运动诱导的低铁状态大鼠骨髓细胞铁摄入的变化

本文观察了运动性低铁状态大鼠骨髓细胞转铁蛋白 (Tf)结合铁和非Tf结合铁摄入的变化。大鼠随机分为 6个月的运动组 (EG)和对照组 (SG)。SG平均每个幼红细胞Tf受体数为 890 15 0± 16 4849个 ,而在EG为 2 17536 0± 46 2 737个 (P <0 0 5 ) ,但受体的解离常数不受运动影响。EG中Tf的内吞平台和胞内铁聚积速度显著高于SG ,胞浆和胞内膜性成分中Tf结合铁和Fe(Ⅱ )摄入增加。EG的胞浆内Fe(Ⅱ )摄入的米氏常数值降低 ;细胞膜性成分中Fe(Ⅱ )摄入的最大速度增加。上述结果表明 ,运动不仅通过增加Tf受体的表达促进Tf结合铁的内吞 ,而且增强非Tf结合铁的内吞途径。尽管这些变化的机制尚不清楚 ,但它们有利于运动时血红素的合成     

Changes of transferrin-free iron uptake by bone marrow erythroblasts in strenuously exercised rats

The effects of strenuous exercise on transferrin-free iron (Fe II) uptake by bone marrow erythroblasts in rats were investigated. Female Sprague-Dawley rats were randomly assigned to one of six groups, three of which underwent 3, 6, or 12 months of strenuous exercise (swimming 2 hr/day, 5 days/week) or their corresponding three control groups. At the end of experiments, bone marrow erythroblasts were isolated for Fe II uptake assay in vitro. It was found that the amounts of iron uptake into cytosole and stroma of the cells of rats in the groups undergoing 3 and 12 months of exercise did not differ from those in their corresponding sedentary groups. In addition, analysis of nonspecific and specific Fe II uptake by cytosole and stroma did not display any significant difference between the exercise and corresponding sedentary groups. However, the amount of Fe II uptake into cytosole and stroma was significantly increased in rats that exercised for 6 months compared with the corresponding controls. Nonspecific iron uptake into stroma was significantly higher in the exercise group than in the sedentary group (0.120 +/- 0.018 vs. 0.049 +/- 0.006 pM/10(6) cells, P < 0.01). The V(max) of the specific iron uptake into stroma was significantly higher (0.326 +/- 0.024 vs. 0.238 +/- 0.037 pM/10(6) cells, P < 0.05) and the K(m) of iron uptake into cytosol lower (0.08 +/- 0.01 vs. 0.21 +/- 0.03 microM, P < 0.001) in the exercise groups than in the control groups. These results indicate that 6 months of strenuous exercise could significantly increase Fe II uptake by the cells, probably by affecting the number and/or affinity of the putative iron carrier and the permeability to iron of cell membrane. The increased ability of cell-free iron accumulation in exercise might be a self-protective mechanism for body cells from the free iron-induced free radical reaction in addition to providing more iron for cell heme synthesis.     

Hemopoiesis in long-term stroma-dependent cultures from lymphoid tissue: Production of cells with myeloid/dendritic characteristics

Summary A long-term stroma-dependent culture system (LTC) has been developed which continuously produces hemopoietic cells providing an in vitro system for the study of cell differentiation. These nonadherent cell populations contain a large subpopulation of dendritic cells (DC). LTC producing DC were easily generated from spleen, but could also be established from bone marrow (BM) and lymph node with less success. It was difficult to establish DC-producing LTC from thymus. The properties of splenic and thymic stroma have been compared. Spleen stroma developed more complicated networks of fibroblasts, endothelial cells, macrophages, and DC. Thymic stromal monolayers were dominated by epithelial cells and fibroblasts, with a lower proportion of macrophages and endothelial cells. They had a relatively sparse structure of cell networks compared with spleen stroma. Cells with dendritiform morphology first appeared in cultures by 2–3 wk. The majority of cells produced were large cells which expressed DC-specific cell surface markers, major histocompatibility complex (MHC) Class II molecules, and the CD80/CD86(B7) costimulator. A high proportion of cells also expressed myeloid cell markers. No T or B lymphoid cells or granulocytes were present in the cultures. LTC continued to produce nonadherent cells resembling myeloid/DC for long periods, even after passage of stromal cells and stem cells at about 3–4 mo. after culture establishment. The LTC system offers potential to study the in vitro differentiation of myeloid/DC.     

