Hybrid resistance to precursor cells of bone marrow stroma]

A focusl of hemopoiesis appearing after the transplantation of a bone marrow fragment of C57BL mice to syngeneic mice (under the kidney capsule) contained more hemopoietic cells than in transplantation to the semisyngeneic (CBA X C57BL) FI recipient. Experiments were conducted with a secondary seeding by intravenous injection of hemopoietic cells of the C57BL transplant genotype into the transplant depopulated by irradiation; it was shown that these differences were caused by lesser dimensions of the hemopoietic microenvironment in the focus in the hybrid organism in comparison with such in the syngeneic system. Thus, the hybrid resistance was expressed not only to the hemopoietic cells, but also to the stromal precursors transferring the hemopoietic microenvironment.     

Influence of the maternal effect on allogenic inhibition of hematopoietic stem cells]

Bone marrow cells (0,5-10(6)) of female mice of CBA or C57BL strains were injected intravenously to lethally irradiated CBA, C57BL/6, (femaleCBA X maleC57BL/6)F1 and (femaleC57BL/6 X maleCBA)F1 mice. Spleen of recipients as assayed for colony count on the 9th day after bone marrow transplantation by the method of Till and McCullouch. Stem cells of CBA mice demonstrated failure of allogenic inhibition in (CBA X C57BL/6)F1 hybrid mice and formed the same number of colonies as in the spleen of syngenic recipients. The level of allogenic inhibition of CBA stem cells transplanted to (C57BL/6 X X CBA)F1 hybrid mice was 50%. Bone marrow cells of C57BL/6 mice formed colonies in spleen of (CBA X C57BL/6)F1 mice at least in 20 times less than in syngenic combination. In the transplantation of bone marrow from C57BL/6 mice to (C57BL/6 X CBA)F1 hybrid mice the allogenic inhibition was less pronounced (77-85%) as compared with the transfer of cells to (CBA X C57BL/6)F1 hybrid mice (95%). The sex of a recipient did not influence the number of formed colonies. The different level of allogenic inhibition of parental stem cells can not be explained by the effect of linkage with sex as the female of reciprocal hybrid mice have identical structure of sex chromosomes (X(CBA)XC57BL/6). The data obtained indicate that the maternal effect affects allogenic inhibition of stem cells in parent--F1 system. It is possible that the maternal influence may be determined by cytoplasmic factors of inheritance which affect the expressivity of recessive genes Hh, controlling the inheritance of specific haematopoietic cell antigens.     

Appearance of foci of hematopoiesis and hematopoietic stem cells in the liver of mice following a single administration of cyclophosphane]

The nidi of hemopoiesis appeared in the liver and the lymph nodes of DBA/2, C57BL/6j and F1 (C57BL/6j X CBA) strains of mice of the 6th day after a single intraperitoneal injection of cyclophosphamide in a dose of 200 mg/kg. This process was accompanied by the appearance of stem hemopoietic cells in the liver. Their maximum number was observed on the 6th and the 9th days. On the 12th day their level decreased, but it still exceeded the one in the animals which received a suspension of normal liver cells. In myelosan exhaustion the pool of the bone marrow and splenic stem cells, the subsequent cyclophosphamide injection failed to induce the appearance of the nidi of hemopoiesis and stem cells in the liver.     

Effect of syngenic lymphocytes on allogenic inhibition of hematopoietic stem cells of embryonic liver]

The transplantation of liver from the embryos and newborn C57BL-6 mice to the lethally irradiated hybrids (CBA X C57BL/6) F1resulted in 90% allogenic inhibition of the colony-forming activity of the donor elements. The degree of allogenic inhibition of liver cells of 19 days old embryos and newborn mice may be changed with the help of syngenic lymphocytes of adult mice or delayed transplantation of cells 72 hrs following the irradiation of recipients but these procedures proved to be ineffective with the liver cells of 13 and 16 days old embryos. A suggestion is put forward to the effect that the allogenic inhibition is based on the active reaction of recipient hybrids (CBAXXC57BL/6) F1 to the stem hemopoietic cells of C57BL/6 mice.     

