Normal Rabbit Bone Marrow Research Articles

FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus

Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on the characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between normal primary rabbit bone marrow (BM) cells and bone marrow cells transformed (BMT) by murine sarcoma virus (MuSV). Primary cells, rather than cell lines, were used for this research because primary cells are similar to normal tissue cells in most of their characteristics. Our results showed dramatic changes in absorbance between the control cells and MuSV124-transformed cells. Various biological markers, such as the phosphate level and the RNA/DNA obtained, based on the analysis of the FTIR-MSP spectra, also displayed significant differences between the control and transformed cells. Preliminary results suggested that the cluster analysis performed on the FTIR-MSP spectra yielded 100% accuracy in classifying both types of cells.

Comparative Pharmacology of Zinc Mesoporphyrin and Tin Mesoporphyrin: Toxic Actions of Zinc Mesoporphyrin on Hematopoiesis and Progenitor Cell Mobilization

The effects of two synthetic heme analogues, zinc mesoporphyrin (ZnMP) and tin mesoporphyrin (SnMP), on in vivo hematopoietic progenitor cell mobilization and in vitro hematopoiesis were examined in rabbit bone marrow. Rabbits received granulocyte colony-stimulating factor (rhG-CSF) for 7 days in order to mobilize increased numbers of erythroid (BFU-E) and myeloid (CFU-GM) progenitors in peripheral blood. Concurrent treatment of rhG-CSF-treated rabbits with ZnMP reduced mobilization of the numbers of BFU-E (76% inhibition, p < 0.0001) and CFU-GM (70% inhibition, p < 0.005) in peripheral blood. In contrast, SnMP administered at the same concentration had no significant suppressive effect on BFU-E and CFU-GM recruitment. Both metalloporphyrins inhibited bone marrow heme oxygenase activity equally in vivo, thus indicating that both compounds enter bone marrow cells. Direct in vitro addition of ZnMP to normal rabbit bone marrow cultures suppressed BFU-E and CFU-GM growth, whereas SnMP had no such effect. These results confirm, in an in vivo system, our earlier in vitro studies and demonstrate that, at the concentrations studied, ZnMP, in contrast to SnMP, displays toxicity for hematopoietic growth and progenitor cell production.

A soluble factor produced by bone marrow natural suppressor cells blocks interleukin 2 production and activity

We previously reported that a population of Fcγ-receptor + (FcγR +) suppressor cells present in normal unstimulated rabbit bone marrow inhibited the growth of autologous rapidly proliferating bone marrow cells devoid of FcγR. It is now reported that the FcγR + bone marrow cells produced a soluble, nondialyzable suppressor factor(s) (SF) which blocked the proliferation of FcγR − bone marrow cells. In addition, the FcγR + cells and SF significantly inhibited spleen cell proliferation in response to concanavalin A (Con A), phytohemagglutinin, and pokeweed mitogen. The bone marrow SF exhibited a dose-dependent suppression of the growth of IL-2-dependent T lymphocytes in the presence of IL-2. SF also completely blocked the production or release of IL-2 by Con A-stimulated T cells. Thus, these bone marrow natural suppressor cells produced a soluble factor, which regulated the growth of rapidly proliferating bone marrow cells and also regulated T cell reactivity by modulating IL-2 production and activity.

Rabbit bone marrow suppressor cells block the production or release of a soluble bone marrow growth factor

Fcγ-receptor (Fc γR)-bearing cells from normal rabbit bone marrow suppressed the constitutive proliferation rates of the remaining, Fc γR −, cells. In the absence of the suppressive influences of Fc γR + cells, cells in the Fc γR − population spontaneously elaborated a soluble growth factor (GF) which induced the proliferation of unseparated bone marrow cells. To examine regulation by Fc γR + bone marrow cells, graded numbers of the Fc γR + cells were mixed with constant numbers of the FcR − cells. At 24 hr, supernates were collected and tested for GF activity. The Fc γR + suppressor cells efficiently and in a dose-dependent fashion blocked GF production or release. The GF was nondialyzable and relatively heat stable. Supernates with GF activity also had colony-stimulating factor activity, but were negative in assays modified from murine interleukin 1 (IL-1) and IL-2 assays. Regulation of GF production or release represents a new function for bone marrow suppressor cells.

Differential activities of rabbit bone marrow suppressor cells.