Endothelial cells create a hematopoietic inductive microenvironment preferential to erythropoiesis in the mouse spleen

The spleen is an erythropoietic organ in mouse. To reconstruct a microenvironment essential for erythropoiesis in vitro, the stroma (MSS31) cell line had been established from a newborn mouse spleens. MSS31 cells exhibited properties of endothelial cells: (a) the cells showed the activity to uptake acetylated low-density lipoprotein (Ac-LDL) and (b) the cells can form a capillarylike structure by a phenotypic modulation in collagen matrices. MSS31 cells selectively supported the proliferation and differentiation of the erythroid progenitor cells by direct cell-to-cell contact in a semisolid medium in the presence of erythropoietin. These layers also supported erythrocyte maturation and enucleation of erythroblasts. This suggests that spleen endothelial cells are a new type of stromal cell with erythropoietic stimulation activity and may have a critical function in the hemopoietic inductive microenvironment of the mouse spleen.     

Functional characterization of two stromal cell lines that support B lymphopoiesis.

Bone marrow stromal cells have well documented effects on the production of B lymphocytes, but whether or not stromal cell signals are involved in the pre-B to B cell transition is unclear. The potential of two stromal cell lines, S10 and S17, in this process was examined. Initial experiments, using a short term liquid culture, indicated that S10 and S17 stroma efficiently supported the generation of clonable B cells (B lymphocyte CFU) from their immediate precursors in fresh bone marrow. The contribution of macrophages and other accessory cells in those experiments was minimized through use of a colony assay system that permits the direct effects of stromal cell signals on single B cell progenitors to be evaluated. The results indicated that soluble mediators from the S10 and S17 lines could support colony formation from fresh or cultured surface Ig- bone marrow cells. Colonies supported by S17 stroma appeared on day 15 and contained cells that expressed the B220 Ag; surface IgM expression was never observed. S10 supported colonies appeared on day 7 and routinely included surface IgM+ cells. Individual colonies were capable of undergoing additional growth when picked and replated directly onto the different stroma. Those colonies replated onto S10 stroma generated surface IgM expressing cells in up to 60% of experiments, but colonies transferred onto the S17 cell line included B cells only 10% of the time. These data demonstrate that stromal cells alone can provide the signals necessary for generating a surface IgM+ B cell from precursors but that not all stromal cell lines are equally efficient at doing so.     

The cellular composition of hemopoietic spleen colonies

The cellular composition of individual hemopoietic spleen colonies has been studied using techniques which tested primarily for cell function rather than cell morphology. Erythroblastic cells were recognized by their capacity to incorporate radioiron, granulocytic cells by their content of peroxidase-positive material, and hemopoietic stem cells by their ability to form spleen colonies in irradiated hosts. It was found that, 14 days after the initiation of spleen colonies, the distribution of these cell types among individual colonies was very heterogeneous, but that most colonies contained detectable numbers of erythroblasts, granulocytes and colony-forming cells. An appreciable proportion of the cells in the colonies could not be identified as any of these three cell types. No strong correlations between numbers of erythroblasts, granulocytes and colony-forming cells in individual colonies were observed, though there was a tendency for colonies containing a high proportion of erythroblasts to contain a low proportion of granulocytes, and for colonies containing a high proportion of granulocytes to contain a higher proportion of colony-forming cells. An analysis of colonies which contained cells bearing radiation-induced chromosomal markers indicated that 83–98% of the dividing cells within 14-day spleen colonies were derived from single precursors.     