Inhibition of the graft-versus-host response by BCGcw-induced suppressor cells or prostaglandin E1

Immunization of C57BL/6 mice with BCGcw stimulated a population of "suppressor cells" which had a decreased capacity to induce the graft-versus-host response. The graft-versus-host response was quantitated using the Simonsen splenomegaly assay. F1 mice (C57BL/6 X CBA) were inoculated intraperitoneally with 1 X 10(8) parental (C57BL/6) or (CBA) spleen cells. The F1 mice were sacrificed 13 days later and the resulting splenomegaly was 3-4 times the normal amount. F1 mice which were injected with parental BCGcw-primed C57BL/6 spleen cells had a 50% inhibition of splenomegaly, whereas BCGcw-primed CBA spleen cells (a strain which does not develop suppressor cells) did not show this inhibition. In vitro results also confirmed that only C57BL/6 mice and not CBA mice developed suppressor cells after BCGcw immunization. A second study showed that X-irradiated (1000 R) BCGcw-primed "suppressor cells" could inhibit splenomegaly caused by the inoculation of normal parental C57BL/6 cells into F1 mice. The mechanism by which BCGcw-primed "suppressor cells" caused this inhibition of splenomegaly was delineated and found to be dependent upon the secretion of prostaglandin (PGE-1). Indomethacin and aspirin, potent inhibitors of prostaglandin synthesis, blocked the activity of C57BL/6 BCGcw "suppressor cells" and splenomegaly resulted. Systemic administration of the prostaglandin (15S)-15-methyl PGE-1 reduced splenomegaly approximately 50% in F1 mice which were injected with C57BL/6 or CBA cells. These results indicated that immunization with BCGcw stimulated a population of "suppressor cells" which could cause a decrease in graft-versus-host response and that the secretion of prostaglandin was responsible for this inhibition.     

Origin of stromal bone marrow mechanocytes according to the results of typing them by isoantigens and chromosomal markers]

The bone marrow of radiochimaeras and heterotopic bone marrow transplants were used to study the origin of precursors of the fibroblasts growing in the monolayer cultures of hemopoietic tissue. In the bone marrow explants of the (C57BL/6 X CBA) F1 mice, in which the CBA bone marrow was transplanted following the lethal irradiation, the fibroblasts grown in the colonies were of recipient origin judging by isoantigens in the reaction of indirect immunofluorescence with the anti-C57BL/6-serum. At the same time in the bone marrow explants from heterotopic transplants (CBA leads to CBA X C57BL/6) the fibroblasts grown in colonies were of donor origin. The cultures of hemopoietic cells of the bone marrow of females heterotopically transplanted in the singenic male (guinea pigs Huston) contained only fibroblasts which were of donor origin judging by sex chromosomes in the metaphase plates of dividing cells. Hence, the bone marrow precursors of fibroblasts do not depend histogenetically on hemopoietic cells and are not replaced at the expense of repopulating cells of the second partner.     

Effect of mouse age on the ability of hematopoietic stem cells to interact with thymus cells]

The effect of the thymus cells of the C57BL/6 mice on the colony forming ability of the stem hemopoietic cells of the embryonic liver and bone marrow of young (3 months) and old (2 years) mice was studied their joint transplantation into the mice (CBAXXC57BL/6) F1. The stimulating effect of the thymus cells on the colony forming ability of the stem hemopoietic cells of different age depends both on the dose of the stem hemopoietic cells of embryonic liver and the dose of T-lymphocytes. A suggestion is put forward that the stimulating effect of the thymus cells on the colony formation is due to their interaction with the stem cells in the G2 phase of the mitotic cycle.     

Hematopoietic tissue reactions associated with the growth of syngeneic transplanted tumors in mice]

Hemangiopericytoma growth in syngeneic male mice F1 (CBA X C57BL/bj) led to regular hemopoietic changes: an increase in the spleen weight, spleen cell count, in the number of colony-forming units (CFU). It also induced augmentation of myelopoiesis in the spleen and leukocytosis with a sharp increase of segmented granulocytes in the circulating blood characterized as the "leukemoid reaction" syndrome. The latter occurred even when the tumour cells were transplanted into the splenectomized host. Although less marked, this leukemoid reaction was also noted in advanced hepatoma transplanted to syngeneic male mice F1 (CBA X C57BL/6j). No leukemoid reaction was observed in these mice after grafting syngeneic strain of urinary bladder carcinoma.     

