Hemopoietic Organs Research Articles

Homing and immunogenicity of murine stromal cells transfected with xenogeneic MHC class II genes

Syngeneic (murine) and xenogeneic (canine) marrow-derived stromal cells were injected intravenously into SCID and normal mice to examine the homing pattern and persistence of these cells in vivo. By in situ hybridization, these stromal cells were detectable in the bone marrow cavity and the spleen 21 days after injection. Xenogeneic cells did not persist in normal mice but persisted in SCID mice. Conditioning of the recipients with irradiation or 5-fluorouracil (5-FU) treatment did not alter these results. In addition, syngeneic murine stromal cells were transfected with the genes for canine MHC class II (DRA + DRB) and transplanted into murine recipients to investigate their homing pattern and immunogenicity. These transfected syngeneic stromal cells did also home to marrow and spleen even in normal recipients. However, these cells led to sensitization of the host towards canine antigens as shown by accelerated skin graft rejection and delayed type hypersensitivity (DTH). Thus, immunodeficient (SCID) mice allow for the homing of xenogeneic stromal cells to hemopoietic organs and for prolonged persistence. In immunocompetent (normal) mice, no xenogeneic stromal cells were identified in spleen and marrow, either because of their inability to home or more likely because of immunological rejection. In contrast, syngeneic stromal cells expressing xenogeneic MHC class II genes did home to spleen and marrow and persisted even though the recipient had become sensitized. Their survival may be due to a loss of expression of the transfected gene. Alternatively, the presentation of these xenogeneic gene products in the hemopoietic organs was such that a cytotoxic response was not induced.(ABSTRACT TRUNCATED AT 250 WORDS)

Homing and immunogenicity of murine stromal cells transfected with xenogeneic MHC class II genes.

Syngeneic (murine) and xenogeneic (canine) marrow-derived stromal cells were injected intravenously into SCID and normal mice to examine the homing pattern and persistence of these cells in vivo. By in situ hybridization, these stromal cells were detectable in the bone marrow cavity and the spleen 21 days after injection. Xenogeneic cells did not persist in normal mice but persisted in SCID mice. Conditioning of the recipients with irradiation or 5-fluorouracil (5-FU) treatment did not alter these results. In addition, syngeneic murine stromal cells were transfected with the genes for canine MHC class II (DRA + DRB) and transplanted into murine recipients to investigate their homing pattern and immunogenicity. These transfected syngeneic stromal cells did also home to marrow and spleen even in normal recipients. However, these cells led to sensitization of the host towards canine antigens as shown by accelerated skin graft rejection and delayed type hypersensitivity (DTH). Thus, immunodeficient (SCID) mice allow for the homing of xenogeneic stromal cells to hemopoietic organs and for prolonged persistence. In immunocompetent (normal) mice, no xenogeneic stromal cells were identified in spleen and marrow, either because of their inability to home or more likely because of immunological rejection. In contrast, syngeneic stromal cells expressing xenogeneic MHC class II genes did home to spleen and marrow and persisted even though the recipient had become sensitized. Their survival may be due to a loss of expression of the transfected gene. Alternatively, the presentation of these xenogeneic gene products in the hemopoietic organs was such that a cytotoxic response was not induced.(ABSTRACT TRUNCATED AT 250 WORDS)

Induced tolerance and chimaerism in human fetuses using coelocentesis: a medical opportunity to avert genetic disease?

Coelocentesis offers a new opportunity for gaining access to the coelomic cavity of human embryos from 28 days post-fertilization (42 days menstrual age). With this technique, cells can be extracted from the cavity for the genetic typing of embryos in early pregnancy. Coelocentesis may also offer a unique opportunity of inducing tolerance to foreign grafts and chimaerism in these human embryos by replacing donor cells into the coelomic cavity. This cavity appears to be closely associated with the fetal haemopoietic system. The optimal age to inject stem cells designed to produce chimaerism may be at 5-6 weeks embryonic age, and these grafted cells may induce tolerance later in gestation. Two successive coelocenteses would be needed, the first to extract fetal cells to type the fetus, and a second within a few days to inject the donor cells into the coelomic cavity. Alternatively, non-invasive methods of diagnosis such as lower uterine pole extramembranous sampling of fetal trophoblast, or the extraction of fetal cells from maternal blood, could be combined with coelocentesis. If tolerance and chimaerism can be established, repeated tissue grafts could be carried out during fetal life and after birth, so that disorders caused by single or multiple gene defects in the haemopoietic system and other organs may be corrected.

