Regulatory role of osteogenic growth polypeptides in bone formation and hemopoiesis

Osteogenic growth polypeptides such as the osteogenic growth peptide (OGP), fragments of the parathyroid hormone (PTH), and insulin-like growth factors (IGF) regulate bone cell activity in vitro and may affect in vivo osteoblastic functions in an autocrine, paracrine, or endocrine manner. Several growth polypeptides capable of regulating osteogenesis circulate in the blood in an inactive form, complexed to parent molecules or binding proteins. During postablation bone marrow regeneration these factors may be activated, released from the blood clot, and together with locally produced polypeptides mediate the initial intramedullary/systemic osteogenic phase of this process. Then osteogenic growth polypeptides expressed by osteoblasts and other stromal cells have the potential to promote the second phase of regeneration that consists of osteoclastogenesis, resorption of the transient intramedullary bone, and hemopoiesis. This is probably an indirect effect inasmuch as these polypeptides can regulate the stromal cell expression of hemopoietic factors such as macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6), and the stem cell factor (SCF). The postablation marrow regeneration model is suitable for studying the expression and activity of osteogenic growth polypeptides and already has been used to assess the effect of aging on these parameters. Clinically, the osteogenic growth polypeptides and marrow regeneration have a potential role in osteoporosis therapy, implant and corrective bone surgery, and bone marrow transplantation.     

TGF-beta1-binding proteins on human bone marrow stromal cells

The stromal cells are integral components of the bone marrow (BM) that provide and respond to cytokines, and offer adhesion elements for hematopoietic cell homing. Steady-state hematopoiesis results from a balance between negative and positive acting cytokines, whose expression is in addition the subject of regulation. TGF-beta1 is present in the BM microenvironment and plays a central role in controlling hematopoiesis, by modulating the synthesis of cytokines and cytokine receptors, as well as cell adhesion molecules. We have recently described the TGF-beta1 receptor system expressed on human BM stromal cells. The consequences of signalling through this system, which can affect stromal cell function, and hence, influence the hematopoiesis, is the subject of this review.     

Addition of a bone marrow "facilitating cell" population increases stem cell-derived cobblestone area formation in impaired long-term bone marrow culture stroma

Treatment of mouse bone marrow (BM) with rabbit anti-mouse brain serum (RAMBS) plus complement (C') depletes several cell types, including T cells and facilitating cells (FCs), that is, cells that facilitate engraftment of sorted allogeneic stem cells (SCs) in vivo. In the present study, treatment of BM with RAMBS+C' resulted in the depletion of approximately half of the late cobblestone area (CA)-forming stem cells as assayed on irradiated long-term bone marrow culture (LTBMC) stroma. In addition, LTBMC of RAMBS+C'-treated BM produced functionally impaired stroma with reduced ability to support CA formation by nontreated exogenous SCs. This stromal impairment was not due to depletion of TCRalphabeta T cells in the BM, because BM cultures from TCR alpha-chain knockout mice supported normal numbers of exogenous CAs. Because CD8+/TCR- cells are enriched for FCs, we tested the effect of adding these cells back to the treated BM prior to culture. The sorted FCs alone did not produce CAs, but did improve the ability of the impaired stroma to support late CA formation by sorted SCs. These studies provide a new model for dissecting the roles of different cellular components of BM in producing functional stroma that supports CA formation by SCs, and show that the number of CAs formed depends on the "quality" of the stroma as well as the number of SCs seeded. These findings further suggest that CD8+/TCR- BM cells may be important for the establishment of functional stroma.     