A population of nonadherent Fc gamma-receptor-bearing (Fc gamma R+) suppressor cells in normal rabbit bone marrow (BM) which inhibited the constitutive proliferation of other BM cells has been previously described. Suppression was by blocking the release of a soluble growth-promoting factor. In the present report, it was found that the Fc gamma R+ BM suppressor cells, which were suppressive to background proliferation, were not suppressive to lymphocyte activation by immune complexes (IC). Adherent BM cells showed suppressive activity toward IC stimulation, but none toward constitutive BM proliferation. IC-induced proliferation was enhanced by the soluble growth factor, but only in the absence of adherent cells. Adherent cells did not affect growth factor enhancement of constitutive proliferation, suggesting that different cell populations were affected. Depletion of the Fc gamma R+ suppressor cells promoted growth of cells which subsequently developed Fc gamma R+, increasing the possibility of interaction with IC, which also preferentially induced Fc gamma R+ cells.

Regulation of constitutive bone marrow cell proliferation by bone marrow suppressor cells.

Bone marrow (BM) cells have previously been shown to suppress specific immune responses of cells from peripheral lymphoid organs. The present report describes a suppressor cell present in normal rabbit BM, which regulated the constitutive proliferation of other BM cells. The suppressor cells were Fc gamma-receptor-positive (Fc gamma R+) complement-receptor-negative, and nonadherent or weakly adherent. Similar suppressive activity was not detected among rabbit spleen cells. Removal of Fc gamma R+ suppressor cells allowed greater than 10-fold increases in the proliferation of Fc gamma R- BM cells. Addition of Fc gamma R+ BM cells to Fc gamma R- cells efficiently blocked proliferation. The suppressor cells acted by inhibiting the elaboration of a soluble growth-promoting factor by cells in the Fc gamma R- population. The growth-promoting factor enhanced proliferation of unseparated rabbit BM cells and peripheral blood mononuclear cells.

Effects of androgens on burst forming units (BFU-E) in normal rabbit bone marrows

The mechanism of action of androgenic steroids on erythropoiesis is not well understood. In order to assess whether the site of action of androgens is on the early erythroid committed stem cell compartment, the in vitro effects of testosterone (T), 5α-dihydrotestosterone (5α-DHT) and 5β-dihydrotestosterone (5β-DHT) on the so-called erythropoietic burst forming unit (BFU-E) in normal rabbit bone marrows were studied. Even though all of the steroids studied increased the number of BFU-E in the presece of Ep, 5β-DHT was the most potent in stimulating BFU-E. Testosterone was moderately effective in increasing BFU-E. Even though 5α-DHT produced a significant increase in BFU-E, it was the least effective of the 3 steroids studied. Preincubation (2 hrs) of normal rabbit bone marrow cells with testosterone followed by removal of T from the culture system resulted in a significant increase in BFU-E when compared with that of non-treated marrow cells in the presence of Ep. These data suggest that testosterone and 5β-DHT and possibly 5α-DHT act on an early uncommitted stem cell, perhaps the CFU-S, to increase the numbers of erythroid committed stem cells to eventually cause an increase in erythropoiesis in combination with Ep.

Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow

The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.

Inhibition of Bone Marrow Erythroid Colony-Forming Cells (CFU-E) by Serum from Chronic Anemic Uremic Rabbits

SummaryThe 5/6th nephrectomized uremic rabbit was demonstrated to develop a marked anemia. Erythropoietic activity in the sera of these chronically uremic anemic rabbits was not detectable. A decrease in the number of erythroid colony-forming cells (CFU-E) was found in chronically anemic uremic rabbit bone marrows and a significant inhibitory effect of the uremic serum on the erythroid colony-forming unit in normal rabbit bone marrow cultures was also seen in the 5/6th nephrectomized rabbit after 35 days. These findings support the hypothesis that an inhibitor of the primary target cell for erythropoietin, the CFU-E, plays an important role in the anemia of chronic renal failure.We acknowledge with thanks the excellent technical assistance of Mr. Jesse Brookins, Mr. Rene Stiaes, and Ms. Patricia Dargon.Human urinary erythropoietin was supplied by the Department of Physiology, University of the Northeast, Corrientes, Argentina. This material was further processed and assayed by the Hematology Research Labo...

Inhibitor of heme synthesis in polycythemic rabbit serum

Sera from hypertransfused polycythemic rabbits were found to significantly inhibit 59Fe incorporation into heme in erythroid cells in normal rabbit bone marrow cultures when compared with that of normal serum controls suggesting a higher concentration of this inhibitor in polycythemic serum. This serum inhibitor delayed the time of peak cumulative heme synthesis in vitro and the delay in peak cumulative heme synthesis was increase with increasing concentrations of polycythemic serum. It is suggested from these studies that this serum inhibitor may be involved in a negative feedback system in the control of erythropoiesis and may act specifically on differentiated nucleated erythroid cells to delay their entry into the cell cycle, consequently inhibiting heme synthesis.