Intermediate filament protein as a marker of uterine stromal cell decidualization

An increase in intermediate filaments has been reported in rat uterine stromal cells undergoing decidualization in vivo and in vitro. In order to identify biochemical correlates of this morphological change, we have identified (two dimensional gel electrophoresis, Western blots, indirect immunofluorescent staining) the constitutive intermediate filament proteins of stromal cells decidualizing in vivo and isolated stroma decidualizing in vitro as vimentin and desmin. Vimentin is common to all uterine stromal cells but increases, proportional to total cell protein, in decidualized stroma. Barely detectable in nondecidualized stroma, desmin, unlike vimentin, increases during decidualization at a rate greater than the increase in total cell protein. Neither the increase in vimentin or desmin is observed in hormonally sensitized, nondecidual stromal cells. Desmin, because it is selectively expressed in decidualizing stroma, could be considered unique enough to serve as a marker of decidual cell differentiation.     

Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.     

An evidence of stromal cell populations functionally linked with epithelial cell populations in the mouse oviduct

The oviductal epithelium consists of two major cell populations, secretory cells and cilial cells. In a previous report, we established clonal cell lines from the epithelium and stroma of an oviduct which allowed us to analyze stromal contribution to epithelial functions. Three stromal cell lines were co-cultured in separated apparatus with 3 epithelial cell lines, respectively. Two stromal cell lines preferentially stimulated mogp-1 expression on secretory cells and the stimulation was additive with estrogen. The lines had no effect on cilial cells. One stromal cell line preferentially stimulated foxj1 expression on cilial cells and the stimulation relieved suppression by estrogen. The line had no effect on secretory cells. Experiments with conditioned media of the stromal cells confirmed the results of co-culture experiments, suggesting that the oviductal stroma contains multiple cell populations preferentially regulating or modulating specific cell populations of the epithelium via diffusible factors.     

Stromal cells and extracellular matrix components in spontaneous canine transmissible venereal tumour at different stages of growth

Stromal cells and extracellular matrix (ECM) components are important for tumour cell behaviour. Little is known about the role of stromal cells and ECM components in the progression and regression of spontaneous canine transmissible venereal tumour (CTVT). In this study, the stromal cell type was determined by immunohistochemical labelling with antibodies to desmin, vimentin and alpha-smooth muscle actin (alpha-SMA) during the progressive and regressive stages of spontaneous CTVT. The distribution of ECM components tenascin-C, chondroitin sulphate and versican were determined immunohistochemically, and hyaluronan distribution was determined using a biotinylated protein complex with specific affinity for hyaluronan. Stromal cells of tumours in both the progressive and regressive stage were positive for vimentin and negative for desmin. The number of stromal cells expressing alpha-SMA was significantly higher (P=0.001) in regressing tumours, than progressing tumours. These results suggest that the modulation of stromal cells that occurs during the regression of CTVT is similar to that occurring during wound healing. Tenascin-C was weakly expressed in the stroma of tumours in the progressive stage and in regions of the regressing tumours with tumour infiltrating lymphocytes (TILs), but intensely expressed in the stroma of tumours in late regressive stage. In addition, tenascin-C was also expressed in the cytoplasm of some tumour cells in the late regressive stage. A strong stromal tenascin-C intensity was significantly associated with regressing tumours (P=0.001). Strong stromal hyaluronan intensity and a high proportion of hyaluronan-positive tumour cells were significantly associated with progressing tumours (P=0.001). This suggests that hyaluronan is involved in the growth of the tumour. There was no significant difference in the expression of chondroitin sulphate and versican in progressing and regressing tumours.     

Macrophage-associated erythropoiesis and lymphocytopoiesis in mouse fetal liver: ultrastructural and ISH analysis

To elucidate the process of fetal liver hematopoiesis, the relationships between stroma and hematopoietic cells involved in maturation were investigated. Cultured mouse fetal liver explants were established for morphological analysis of the interactions between fetal liver stroma and hematopoietic cells ex vivo. Fetal liver stroma comprised epithelial cells and macrophages, which occupied most of the culture surface. Macrophages proliferated extensively in primary culture, but almost disappeared after 3 passages. Close morphological and functional relationships were established between macrophages and hemopoietic cells, whereas epithelial cells did not interact with blood cells. Scanning electron microscopy revealed that macrophages were in close contact with erythroblasts and formed a three-dimensional network. In each erythroblastic island, 2-3 lymphocytes were also in contact with the macrophages; erythroblasts, lymphocytes and macrophages formed close, firm associations through their cytoplasmic membranes. This cell orientation was confirmed by transmission electron microscopy of fetal liver in vivo. In situ hybridization revealed that the macrophages expressed jagged-1, an important ligand of the Notch signaling system in hematopoiesis.     