Differential use of very late antigen-4 and -5 integrins by hematopoietic precursors and myeloma cells to adhere to transforming growth factor-beta1-treated bone marrow stroma

The very late antigen (VLA)-4 and VLA-5 integrins mediate hematopoietic progenitor cell attachment to bone marrow (BM) stroma. Transforming growth factor-beta1 (TGF-beta1) is a cytokine present in the BM microenvironment that has been shown to regulate the synthesis of adhesion elements in several cell types. We have investigated whether TGF-beta1 action on human BM stromal cells affected the adhesion of progenitor cells involving integrins VLA-4 and VLA-5. Two precursor cell lines, pre-B Nalm-6 and the multipotential UT-7, attached to untreated primary stroma and to the human BM stromal cell line Str-5 preferentially using VLA-4. However, treatment of the stroma with TGF-beta1 resulted in a significant reduction in the participation of VLA-4 in mediating precursor cell adhesion to stroma and a concomitant increase in the utilization of VLA-5. This effect was not exclusive of normal BM stroma. Treatment with TGF-beta1 of stroma from multiple myeloma BM samples produced a substantial increase in VLA-5 use by the myeloma cell line NCI-H929 to adhere to this stroma. The differential use of VLA-4 and VLA-5 correlated with an increase in fibronectin surface expression by stromal cells in response to TGF-beta1. Adhesion assays to purified fibronectin using Nalm-6 cells showed a predominant utilization of VLA-4 at low concentrations of this ligand, whereas higher concentrations resulted in a preferential use of VLA-5. These results indicate that regulation of fibronectin expression on BM stromal cells by TGF-beta1 results in a modulation of the pattern of integrins used by the precursor and myeloma cells to adhere to BM stroma, which could have important consequences on the proliferation and differentiation of hematopoietic precursor cells as well as on the localization and growth of myeloma cells.     

Characterization of a B cell progenitor present in neonatal bone marrow and spleen but not in adult bone marrow and spleen

The neonatal period marks an important time in mammalian immunologic development, yet it is often ignored in studies of lymphocyte development. We identified a cell population with the phenotype heat stable Ag (HSA)low lin- CD43low that contained B cell progenitors at a high frequency in the neonatal bone marrow and spleen. Although cells with a similar phenotype can be identified in the bone marrow and spleen of adult animals, these populations showed a greatly reduced frequency of B cell progenitors. B lineage cells were detected after 7 days in culture at a frequency of 1:15 when HSAlow lin- CD43low cells from neonatal bone marrow were cultured on stromal cells and IL-7 under limiting dilution conditions. Under similar conditions, the equivalent population in adult bone marrow had a frequency of B cell progenitors that was less than 1:2000. The expression of terminal deoxynucleotidyl transferase in freshly sorted neonatal HSAlow lin- CD43low cells suggested that cells committed to the lymphocyte lineage were present in this population. These data suggested that the HSAlow lin- CD43low population of cells represents a pool of B lineage precursors that may be responsible for filling the immune compartment early in neonatal life.     

Infection of human marrow stroma by human immunodeficiency virus-1 (HIV-1) is both required and sufficient for HIV-1-induced hematopoietic suppression in vitro: demonstration by gene modification of primary human stroma

Patients with human immunodeficiency virus-1 (HIV-1) infection often present with bone marrow (BM) failure that may affect all hematopoietic lineages. It is presently unclear whether this failure reflects a direct viral impairment of the CD34+ hematopoietic progenitor cells or whether the virus affects the BM microenvironment. To study the effects of HIV-1 on the BM microenvironment, we examined the stromal cell monolayers in long-term BM culture (LTBMC), which are the in vitro equivalent of the hematopoietic microenvironment. We assessed the hematopoietic support function (HSF) of human stromal layers by determining the cellular proliferation and colony-forming ability of hematopoietic progenitors from BM cells grown on the stromal layers. We show that the HSF is reduced by in vitro infection of the human stromal cell layer by a monocytotropic isolate of HIV-1 (JR-FL). There is no loss of HSF when the stromal cell layer is resistant to HIV-1 replication, either using murine stromal cell layers that are innately resistant to HIV-1 infection or using human stromal cells genetically modified to express a gene that inhibits HIV-1 replication (an RRE decoy). Decreased HSF was seen using either human or murine hematopoietic cells, if the stromal cells were human cells that were susceptible to HIV-1 infection. These in vitro studies implicate HIV-1 replication in the stroma as the essential component causing decreased hematopoietic cell production in HIV-1 infection.     