Studies on an inhibitor of erythropoiesis. II. Inhibitory effects of serum from uremic rabbits on heme synthesis in rabbit bone marrow cultures (38483).

The effects of sera from anephric rabbits on the rate of heme synthesis in erythroid cells were studied in vitro in rabbit bone marrow cultures. Sera from nephrectomized rabbits significantly (p smaller than 0.05) inhibited 59-Fe incorporation into heme in normal rabbit bone marrow cultures incubated with erythropoietin (ESF) when compared with the response in control culutres with normal serum and ESF. This inhibitory activity increased exponentially with increasing concentrations of the serum in cultures suggesting that a higher concentration of the inhibitor is present in uremia and may be the result of the failure to excrete the inhibitor. This suggests that the retention of this inhibitor may play an important role in the mechanism of the anemia of uremia. The inhibitor of heme synthesis is a low molecular weight substance.

Studies on an inhibitor of erythropoiesis. I. Effects of sera from normal and polycythemic rabbits on heme synthesis in rabbit bone marrow cultures.

SummaryThe effects of sera from normal and polycythemic rabbits on the rate of heme synthesis in erythroid cells were studied in vitro in rabbit bone marrow cultures. Sera from normal or hypertransfused rabbits significantly inhibited 59Fe incorporation into heme in normal rabbit bone marrow cultures when incubated alone or with erythropoietin (ESF) when compared with the response in controls with newborn calf serum alone or with ESF. A significantly greater inhibitory effect was seen with sera from polycythemic than from normal animals. This inhibitory activity increased exponentially with increasing concentrations of the test sera in the cultures, and was also directly proportional to the increase seen in the hematocrit of the polycythemic rabbits. These results suggest that the inhibitor in both normal and polycythemic sera is tissue-specific and may control the rate of heme synthesis and erythroid cell proliferation. This inhibitor is a low-molecular-weight substance.

Proliferative response of normal rabbit and mouse bone marrow cells in vitro

Bone marrow cells from both normal rabbits and mice are stimulated by exposure to antigens in vitro. Responsiveness is not removed by prior injection of the antigen into the animals. The specificity of this phenomenon is doubtful in view of a lack of additive effects between antigens, unrelatedness to immunogenicity of the antigen, and occurrence in cells from immunologically immature rabbits.

Cells Involved in the Immune Response

Abstract Normal bone marrow antigen-reactive cells could be stimulated to commence the sequence of intracellular reactions characteristic of the afferent limb of the immune response by incubation with antigen in vitro in cell culture. Injection of these in vitro-stimulated bone marrow cells, along with antigen, into irradiated immunoincompetent recipients resulted in a reconstitution of the host's immune competence that was characterized by plaque-forming capacity in the spleen cells 7 days later. Recipients of cells irradiated following in vitro incubation with the antigen gave normal immune responses, whereas irradiation of the cells before in vitro incubation with the antigen completely suppressed the capacity of these cells to transfer immunocompetence. Bone marrow cells irradiated in vitro could exclude the trypan blue dye but failed to undergo blastogenesis and mitosis when cultured in the presence of phytohemagglutinin. Data in support of the clonal selection theory, with respect to the antigen reactive cells (ARC) present in the normal rabbit bone marrow, were also presented. Precommitment of the ARC to one antigen could not be subverted even if the cells were incubated for 24 hr in vitro in the presence of a second non-cross-reacting antigen in high concentration. Therefore, the ARC is unipotential with respect to antigenic specificity.

In vitro induction of specific unresponsiveness of immunologically reactive, normal bone marrow cells.

Normal rabbit bone marrow cells have been studied according to their immunological reactivity in vitro. The test system involved stimulation by antigen after the subsequent stimulation into cellular proliferation by measuring the uptake of tritium-labeled thymidine. Specific separation of immunological reactivity was obtained by filtration of cells through antigen-coated bead columns. All experimental evidence supported the view that this separation was due to the existence of preformed antibody molecules on the outer cell surface of the antigen-recognizing cells. The response to antigenic stimulation was shown to be strictly dose related and, using supraoptimal concentrations of one antigen, no increased DNA synthesis was recorded. That this state of unresponsiveness represented a state of immunological paralysis was indicated by the normal response of these cells to stimulation by a second antigen in optimal concentration. Thus both methods, cell separation on antigen-coated columns or induction of specific unresponsiveness by antigen in vitro, can produce a cell population specifically devoid of cells reactive against a given antigen.

Effect of splenic X-irradiation on bone marrow function: experimental studies of a serum mitotic inhibitor in rabbit and in patients with chronic myelocytic leukaemia.