Effect of thymocytes on the quantity of polypotent hematopoietic mast cells in the spleen of sublethally irradiated mice]

With the aid of Till and McCulloch's method, 5 times 10(-6) thymic cells were found to cause an increase in the number of hemopoietic endogenous spleen colonies in syngeneic donor-recipient combination. Thymic cells of C57BL mice had no effect on the number of endogenous colonies in the spleen. 40 times 10(-6) thymic cells administered 24 hr after sublethal irradiation caused an increase in the number of colony forming units in the spleen within 14 days. Possible ways of the thymus effect on hemopoiesis are discussed.     

Experimental Models for Prevention of Graft-versus-Host Reaction in Bone Marrow Transfusion

Induction and suppression of splenomegaly and cytotoxicity against C57BL/6 cells were studied in (AKR × C57BL/6) F1 hybrid adult mice after the transfer of AKR lymphoid and bone marrow cells. 1) Splenomegaly and cytotoxicity were dissociated in the developmental stages of the graft-versus-host reaction. When lymphoid and bone marrow cells of normal AKR mice were injected into F1 recipients, splenomegaly was prominent on days 5 and 7, but cytotoxicity of spleen cells was not detected. Splenomegaly became less prominent but the cytotoxicity became detectable on day 14 after the injection. 2) Cytotoxic activity of spleen cells of F1 recipients was suppressed by the treatment of AKR donors with C57BL/6 lymphoid cells in Freund's complete adjuvant. Splenomegaly, however, was substantially enhanced by such a treatment of the donors. On the other hand, induction of the cytotoxic activity was facilitated by the treatment of donors with C57BL/6 skin grafts. 3) F1 hybrid mice could be protected from the graft-versus-host reaction by the injection of AKR anti-C57BL/6 serum or pretreatment of AKR donors with sonicated cellular antigens of C57BL/6.     

Visualization, fate, and pathogenicity of antigen-specific CD8+ T cells in the graft-versus-host reaction.

To follow the fate of alloreactive T cell effectors in graft-vs-host disease, Ld-specific CD8+ T cells from C57BL/6 2C TCR-transgenic donors were transplanted into sublethally irradiated (750 cGy) Ld+ or Ld- recipients. In Ld- C57BL/6 or (BALB/c-dm2 x C57BL/6)F1 recipients, naive 2C T cells engrafted and survived long term, but did not acquire effector function. In Ld+ (BALB/c x C57BL/6)F1 recipients, 2C T cells engrafted, expanded, became cytolytic, destroyed host B cells and double-positive thymocytes, and later disappeared. Despite marked damage to lymphoid and hemopoietic cells by 2C T cells, no significant pathology was detected in other organs, and recipients survived. Ld+ (BALB/c x C57BL/6)F1 recipients died when LPS/endotoxin was administered on day 7 after cell transfer, while Ld- (BALB/c-dm2 x C57BL/6)F1 recipients survived. Our findings show that under certain conditions, a CD8+ T cell population recognizing an extremely limited repertoire of Ags can initiate graft-vs-host disease.     

Three methods for producing fertile hemopoietic chimeras in mice

Three methods for producing semiallogeneic (F1----parental) hemopoietic chimeras with retained or regained fertility are detailed here. Prenatal (PN) chimeras were produced by injecting F1 ([BALB/c female x C3H/HeJ male] or [CBA/J female x C57BL/6 male]) fetal liver (days 13-18) or adult bone marrow cells (10(6)-10(7) cells/20 microliters/embryo) into the yolk-sac cavities of days 13-17 gestation BALB/c or CBA/J embryos, respectively, and allowing them to be born naturally. Neonatal (NN) chimeras were made by introducing F1 bone marrow cells (1-2 x 10(7) cells/0.25 ml) into newborn (less than 24 hr old) female mice through the anterior facial vein. Female mice were raised to maturity in both cases. Ovary-transplanted (OT) chimeras were made by first irradiating (9.5 Gy) and repopulating young female adult mice with 10(7) F1 bone marrow cells, followed by bilateral orthotopic transplantation of syngeneic ovarian tissue six weeks later. Females reconstituted with the above three methods were mated with normal syngeneic males and sacrificed at 11-16 days of pregnancy to evaluate hemopoietic chimerism. This was determined in all cases by a radioautographic evaluation of the extent of donor H-2 phenotype marker expression on splenic small lymphocytes, after an indirect labelling of single-cell suspensions with monospecific antibody and [125I]protein-A. Results indicate that hemopoietic chimerism was best in the PN group (0.3-78.1%, mean = 27.1); intermediate in the OT group (5.8-38.2%, mean = 18.1); and low in the NN group (0-14%, with one exception, which was 83.6%). Observed fertility was best for BALB/c host PN chimeras.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inheritance of hydronephrosis in the inbred mouse strain DDD