Immortalization and characterization of bone marrow stromal fibroblasts from a patient with a loss of function mutation in the estrogen receptor-alpha gene

A male patient with abnormal postpubertal bone elongation was shown earlier to have a mutation in both alleles of the estrogen receptor, resulting in a nonfunctional gene. Marrow stromal fibroblasts (MSFs) derived from this patient were called HERKOs (human estrogen receptor knock outs), and in order to obtain continuous HERKO cell lines, they were immortalized using a recombinant adenovirus-origin-minus SV40 virus. MSFs are unique cells because they support hematopoesis and contain a mixed population of precursor cells for bone, cartilage, and fat. Three established cell lines (HERKO2, HERKO4, and HERKO7) were characterized and compared with the heterogeneous population of nonimmortalized HERKOs for their osteogenic potential. We performed Northern analysis of matrix genes implicated in bone development and metabolism and an in vivo bone formation assay by transplanting the cells subcutaneously into immunodeficient mice. All three HERKO lines expressed high amounts of collagen 1A1, osteopontin, osteonectin, fibronectin, decorin, biglycan, and alkaline phosphatase. Except for osteopontin, expression of these genes was slightly lower compared with nonimmortalized HERKOs. In the in vivo bone formation assay, the heterogeneous population of nonimmortalized HERKOs formed bone with high efficiency, while the HERKO lines induced a high-density, bone-like matrix. Finally, all HERKO cell types secreted high levels of insulin-like growth factor I and interleukin-6 into the culture medium relative to cells of normal human subjects. In summary, these lines of HERKO cells retain several of the phenotypic traits of MSFs after immortalization, including matrix and cytokine production, and provide a valuable source of a unique human material for future studies involving estrogen action in bone and bone marrow metabolism.     

Identification of an IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF). I. Production of the non-IL-7 component by bone marrow stromal cells from IL-7 gene-deleted mice

Mouse bone marrow (BM) stromal cell conditioned medium (CM) from our long-term lymphoid culture system selectively induces the in vitro proliferation and presumptive differentiation of pre-pro-B cells (B220+, HSA-, TdT- or TdT+, c[mu-]) from adult rat, mouse, and human BM. However, the responsible growth factor(s) has not yet been identified. Inasmuch as IL-7 is one of the cytokines most closely associated with early B-lineage development, we utilized BM adherent cells and stromal cell lines from IL-7 gene-deleted (-/-) mice in combination with rIL-7 and anti-IL-7 mAb to investigate its possible regulatory role in our culture system. The results show that, although rIL-7 and IL-7 (-/-) CM each can maintain the viability of freshly harvested pre-pro-B cells in vitro, neither induces them to proliferate and/or differentiate, even in the presence of recombinant stem cell factor (rSCF) and/or recombinant insulin-like growth factor (rIGF). The results also show that anti-IL-7 mAb fails to neutralize the pre-pro-B cell growth-stimulating activity in IL-7 (+/+) CM. Yet rIL-7 enables IL-7 (-/-) CM to induce proliferation of pre-pro-B cells, and to "prime" them to respond directly to monomeric IL-7. Furthermore, anti-IL-7 mAb adsorbs the pre-pro-B cell growth-stimulating activity from both IL-7 (+/+) CM and rIL-7-supplemented IL-7 (-/-) CM; but rIL-7 does not restore this activity. Lastly, both pre-pro-B cell growth-stimulatory activity and IL-7 are quantitatively recovered by ultrafiltration in the 50 to 100 kDa, rather than the 10 to 50 kDa, apparent molecular mass fraction. These results suggest that the pre-pro-B cell growth-stimulating activity in our culture system is the property of a self-associating complex of IL-7 and a second BM stromal cell-derived cofactor.     