Summary. The effect of a selective splenic irradiation on bone marrow function was studied in rabbits given a single splenic exposure with 300 r. of X‐rays. Serum collected shortly thereafter decreased mitotic activity in suspensions of normal rabbit bone marrow. This in vitro inhibitory effect was only transiently demonstrable in the serum appearing 2 hours after irradiation and disappearing 3 hours later.Five patients with chronic myelocytic leukaemia in relapse receiving splenic radiotherapy in large single doses of 200–400 r. (surface dose) were studied along these lines. Serum collected 3 hours after X‐ray treatment was tested with suspensions of normal bone marrow and was found to decrease erythroblastic mitotic activity significantly below the control values observed with serum collected before irradiation. The magnitude of this in vitro effect was to some extent related to the spleen size and the radiation dose. No differences were observed between bone marrow suspensions incubated with pre‐irradiation leukaemic serum and normal control serum.

Properties of Deoxyribonuclease III from Mammalian Tissues

A DNA exonuclease, DNase III, has been purified 680-fold from normal rabbit bone marrow. The enzyme has a pH optimum at 8.5, requires Mg++ or Mn++ as a cofactor, and has a molecular weight of 52,000 ± 5,000. It hydrolyzes denatured DNA four times more rapidly than native DNA with no apparent base specificity, but it does not attack RNA. DNA is degraded from the 3′ end with the release of 5′-mononucleotides and dinucleotides. DNase III is localized in the cell nucleus, and it appears to be the predominant DNA exonuclease in many different mammalian tissues.

Cells involved in the immune response. X. The transfer of antibody-forming capacity to irradiated rabbits by antigen-reactive cells isolated from normal allogeneic rabbit bone marrow after passage through antigen-sensitized glass bead columns.

The antigen-reactive cells in normal rabbit bone marrow could be isolated from a suspension of marrow cells by passage of the cells through an antigen-sensitized glass bead column. The cells which passed through the column were deficient in antigen-reactive cells directed to the antigen used to sensitize the glass beads, whereas the cells eluted from the column could transfer antibody-forming capacity to irradiated recipients only with respect to the specific sensitizing antigen. Separation of the bone marrow antigen-reactive cells could not be achieved by passage of the cells through nonsensitized glass bead columns or in the presence of excess free antigen in the column. Cells which were retained by, and later eluted from, the antigen-sensitized glass bead columns were mostly small mononuclear cells, whereas cells which passed through the columns were morphologically similar to the original unfractionated bone marrow cell suspension. The data indicate the presence of an antibody or antibody-like structure, with defined immunological specificity, on the surface of the normal bone marrow antigen-reactive cell.

Cells involved in the immune response. VII. The demonstration, using allotypic markers, of antibody formation by irradiation-resistant cells of irradiated rabbits injected with normal allogeneic bone marrow cells and sheep erythrocytes.

Bone marrow cells obtained from rabbits of one allotype were injected into irradiated rabbits of a different allotype. The recipients were also injected with sheep red blood cells, and their spleen cells were tested for plaque-forming capacity 7 days later. Spleen cells of all recipients gave large numbers of plaques as did spleen cells incubated with antiserum, directed toward donor allotype. However, incubation of the recipient spleen cells with antiserum directed toward recipient allotype completely suppressed plaque formation. These results demonstrate that antibody-formation in irradiated recipients of transferred lymphoid cells is a property of the recipient animal and that the antibody-forming cell is relatively irradiation-resistant. It was also demonstrated that only viable normal bone marrow cells are capable of transferring antibody-forming capacity to irradiated recipient rabbits. Neither sonicates nor heat-killed preparations of normal rabbit bone marrow cells possessed this capacity.

Cells involved in the immune response. VI. The immune response to red blood cells in irradiated rabbits after administration of normal, primed, or immune allogeneic rabbit bone marrow cells.

Irradiated rabbits given allogeneic bone marrow cells from normal adult donors responded to an injection of sheep red blood cells by forming circulating antibodies. Their spleen cells were also capable of forming many plaques using the hemolysis in gel technique, and were also capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine upon exposure to the specific antigen in vitro. However, irradiated rabbits injected with allogeneic bone marrow obtained from rabbits injected with sheep red blood cells 24 hr prior to sacrifice (primed donors) were incapable of mounting an immune response after stimulation with sheep red cells. This loss of reactivity by the bone marrow from primed donors is specific for the antigen injected, since the immune response of the irradiated recipients to a non-cross-reacting antigen, the horse red blood cell, is unimpaired. Treatment of the bone marrow donors with high-titered specific antiserum to sheep red cells for 24 hr prior to sacrifice did not result in any diminished ability of their bone marrow cells to transfer antibody-forming capacity to sheep red blood cells. The significance of these results, with respect to the origin of the antigen-reactive and antibody-forming cells in the rabbit, is discussed.