The mode of inheritance of hydronephrosis was investigated by crossing inbred DDD mice having a high incidence of hydronephrosis and C57BL/6 mice having normal kidneys. In the males, incidences of hydronephrosis in F1 animals were intermediate between the two parental strains at a rate of 32.6% in (DDD x C57BL/6)F1 and 23.4% in reciprocal F1. The same tendency was observed in F2 male animals. In BCF1 males, the number of affected mice was higher in (C57BL/6 x DDD) F1 x DDD (72.4%) than in (DDD x C57BL/6)F1 x C57BL/6 (11.1%). A few affected mice were found among the females of hybrids F1, F2 and BCF1. These results suggested that hydronephrosis in the DDD strain of mice was controlled by polygenes, and that male hormones may have some effect on the occurrence of hydronephrosis.     

Major histocompatibility complex-dependent cytotoxic T lymphocyte repertoire and functional avidity contribute to strain-specific disease susceptibility after murine respiratory syncytial virus infection

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.     

Long-term maintenance of multilineal hemopoiesis in collagen gel culture.

We examined the long-term maintenance of multilineal hemopoiesis in a collagen gel culture of mouse bone marrow cells. When cells were inoculated into the gel, stromal cells formed foci that were composed of sinusoidlike capillary structures, fibroblastic cells, adipocytes and macrophages. Many small hemopoietic foci similar to granulocyte-macrophage colonies (CFU-GM) appeared within a week and disappeared after two weeks. Several large hemopoietic foci appeared after two to three weeks of culture, without a second challenge of marrow cells. These large hemopoietic foci were composed mainly of myeloid cells. Megakaryocytes and mast cells were also observed. When erythropoietin (EPO) was added to the culture at the beginning, the erythroid focus appeared after 3 weeks and the number of megakaryocytes was greater than that in the culture without EPO. However, when EPO was added to the cultures after 6 or 12 weeks, erythroid cells appeared after 1 week and the number of megakaryocytes increased. This hemopoiesis lasted more than 6 months.     

Long-term cultures of chicken bone marrow cells

We report an adaptation to cultures of chicken bone marrow cells of the Dexter culture technique for obtaining long-term hemopoiesis in vitro. Cells were seeded in DMEM supplemented with fetal calf serum (20%) and hydrocortisone (10(-6) M) with or without chicken serum (1%). Cultures were incubated at 37 degrees C and fed every 2 weeks. An adherent cell layer composed of macrophages, fibroblasts, and adipocytes became established, over which hemopoietic cells formed foci and were released into the supernatant. Granulocytes and monocytes-macrophages differentiated in a constant proportion until Week 6, whereafter differentiation became progressively restricted to the monocytic lineage. As demonstrated by the generation of colony-forming cells, hemopoiesis was maintained for either 12 or 28 weeks.     

Phenomenon of parental resistance and its genetic regulation]

Lymphocytes of mice F1 (CBA X M523) and F1 (A X M523) transplanted to 1000 R irradiated CBA or A mice responded to the test antigens--SRBC or S. typhi Vi-antigen--by formation of 100--1000 times less antibody forming cells than in syngeneic recipients. An intermediate result is achieved when the lymphoid cells are transplanted to the irradiated M523 mice. Lymphocytes of mice F1 (A X CBA), F1 (CBA X C57Bl/6), or F1 (A X A.CA) developed a similar immune response in the irradiated syngeneic mice and in both parental lines. The ability of parental line M523 to respond to SRBC was the same as in the other lines studied when examined in situ or in adoptive transfer experiments. The stem hemopoietic cells of mice F1 (CBA X M523) develop in the spleen of CBA mice 2--2.5 times less hemopoietic colonies than in the spleen of syngeneic animals. A conclusion was drawn that mutation M523 in CBA mice inhibited the proliferation and differentiation of hemopoietic and lymphoid cells in the irradiated nonsyngeneic recipients.     