Conditionally immortalized murine bone marrow stromal cells mediate parathyroid hormone-dependent osteoclastogenesis in vitro

PTH recruits and activates osteoclasts to cause bone resorption. These actions of PTH are thought to be mediated indirectly via type 1 PTH/PTH-related peptide receptors (PTH1Rs) expressed by adjacent marrow stromal or osteoblastic cells, although some evidence suggests that PTH may act directly on early hematopoietic osteoclast progenitors. We have established clonal, conditionally immortalized, PTH-responsive, bone marrow stromal cell lines from mice that harbor both a transgene encoding a temperature-sensitive mutant of the simian virus 40 large T antigen and deletion of a single allele of the PTH1R gene. Of 60 stromal cell lines isolated, 45 expressed functional PTH1Rs. During coculture with normal murine spleen cells, 5 of 42 such cell lines could support formation of tartrate-resistant acid phosphatase-positive, multinucleated cells (TRAP+ MNCs) in response to 1,25-dihydroxyvitamin D3, but only 2 of these did so in response to PTH. One of these, MS1 cells, expressed numerous cytokines and proteins characteristic of the osteogenic lineage and showed increased production of interleukin-6 in response to PTH. MS1 cells supported dose-dependent induction by rat (r) PTH-(1-34) (0.1-100 nM) of TRAP+ MNCs that expressed calcitonin receptors and formed resorption lacunae on dentine slices. This effect of PTH, which required cell to cell contact between MS1 and spleen cells, was mimicked by coadministration of cAMP analog and phorbol ester but only partially by either agent alone. The carboxyl-terminal fragment rPTH-(53-84) also induced osteoclast-like cell formation, but the maximal effect was only 30% as great as that of rPTH-(1-34). Importantly, rPTH-(1-34) induced TRAP+ MNC formation even when PTH1R-/- osteoclast progenitors (from fetal liver of mice homozygous for ablation of the PTH1R gene) were cocultured with MS1 cells. We conclude that activation of PTH1Rs on cells of the osteoclast lineage is not required for PTH-(1-34)-induced osteoclast formation in the presence of appropriate PTH-responsive marrow stromal cells. MS1 cells provide a useful model for further study of PTH regulation of osteoclastogenesis.     

Interaction between a murine myeloma cell line and bone marrow stromal cells

Intravenous transplantation of an in vitro maintained murine myeloma cell line, 5T33, results in progressive growth in the bone marrow of C57Bl/KaLwRiJ mice. Concurrent with the growth of the tumor in vivo, the bone marrow stromal cells are inhibited, as assayed by their ability to form stromal cell foci and long-term monolayers in vitro. Inhibition of normal mouse marrow stromal cell growth also occurs when 5T33 cells are added to the marrow cells in vitro, and contact between the marrow and 5T33 cells is not necessary to achieve inhibition, indicating secretion of one or more diffusible inhibitory factors.     

Immunophenotypic characterization of stromal cells in aspirated human bone marrow samples

The presence of stromal cells was investigated in aspirated bone marrow prepared by the same method as that used for the initiation of human long-term bone marrow culture (hLTBMC). In previous studies, we performed immunocytochemical staining of cytocentrifuge cell preparations using a panel of antibodies with which we characterized stromal cell populations in hLTBMC. This approach allowed morphological as well as immunophenotypic assessment of cells of interest. Morphologically distinctive cell populations expressing vascular cell adhesion molecule-1 and low-affinity nerve growth factor receptor (NGFR) were observed to be present, but no cells expressing alpha-smooth muscle actin were found. Few macrophages were present, consistent with the origin of hLTBMC stroma-adherent macrophages from monocytes and their precursor cells rather than from mature macrophages among the culture-initiating cells. In the absence of double immunostaining, it was not possible to deduce whether CD34+ cells, which were present in varying numbers in the cytocentrifuge preparations, included stromal as well as primitive hematopoietic cells. In addition to single cells, multicellular tissue fragments containing a variety of stromal cell types were detected in many samples. Their presence raises the possibility that at least some components of hLTBMC stroma may arise by explant growth from complex tissue fragments containing vascular and fibroblastic elements. Overall, our results indicate that demonstration of a variety of stroma-associated antigens, in particular NGFR, provides a useful new tool for identifying stromal elements in aspirated bone marrow.     