Presentation of galectin-1 by extracellular matrix triggers T cell death

Apoptotic elimination of T cells at sites of inflammation or infiltration into tumors limits an effective immune response. T cell apoptosis can be initiated by a variety of triggers, including galectin-1, a soluble, secreted lectin that binds to oligosaccharide ligands on cell surface glycoproteins, or to oligosaccharide ligands on extracellular matrix glycoproteins in tissue stroma. Although galectin-1 has no transmembrane domain and is secreted from cells that make it, it is not clear if galectin-1 functions as a soluble death trigger in vivo. We examined the ability of stromal cells secreting galectin-1 to kill T cells. Although the stromal cells synthesized abundant galectin-1, the majority of the galectin-1 remained bound to the cell surface, and stromal cell-associated galectin-1 killed bound T cells. In contrast, insufficient amounts of functional galectin-1 were released from the stromal cells into the media to kill T cells in the absence of contact with stromal cells. However, when stromal cells were grown on Matrigel, a mixture of extracellular matrix proteins, or on permeable membranes above Matrigel, secreted galectin-1 bound to Matrigel and killed T cells without stromal cell contact. Ten-fold less galectin-1 on Matrigel was sufficient to kill adherent T cells compared with soluble galectin-1. These results demonstrate that galectin-1 in extracellular matrix is able to directly kill susceptible T cells. Because increased galectin-1 deposition in tumor stroma occurs with tumor progression in various types of cancer, galectin-1 in stroma may act locally in the apoptotic elimination of infiltrating T cells during an immune response.     

Multilineage hemopoiesis induced by cloned stromal cells

Long-term hemopoiesis in culture depends upon the presence of an adherent layer composed of a variety of stromal cells. A subtype of endothelial-adipocytes from the bone marrow stroma (clone 14F1.1) was previously shown to induce long-term myelopoiesis and renewal of pluripotent stem cells. One of a series of stromal cell lines and clones from mouse thymus stroma (STAC-1.2) has now been found to support long-term hemopoiesis. These marrow- and thymus-derived stromal cell clones also have lymphopoietic activities: precursor T cells, or pre-B cells accumulated in co-cultures of thymus cells and the stromal clones, as indicated by cell surface markers, T cell receptor and immunoglobulin gene rearrangements. The predominance of a cell type in these cultures depended upon the serum used to supplement the medium. Recombinant interleukin 2 (IL-2) and the 14F1.1 clone synergistically promoted the proliferation of thymocytes, while a thymus hormone, THF-gamma 2, shifted the population to a relatively mature phenotype. It is proposed that one major function of stromal cells, whether from the bone marrow or thymus, is to restrain the maturation flow and preferentially support the accumulation of cells at early differentiation stages.     

Morphology of interactions of the haematopoietic microenvironment with the haematopoietic cells in erythroid spleen colonies

The morphology of the interactions of the cells of the haematopoietic microenvironment with haematopoietic cells was studied in exogenous erythroid spleen colonies formed in mice, on the fifth and eighth day after their irradiation with a lethal dose of gamma rays and bone marrow transplantation. The characteristic type of stromal cell interacting with less mature cells of the erythroid series was a dark, branching reticular cell. The typical structural interaction of the reticular cells with erythroblasts was the formation of very long, fine cytoplasmic processes by the reticular cells. The processes were in close contact with the erythroblasts and formed a three-dimensional network stretching long distances from the nucleus of the reticular cells. Other cells of the haematopoietic microenvironment, in contact chiefly with poly- and ortochromic erythroblasts, were macrophages and the two together formed typical erythroblastic islands. In places, the macrophages and erythroblasts formed close, firm contacts by means of their cytoplasmic membranes. These morphological observations support the conception that close functional cooperation exists between the cells of the haematopoietic microenvironment and the haematopoietic cells in differentiation and proliferation processes in the haematopoietic tissue of spleen colonies.     