Secreted and Membrane-Bound Isoforms of T1, an Orphan Receptor Related to IL-1-Binding Proteins, Are Differently Expressed in Vivo

The murine T1 gene encodes a membrane-bound glycoprotein (T1-M), highly similar to interleukin-1 (IL-1) receptor type I, and a soluble variant (T1-S) representing its isolated extracellular domain. In vivo, the expression pattern of both T1 isoforms differs drastically. The T1-M receptor is abundantly expressed in single cells of the major hemopoietic organs (embryonic liver, spleen, bone marrow). It is restricted to few hemopoietic cell types throughout ontogenesis. By contrast, the soluble T1-S protein is predominantly expressed in selected nonhemopoietic embryonic tissues (developing skin, bone, and retina) and deposited in extracellular matrix. Despite the similarity of the T1 ligand-binding domain to all IL-1-binding proteins, it does not exhibit affinity to either IL-1α or -β. Thus, T1-M likely represents a novel orphan receptor of selected hemopoietic cells. The matrix-associated T1-S variant might act to create a reservoir of the putative T1 ligand in some differentiating tissues.

Immortalized hemopoietic cells with stem cell properties

We have established a hemopoietic cell line, BL3, that possesses a rearranged retroviral genome, which we used as a genetic tag for their engraftment into lethally irradiated mice. Analysis of recipients up to 7 months after engraftment indicates that the marker was present in differentiated cells of various hemopoietic organs, in colony-forming cells, pre-CFU-S-forming cells, and in organs of secondary recipients from bone marrow cells of primary recipients. BL3 cells are Thy-1 +, Sca-1 +, B220 −, Mac-1 -, and Gr-1 −. They express GATA-1 and are able to develop “cobblestones” with stromal cells. They do not express and respond to several hemopoietic growth factors known to facilitate marrow recovery. However, they are negatively regulated by TGFO and can be stimulated by mitogenstimulated spleen cell-conditioned medium. We conclude that BL3 cells possess properties of hemopoietic stem cells, including their capability to contribute to long-term repopulation.

Diseases of blood and hemopoietic organs in persons involved in the accident elimination at the Chernobly Nuclear Power Station

The sickness rate of blood and hemopoietic organs of eliminators of the accident at the Chernobyl nuclear station is estimated. The necessary information is obtained from the data base of the Branch record. The general sickness rate of this type of diseases is 2,860,3 to 1000 observed persons. In women anemias are 6 times more frequent. The malignant tumors of hemopoietic and lymphatic tissue are revealed in 6 patients. The dependence on the irradiation dose is not established.

Inhibition of in vitro erythropoiesis by soluble mediators in Plasmodium chabaudi AS malaria: lack of a major role for interleukin 1, tumor necrosis factor alpha, and gamma interferon

By using erythropoietin-dependent proliferation of splenic erythroid cells as an in vitro erythropoiesis model system, we demonstrate that spleen cells from Plasmodium chabaudi AS-infected C57BL/6 mice potently inhibited erythroid cell proliferation. Inhibitory activity was detected in spleen cell conditioned media (SPCM) prepared from infected mice but not from uninfected mice. The inhibitory activity in SPCM was characterized as being heat sensitive, macromolecular, and host derived. The inhibitory activity was not reversed by increasing the erythropoietin concentration and was found to be specific for the late erythroid lineage. Mouse strains, which differ in their resistance to P. chabaudi AS infection, produced and responded to the inhibitory activity to a similar extent. Putative immune mediators, interleukin 1 alpha, interleukin 1 beta, and gamma interferon, were found to be potent inhibitors of erythroid cell proliferation. However, antibody neutralization experiments failed to demonstrate a major role for these cytokines in the inhibitory activity of SPCM. Our results suggest that the elaboration of inhibitor(s) of erythropoiesis in hemopoietic organs of Plasmodium-infected mice may impair erythroid regeneration. The identity of the inhibitory mediator(s) is presently unknown but is distinct from interleukin 1, tumor necrosis factor alpha, and gamma interferon.