Prolonged expression of c-fos suppresses cell cycle entry of dormant hematopoietic stem cells

The proto-oncogene c-fos was transiently upregulated in primitive hematopoietic stem (Lin-Sca-1(+)) cells stimulated with stem cell factor, interleukin-3 (IL-3), and IL-6. To investigate a role of the c-fos in hematopoietic stem cells, we used bone marrow (BM) cells from transgenic mice carrying the c-fos gene under the control of the interferon-alpha/beta-inducible Mx-promoter (Mx-c-fos), and fetal liver cells from c-fos-deficient mice. Prolonged expression of the c-fos in Lin-Sca-1(+) BM cells inhibited factor-dependent colony formation and hematopoiesis on a stromal cell layer by keeping them at G0/G1 phase of the cell cycle. These Lin-Sca-1(+) BM cells on a stromal layer entered into the cell cycle whenever exogenous c-fos was downregulated. However, ectopic c-fos did not perturb colony formation by Lin-Sca-1(+) BM cells after they entered the cell cycle. Furthermore, endogenous c-fos is not essential to cell cycle progression of hematopoietic stem cells because the factor-dependent and the stroma-dependent hematopoiesis by Lin-Sca-1(+) fetal liver cells from c-fos-deficient mice was not impaired. These results suggest that the c-fos induced in primitive hematopoietic stem cells negatively controls cell cycle progression and maintains them in a dormant state.     

Tamoxifen attenuates glucocorticoid actions on bone formation in vitro

Tamoxifen is a synthetic estrogen analog which may regulate osteogenesis in vivo by virtue of its antiglucocorticoid properties. We have examined tamoxifen regulation of glucocorticoid-induced osteogenesis in two different in vitro bone systems: the chicken periosteal osteogenesis model (CPO) and rat bone marrow stromal cells (RBMC). Hormone uptake studies were conducted with the osteosarcoma cell line, ROS 17/2.8. In the CPO model, alkaline phosphatase (AP) activity and collagen synthesis were stimulated by the glucocorticoid dexamethasone (Dex; 0.1 microM). These Dex-mediated effects were inhibited by increasing concentrations of tamoxifen (10-100 microM). Similarly, in the RBMC model, Dex-dependent (0.01 microM Dex) mineralized tissue formation and AP activity were blocked by tamoxifen (0.1 microM). Although tamoxifen inhibited Dex-mediated increases of AP activity in ROS 17/2.8 cells, it did not inhibit uptake of 3H-Dex or of 3H-estrogen. Northern analyses showed that tamoxifen did not affect messenger RNAs (mRNAs) for AP. Tamoxifen did seem to reduce mRNA for collagen type I, but not bone sialoprotein, osteopontin, and osteocalcin. Dex-induced increases for all proteins mRNAs in the RBMC model were not reduced by tamoxifen. Similarly, tamoxifen had no effects on cellular proliferation. We conclude that tamoxifen has no direct effect on gene expression of bone-related proteins of osteoblastic cells. Further, in the ROS 17/2.8 cell line, the antiglucocorticoid properties of tamoxifen do not appear to be mediated through either Dex or estrogen receptors.     

Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice

The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP-deficient mutant mice. The three genotypes (+/+, +/-, -/-) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3-5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.     

The production of granulocyte colony-stimulating factor by stromal bone marrow populations]

Passaged bone marrow fibroblasts (PBMF) fail to maintain long-term hemopoiesis in culture unlike stromal adhesive layers of long-term culture of the bone marrow (LTCBM). What differs PBMF from LTCBM stromal cells in terms of the above ability we attempted to find out using immunocytochemical microscopy and flow cytometry. There were no differences as to protein production of the extracellular matrix, production of granulocytic-macrophagal colony-stimulating factor, stem cell factor, transforming growth factor beta, interleukin-1 beta and interleukin-6. The difference lies in inability of PBMF to produce granulocytic colony-stimulating factor (GCSF). It is evident that production of GCSF by stromal cells of the bone marrow is essential for maintenance of myelopoiesis in LTCBM.     