Differentiation of germ cells in seminiferous tubules transplanted to testes of germ cell-deficient mice of W/Wv and Sl/Sld genotypes

(WB X C57BL/6)F1-W/Wv (hereafter, WBB6F1-W/Wv) mice and (WC X C57BL/6)F1-Sl/Sld (hereafter, WCB6F1-Sl/Sld) mice are sterile due to the deficient spermatogenesis in the testes. The cause of deficient spermatogenesis in WBB6F1-W/Wv mice is considered to be a defect in germ cells themselves, whereas that in WCB6F1-Sl/Sld mice is considered to be a defect in tissue environment necessary for differentiation of germ cells. Seminiferous tubules isolated from cryptorchid testes of C57BL/6- +/+ mice were transplanted into the testes of WBB6F1-W/Wv and WCB6F1-Sl/Sld mice to clarify that the extratubular environment of these mice was intact or not. Type A spermatogonia in the transplanted tubules normally differentiated into spermatids, suggesting that the extratubular environment is intact in both WBB6F1-W/Wv and WCB6F1-Sl/Sld mice.     

The responses of hemopoietic precursor cells in mice to bacterial cell-wall components

The influence upon differnt cellular and humoral parameters of hemopoiesis of three structurally unrelated, highly purified bacterial cell-wall components (BCWC) was investigated. The spleens of C57BL/6 mice assayed 6 days after the injection of either lipid A or outer-membrane lipoportein, but not murein, showed a marked increase in granulocyte-macrophage, eosinophil, and megakaryocyte progenitor cell levels. The number of pluripotent hemopoietic stem cells (CFU-S) also increased in the spleens of mice treated with either lipid A or lipoprotein. Similar results were obtained following the injection of lipoprotein or lipid A into CBA or C57BL/6.nu mice. Genetically anemic W^f/W^f mice were found to have spontaneously elevated numbers of splenic progenitor cells, which increased further after the injection of lipid A. The proportions of the different splenic progenitor cell types were similar in both untreated and lipid A treated W^f/W^f mice, and in normal littermate controls. When tested in vitro, unfractionated or partially purified post-lipid A serum was found to stimulate the growth of granulocyte-macrophage progenitor cells (GM-CFC), but no detectable stimulation of eosinohphil, megakryocyte, or erythroid progenitor cells was observed. The data suggest that the rise in splenic levels of the different progenitor cells is not mediated by the corresponding types of CSF, but more likely by proliferation and differentiation of CFU-S.     

Experimental models for prevention of graft-versus-host reaction in bone marrow transfusion. I. Selective suppression and augmentation of splenomegaly and cytotoxicity.

Induction and suppression of splenomegaly and cytotoxicity against C57BL/L cells were studied in (AKR X C57BL/6) F1 hybrid adult mice after the transfer of AKR lymphoid and bone marrow cells. 1) Splenomegaly and cytotoxicity were dissociated in the developmental stages of the graft-versus-host reaction. When lymphoid and bone marrow cells of normal AKR mice were injected into F1 recipients, splenomegaly was prominent on days 5 and 7, but cytotoxicity of spleen cells was not detected. Splenomegaly became less prominent but the cytotoxicity became detectable on day 14 after the injection. 2) Cytotoxic activity of spleen cells of F1 recipients was suppressed by the treatment of AKR donors with C57BL/6 lymphoid cells in Freund's complete adjuvant. Splenomegaly, however, was substantially enhanced by such a treatment of the donors. On the other hand, induction of the cytotoxic activity was facilitated by the treatment of donors with C57BL/6 skin grafts. 3) F1 hybrid mice could be protected from the graft-versus-host reaction by the injection of AKR anti-C57BL/6 serum or pretreatment of AKR donors with sonicated cellular antigens of C57BL/6.