Characterisation of immunoglobulin-binding leucocytes in carp ( Cyprinus carpio L.)

This study demonstrates the immunoglobulin(Ig)-binding capacity of Ig-positive carp macrophages employing immunofluorescence and immunogold methods. These methods allow for the characterisation of the Ig-binding cells. After internalisation of fluorescent- or gold-labelled Ig (30 min at room temperature), most macrophages from the hindgut were able to bind added purified carp Ig, which could be demonstrated clearly with a second fluorescent or gold label. In pronephros, an important haemopoietic organ in fish, a limited number of monocyte-like cells also showed Ig binding. Pronephros macrophages and neutrophilic granulocytes appeared to be Ig-negative. The use of goat anti-mouse Ig gold particles bound by carp anti-goat antibodies revealed that, in addition to hindgut macrophages and pronephric monocyte-like cells, some lymphoid cells in both hindgut and pronephros cell suspensions were also able to bind Ig. The classic erythrocyte-antibody rosette assay resulted in a limited number of small rosettes in cell suspensions from both organs.

Establishment of a hepatocytic epithelial cell line from the murine fetal liver capable of promoting hemopoietic cell proliferation.

Although the fetal liver has been thought to be the main hemopoietic organ in the embryonal period, whether or not hepatocytes play a major role in hemopoiesis remains obscure. We have established an epithelial cell line from the murine fetal liver, which can support hemopoiesis in vitro. The proliferation of the epithelial cells was promoted synergistically by both epidermal growth factor (EGF) and insulin. The cells were identified as epithelial cells by the presence of desmosomes and tight junctions. Cytoplasmic organelles including small mitochondria and dilated Golgi apparati as well as intercellular canalicular structures similar to bile canaliculus also helped in confirming the hepatic origin of the cell line (designated as FHC). The cells in the primary culture were positive for both alpha-fetoprotein and albumin, indicating the hepatocytic nature of the cell line. Cloned FHC cells were demonstrated to have the ability to maintain hemopoietic progenitors in fetal liver and adult bone marrow in the coculture, and among them, FHC-4D2 clone displayed the greatest activity. Hemopoiesis-supporting function could also be seen even when bone marrow cells were separated from FHC-4D2 cells by nitrocellulose membrane. Column chromatography revealed three distinct peaks of hemopoietic activities with different molecular sizes in the supernatant of FHC-4D2. Neutralization test with antibodies and proliferative response to interleukin-3 (IL-3)/granulocyte-macrophage colony stimulating factor (GM-CSF)-responding IC2 cells demonstrated that the hemopoietic activities were attributed to GM-CSF and macrophage colony stimulating factor (M-CSF). Transcripts of GM-CSF and M-CSF were readily detectable in Northern blot analysis, whereas no messages for IL-3, IL-6, CSF for granulocytes (G-CSF) or erythropoietin (EPO) were identified. Therefore, this is the first report on the fetal hepatocyte cell line capable of supporting hemopoiesis.

Murine granulocyte-macrophage and mast cell colony formation promoted by nerve growth factor.