Membrane-associated chondroitin sulfate proteoglycan and fibronectin mediate the binding of hemopoietic progenitor cells to stromal cells.

The initial step in hemopoiesis is the binding of progenitor cells to stroma. What mediates this binding at the molecular level is not entirely clear. We have previously reported that the cell line FDCP-1, a factor-dependent hemopoietic progenitor cell, actively synthesizes a membrane-associated chondroitin sulfate (CS) proteoglycan (MA-PG) which is unstable. After the binding of the progenitor cell to stromal, the stability of the MA-PG is enhanced, suggesting its involvement in the binding of progenitor cells to the stroma. Since stromal cells possess pericellular fibronectin (FN), we examined the possibility that binding to stromal cells may involve interactions between MA-PG of FDCP-1 on the one side and pericellular FN in stromal cells on the other side. To examine this hypothesis, we developed a cell adherence assay to measure the binding of FDCP-1 cells to a monolayer of stromal cells or to FN-coated dishes. Cell binding was inhibited by a monoclonal antibody against CS as well as by free CS and heparin, suggesting the involvement of MA-PG in the binding. Pretreatment of FDCP-1 cells with chondroitinase ABC, which selectively removes the CS portion of the MA-PG, also affects binding to the stromal cells. The binding was also inhibited by a pentapeptide (GRGDS) which competes with the cell-binding domain of FN as well as by a monoclonal antibody anti-FN. We conclude that interactions between MA-PG and a putative integrin-like molecule in FDCP-1 and the heparin and the cell binding domains in pericellular FN in the stromal cells contribute to the stabilization of progenitor-stromal cell binding which originally comes about by homing receptors of progenitor cells.     

Increased expression of transferrin receptor on membrane of erythroblasts in strenuously exercised rats.

This study investigated the effects of strenuous exercise on transferrin (Tf)-receptor (TfR) expression and Tf-bound iron (Tf-Fe) uptake in erythroblasts of rat bone marrow. Female Sprague-Dawley rats were randomly assigned to either an exercise or sedentary group. Animals in the exercise group swam 2 h/day for 3 mo in a glass swimming basin. Both groups received the same amount of handling. At the end of 3 mo, the bone marrow erythroblasts were freshly isolated for Tf-binding assay and determination of Tf-Fe uptake in vitro. Tissue nonheme iron and hematological iron indexes were measured. The number of Tf-binding sites found in erythroblasts was approximately 674,500 +/- 132,766 and 1,270,011 +/- 235,321 molecules/cell in control and exercised rats, respectively (P < 0. 05). Total Fe and Tf uptake by the cells was also significantly increased in the exercised rats after 30 min of incubation. Rates of cellular Fe accumulation were 5.68 and 2.58 fmol. 10(6) cells(-1). min(-1) in the exercised and control rats, respectively (P < 0.05). Tf recycling time and TfR affinity were not different in exercised and control rats. Increased cellular Fe was mainly located in the stromal fraction, suggesting that most of accumulated Fe was transported to the mitochondria for heme synthesis. The findings demonstrated that the increased cellular Fe uptake in exercised rats was a consequence of the increased TfR expression rather than the changes in TfR affinity and Tf recycling time. The increase in TfR expression and cellular Fe accumulation, as well as the decreased serum Fe concentration and nonheme Fe in the liver and the spleen induced by exercise, probably represented the early signs of Fe deficiency.     

