease-resistant donors. For potential clinical application, it would be important to avoid the morbidity and mortality associated with lethal conditioning and achieve mixed chimerism using less than complete recipient ablation. We report here for the first time that stable chimerism achieved in NOD mice using a sublethal radiation-based conditioning approach is sufficient to prevent beta-cell destruction and abrogate insulitis in prediabetic NOD mice. The percentage of NOD mouse recipients (8 wk of age) that engrafted with donor bone marrow correlated with the dose of irradiation and number of bone marrow cells transplanted. Engraftment of B10.BR bone marrow occurred in > or = 94% of animals receiving > or = 750 cGy of total body irradiation before bone marrow transplantation and > or = 30 x 10(6) bone marrow cells, while reproducible engraftment did not occur at radiation doses of less than 700 cGy and cellular doses of less than 30 x 10(6) bone marrow cells. All chimeric animals remained free of diabetes (n = 38) for 10 mo following bone marrow transplantation. Moreover, in all animals examined, no insulitis was present from 12 to 36 wk following reconstitution. In striking contrast, 61% (22 of 36) of NOD recipients that were conditioned but did not receive bone marrow developed acute diabetes by 12 mo. Insulitis was present in all remaining animals. These results suggest that allogeneic chimerism achieved using a sublethal conditioning approach can prevent the onset of diabetes and even reverse preexisting insulitis in NOD mice.