The nerve growth factor (NGF) is widely distributed in the target tissues of sympathetic neurons. Hemopoietic organs such as the bone marrow (BM) and spleens are known to be innervated by noradrenergic sympathetic neurons. Some of their constitutive cells express NGF receptors (lymphocytes and stroma cells) and its biologic effects have been extensively studied in the immune system and inflammatory responses. However, the effects of NGF on hemopoiesis have been little examined. Recently, we demonstrated that NGF promoted mast cell colony formation from murine BM cells (BMC) or BMC-derived cultured mast cells and induced the phenotypic changes in standard hemopoietic assays. Besides, we demonstrated NGF-enhanced murine neutrophil survival and functional properties. In this study, in order to investigate NGF activities on neutrophil differentiation, we examined granulocyte-macrophage (GM) colony formation from murine BMC or spleen cells (SC) in the methylcellulose culture. We also assessed mast cell colony formation. GM colonies were counted on day 5 and mast cell colonies were counted on day 20 in culture. Although NGF alone (50 ng/ml) neither supported GM nor mast cell colony formation, addition of various doses of NGF to the suboptimal dose of pokeweed mitogen-stimulated SC-conditioned medium (2.5%) or interleukin 3 (50 U/ml), well-known colony-stimulating factors, increased the number of GM and mast cell colonies from both BMC and SC in a dose-dependent manner. These colony formation-enhancing effects of NGF were inhibited by the addition of neutralizing sheep anti-NGF antibodies. The results suggest that NGF may act to develop granulopoiesis including neutrophil and mast cell differentiation in cooperation with hemopoietic factor(s) during inflammatory processes.

Alpha-irradiation of haemopoietic tissue in pre- and postnatal mice: 2. Effects of mid-term contamination with 239Pu in utero.

The distribution of 239Pu in various tissues of foetal and postnatal offspring of pregnant mice, injected i.v. at 13 days gestation with 30 kBq 239Pu/kg (in some cases with 10 or 100 kBq/kg), together with the numbers of haemopoietic progenitors in the bone marrow, spleen and liver, were measured through to 1 year post-partum. The quality of the haemopoietic microenvironment in these mice was also measured using the renal-capsule implant method. The largest radiation dose received by any haemopoietic organ was that in the liver, amounting to 10-14 mGy, as reported previously. In spite of normal numbers of haemopoietic spleen colony-forming cells (CFC-S) in the liver and seeding, at birth, into the bone marrow where the level of plutonium was minimal, a long-term deficit in their number rapidly developed. The development of the stromal microenvironment, however, was also deficient, suggesting that the dose of alpha-irradiation to the foetal liver was sufficient to cause sublethal damage in those cells destined to become the precursors of the supportive haemopoietic microenvironment in bone marrow and spleen. The results of this study suggest that although the placenta affords significant shielding to the tissues of the developing foetus from maternal contamination, the long-term effects on haemopoiesis are comparable to those in mice contaminated as adults. This further implies that the developing haemopoietic tissues are exquisitely sensitive to 239Pu contamination.

Histiocytic cytophagic panniculitis: Report of a case in a 12-year-old girl

We report a 12-year-old girl with nodular skin lesions, fever, pancytopenia, coagulation defects and a fatal outcome within 4 months. Histopathology revealed proliferation of histiocytes with haemophagocytosis in skin, perivisceral fat and haemopoietic organs. The symptoms were most consistent with cytophagic histiocytic panniculitis.

Expression of a divalent cation-dependent erythroblast adhesion receptor by stromal macrophages from murine bone marrow

Stromal macrophages in haemopoietic organs express novel surface receptors that are implicated in trophic interactions with developing blood cells. Macrophages isolated from foetal liver bind erythroblasts (Eb) by a divalent cation-dependent receptor (EbR), whereas stromal macrophages in adult bone marrow and lymphoid organs express a lectin-like receptor, sialoadhesin, which interacts with sialylated structures on sheep erythrocytes and murine haemopoietic cells. In order to learn more about the regulation of these haemagglutinins, we examined binding of Eb by stromal macrophages that had been isolated from adult murine tissues or generated in Dexter-type cultures of bone marrow. Macrophages were purified from bone marrow by collagenase digestion, adherence to a substratum and detachment of clustered haemopoietic cells, and tested for their ability to bind Eb from foetal liver or anaemic adult spleen. Freshly isolated bone marrow macrophages bound Eb mainly by a divalent cation-dependent activity that was not inhibited by neuraminidase treatment of Eb or by specific anti-sialoadhesin monoclonal antibodies, although these macrophages express sialoadhesin and Eb bear a potential ligand for this receptor. Macrophages obtained by digestion from other adult lymphoid tissues also bound Eb by a divalent cation-dependent activity, whereas blood monocytes and lavaged peritoneal macrophages failed to do so. Peritoneal macrophages could be induced to express high levels of sialoadhesin by cultivation in homologous mouse serum, but such macrophages did not acquire sialoadhesin-independent EbR activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Idiopathic glomerulopathy in a young ICR mouse