Senescent B lymphopoiesis is balanced in suppressive homeostasis: decrease in interleukin-7 and transforming growth factor-beta levels in stromal cells of senescence-accelerated mice

The suppression of the B cell population during senescence has been considered to be due to the suppression of interleukin-7 (IL-7) production and responsiveness to IL-7; however, the upregulation of transforming growth factor-beta (TGF-beta) was found to contribute to B cell suppression.To investigate the mechanism of this suppression based on the interrelationship between IL-7 and TGF-beta during senescence, senescence-accelerated mice (SAMs), the mouse model of aging, were used in this study to elucidate the mechanisms of B lymphopoietic suppression during aging. Similar to regular senescent mice, SAMs showed a decrease in the number of IL-7-responding B cell progenitors (i.e., colony-forming unit pre-B [CFU-pre-B] cells in the femoral bone marrow [BM]). A co-culture system of B lymphocytes and stromal cells that the authors established showed a significantly lower number of CFU-pre-B cells harvested when BM cells were co-cultured with senescent stromal cells than when they were co-cultured with young stromal cells. Interestingly, cells harvested from a senescent stroma and those from the control culture without stromal cells were higher in number than those harvested from a young stroma, thereby implying that an altered senescent stromal cell is unable to maintain self-renewal of the stem cell compartment. Because TGF-beta is supposed to suppress the proliferative capacity of pro-B/pre-B cells, we added a neutralizing anti-TGF-beta antibody to the co-culture system with a pro-B/pre-B cell-rich population to determine whether such suppression may be rescued. However, unexpectedly, any rescue was not observed and the number of CFU-pre-B cells remained unchanged when BM cells were co-cultured with senescent stromal cells compared with the co-culture with young stromal cells, which essentially showed an increase in the number of CFU-pre-B cells (P < 0.001 in 5 microg/ml). Furthermore, TGF-beta protein level in the supernatant of cultured senescent stroma cells was evaluated by enzyme-linked immunoabsorbent assay, but surprisingly, it was found that TGF-beta concentration was significantly lower than that of cultured young stromal cells. Thus, TGF-beta activity was assumed to decline particularly in a senescent stroma, which means a distinct difference between the senescent suppression of B lymphopoiesis and secondary B lymphocytopenia. Concerning proliferative signaling, on the other hand, the level of IL-7 gene expression in cells from freshly isolated BM decreased significantly with age. Therefore, the acceleration of proliferative signaling and the deceleration of suppressive signaling may both be altered and weakened in a senescent stroma (i.e., homeosuppression).     

Vascular cell adhesion molecule-1 expression and hematopoietic supportive capacity of immortalized murine stromal cell lines derived from fetal liver and adult bone marrow

Ontogeny-specific differences in hematopoietic behavior may be influenced by unique adhesive interactions between hematopoietic cells and the microenvironment, such as that mediated by vascular cell adhesion molecule-1 (VCAM-1, CD 106). Although VCAM-1 is variably expressed during vertebrate development, we hypothesized that VCAM-1 expression might be linked to the enhanced capacity of the fetal liver microenvironment to support hematopoiesis. To test this we used immortalized murine stromal cell lines derived from midgestation fetal liver and adult bone marrow to compare the functional expression of VCAM-1. Molecular analysis of VCAM-1 expression was performed on stromal cell lines using Northern blot analysis, immunoprecipitation studies, and solid-phase enzyme-linked immunosorbent assay. Hematopoietic studies were performed by coculturing fetal liver cells with stromal cell lines, and the functional readout was determined by high-proliferative potential colony-forming cell (HPP-CFC) adherence assays. In contrast to our initial hypothesis, we observed greater expression of VCAM-1 messenger ribonucleic acid and protein on an adult marrow stromal cell line. In functional studies, anti-VCAM-1 antibody inhibited the binding of nearly half of the HPP-CFCs to adult marrow stroma but had a minimal effect on their binding to fetal liver stroma, despite the greater adherence of HPP-CFCs to fetal stroma. We conclude that VCAM-1 influences the hematopoietic supportive capacity of immortalized murine stroma derived from adult bone marrow. Our studies suggest that cellular interactions other than those mediated by VCAM-1 are involved in the increased adhesive capacity of immortalized murine stroma derived from fetal liver.