Idiopathic glomerulopathy of the kidneys was found in a female CRJ: CD-1 (ICR) mouse which died at the age of 33 days. The glomerular lesions were characterized by hyalinization of the glomeruli. There was thickening of the glomerular capillary walls and enlargement of the mesangial areas due to deposits of PAS-positive hyaline material. The hyaline material was distinguished from amyloid, fibrin, acid mucopolysaccharide and immunoglobulins by histochemical and immunohistochemical methods. The basement membrane of the glomerular capillaries was partially thickened, double-contoured and discontinuous. Neither cellular proliferation nor inflammatory cell infiltration was observed in the glomeruli. Ultrastructurally, low electron-dense, amorphous deposits were present in the subendothelial regions and basement membrane of the glomerular capillaries as well as in the mesangium. Atrophy of haemopoietic organs was accompanied by the above lesions.

Ontogenic Appearance of MHC Class I (B‐F) Antigens During Chicken Embryogenesis

Expression of chicken MHC class (B-F) antigens during ontogeny was determined by binding of anticlass antibody and appearance of B-F transcripts by Northern blotting in chicken organs during embryogenesis until 2 weeks after hatching. MHC class transcripts first become detectable in day 6.5 of embryogenesis. B-F cell-surface expression first becomes detectable in hemopoietic organs by day 10‑12 of embryogenesis and somewhat later in nonhemopoietic organs. Flow cytometry analysis of hemopoietic cells throughout embryogenesis revealed B-Fhi and B-F1 cell populations. The percentage of B-F cells in spleen and bone marrow decreased around hatching, which could reflect either cell flows in these organs during this period or the sensitivity of hemopoietic cells to hatching stress.

Effects of <i>Toxocara canis </i>Infection on Hemopoietic Stem Cells and Hemopoietic Factors in Mice

The effects of Toxocara canis infection on hemopoietic stem cells and hemopoietic factors were examined in mice. Severe eosinophilia was observed with a peak 14 days after infection. When the numbers of hemopoietic stem cells in peripheral blood, spleen, and bone marrow were examined by spleen colony assay (CFU-S), those in peripheral blood and spleen increased in parallel with peripheral blood eosinophilia. On the other hand, CFU-S in bone marrow did not alter significantly throughout the course of infection. Interleukin (IL)-3, which is known as multi-colony-stimulating factor and is involved in the growth/differentiation of various blood cells including stem cells, was produced by spleen cells of infected mice. The time course study showed that concanavalin A stimulated IL-3 production peaked on day 7 after infection, whereas that with excretory secretory antigen peaked on day 14. Even without stimulation, spleen cells obtained on day 21 after infection produced IL-3 spontaneously. IL-5, which is known to have eosinophil differentiation factor activity, was also produced by spleen cells obtained on day 13 after infection. These results suggest that in response to increased demand for eosinophils, hemopoietic stem cells migrate into various extramedullar hemopoietic organs where they grow/differentiate into mature eosinophils, depending on the hemopoietic factors.

Extramedullary eosinophilopoiesis in the liver ofSchistosoma japonicum-infected mice, with reference to hemopoietic stem cells

Extramedullary hemopoiesis recognized as hemopoietic foci increased in the liver of Schistosoma japonicum-infected mice in parallel with kinetic changes in periovular granuloma formation. At the peak of the response, about 65% of the hepatic hemopoietic foci were of eosinophil lineage. When S. japonicum-infected mice were irradiated, hepatic hemopoietic foci rapidly disappeared within 3 days, whereas inflammatory cells in the periovular granulomas slowly reduced in number. When the number of hemopoietic stem cells in the liver were examined by spleen-colony assay, kinetic changes in the number of hemopoietic stem cells in hepatic nonparenchymal cells paralleled those of hepatic hemopoietic foci. Hemopoietic stem cells were rare in the granuloma cells. These results indicate that in response to the increased demand for eosinophils and other inflammatory cells, the liver acts as an extramedullary hemopoietic organ in which inflammatory cells are generated from hemopoietic stem cells.

Measurement of Homing Receptors on the Surface of Leukemic and Nonleukemic Cell Lines

Selective homing of hemopoietic progenitor cells to hemopoietic organs is the initial event in hemopoiesis. At a molecular level, it is mediated by a membrane receptor on the surface of hemopoietic progenitor cells. The receptor molecule then binds selectively to a glycoconjugate on the surface of bone marrow stromal cells. The molecular nature of this receptor has been shown previously to be a lectin with specificity for an as yet unknown configuration of galactosyl and mannosyl residues of the glycoconjugate. Normal murine progenitor cell lines, FDCP-1 and B6SUT, were found to possess this homing receptor on their cell membrane, and they also bind well to hemopoietic stroma. However, when three human leukemic cell lines, U937, HL60 and K562, were probed for the presence of this receptor, they were found to be deficient in the homing protein. Further, these cells showed little ability to bind the hemopoietic stromal cell line, GB1/6. These findings suggest that leukemic cells experience a loss, or at least alteration, of homing receptor molecule during leukemic transformation. This can result in a diminished ability to bind to marrow stroma. The lack of homing function may be of pathogenetic significance in the lack of differentiation of leukemic cells and may also contribute to the dissemination of leukemic cells in the circulation.

Factors influencing the time and site of leukemic transformation of factor-dependent cells after injection into irradiated recipient mice.

After cells of the non-tumorigenic-factor-dependent line FDC-PI are injected into irradiated DBA/2 mice a progressive increase occurs in the number of engrafted FDC-PI cells and eventually leukemic transformation occurs. Step-wise increase in the number of cells injected led to an increasingly rapid accumulation of untransformed FDC-PI cells in the hemopoietic organs and some shortening of the pre-leukemic phase. FDC-PI cells explanted from pre-leukemic mice differed from primary FDC-PI cells in that they were able to undergo leukemic transformation in non-irradiated recipients after short latent periods. Pre-leukemic populations contained FDC-PI variants with an improved ability to proliferate in non-irradiated tissues. Co-injection of normal marrow cells delayed the leukemic transformation of injected FDC-PI cells. The accelerating effect of prior irradiation of the recipient on leukemia development was also abrogated when the injection of FDC-PI cells was delayed by several weeks. No specific site of transformation could be determined in mice with very early leukemias. Proliferation of untransformed FDC-PI cells and the emergence of variants with improved adaptation to in vivo conditions appear to be important and possibly necessary steps in the pathogenesis of the disease. Whether the host contributes actively to the final transformation process remains speculative.

Ontogeny and tissue distribution of leukocyte-common antigen bearing cells during early development of Xenopus laevis

To analyze the ontogenic emergence of leukocytes during early development, a mouse monoclonal antibody (IgG1), designated as XL-1, was produced against the peritoneal macrophages of adult Xenopus laevis. The XL-1 determinant was expressed on all types of leukocytes, including lymphocytes, granulocytes, thrombocytes and macrophages, but not on erythrocytes of either larvae or adults. Immunohistochemical observations of the hemopoietic organs revealed that the XL-1+ cells with granulocyte and/or macrophage morphology appeared at st.36-37 in the liver, at st.44-45 in the mesonephric and the thymus rudiments, and at st.47 in the spleen. The XL-1 determinant was expressed on the precursor cells of T lymphocytes in the thymus rudiments at st.46-47, on the pre-B cells in the liver rudiments at st.47, and on lymphocytes in the spleen at st.48-49. A few XL-1+ cells were present in the ventral blood island of the st.35/36 embryos, where differentiating erythrocytes had predominated since st.28. XL-1+ cells with a macrophage-like morphology were found in several locations of the mesenchyme in the st.32 embryos, before the establishment of vascularization at st.33/34 and far earlier than the emergence of lymphocytes.