Interleukin 4, interferon-gamma, and prostaglandin E impact the osteoclastic cell-forming potential of murine bone marrow macrophages

Interleukin 4 (IL-4) is an immune cytokine that inhibits bone resorption in mice and suppresses osteoclastic cell formation in vitro through an undefined mechanism. In this report, we have established the cellular identity of the IL-4 target cell using a variety of bone marrow/stromal cell coculture methods. Initially, we found that the majority of IL-4's inhibition of osteoclastic cell formation was due to its effect on bone marrow cells, not stromal cells. Consequently, bone marrow macrophages were used as osteoclastic cell progenitors after they had been transiently exposed to IL-4 (48 h), before the addition of stromal cells, 1,25-dihydroxyvitamin D3, and dexamethasone. In this circumstance, IL-4 impaired subsequent osteoclastic cell formation, suggesting that the macrophage may be potentially targeted by many factors known to influence osteoclast formation. Consequently, we discovered that interferon-gamma (IFN gamma), prostaglandin E (PGE), and cell-permeant cAMP analogs also impacted osteoclastic cell formation when used to selectively treat bone marrow macrophages. IFN gamma suppressed osteoclastic cell formation, whereas PGE and cAMP analog treatment led to the formation of significantly enlarged osteoclastic cells. Importantly, PGE antagonized the inhibitory effects of both IL-4 and IFN gamma on the osteoclastic cell-forming potential of bone marrow macrophages. Collectively, these findings establish bone marrow macrophages as osteoclastic cell precursors with the degree of their commitment to the osteoclast pathway sensitive to the effects of soluble mediators, including IL-4, IFN gamma, and PGE.     

Associations between TGF-beta1 receptors in human bone marrow stromal cells

Bone marrow stromal cells are required for sustained haemopoiesis. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine present in the bone marrow microenvironment which regulates the expression of several cytokines, cytokine receptors and cell adhesion elements. The TGF-beta receptors type I and II, and endoglin, mediate TGF-beta1 binding to the membrane of human bone marrow stromal cells. [125I]TGF-beta1-affinity labelling experiments showed that three different anti-endoglin monoclonal antibodies co-immunoprecipitated a 68 kD TGF-beta1-labelled polypeptide together with TGF-beta1/endoglin complexes. Here, we have shown that the 68 kD receptor corresponds to the type I receptor, indicating that endoglin and the type I receptor associate on the membrane of these cells upon ligand binding. The expression of endoglin by stromal cells was found to be up-regulated by TGF-beta1, but not by IL-1beta. The association of endoglin with signalling components of the TGF-beta receptor system on the membrane of bone marrow stromal cells might modulate TGF-beta1 access to the signalling pathways, and therefore it could regulate TGF-beta1-mediated stromal cellular responses.     

Apoptosis during B lymphopoiesis in mouse bone marrow

To evaluate the magnitude of cell death and the critical stages at which it occurs during B lymphopoiesis in mouse bone marrow (BM), we have examined the kinetics of apoptosis at defined stages of B cell differentiation. FACS-sorted B220+ BM cells exhibited a low incidence of morphologically apoptotic cells by electron microscopy. In freshly prepared BM suspensions, the incidence of hypodiploid cells detected by multiparameter flow cytometry was greater among large dividing B220+ surface IgM- (sIgM-) precursor B cells and sIgM(low) immature B lymphocytes than among terminal deoxynucleotidyl transferase+ (TdT+) pro-B cells, small nondividing B220+ sIgM- precursors, and surface IgD+ mature B lymphocytes. During short-term culture, apoptotic cells, identified by both DNA content and in situ DNA strand break labeling, increased linearly with time without macrophage ingestion, providing an assay for the rate of entry into apoptosis. B220+ B lineage cells accumulated in apoptosis more rapidly than cells of other lineages. The apoptotic rate was greater among B220+ sIgM- precursor cells than sIgM+ B cells, and was highest among B220+ mu- pro-B cells. Coculture with stromal cells reduced the apoptotic rate of B220+ sIgM- precursors to a greater extent than that of sIgM+ B lymphocytes. The results lead to estimates of the actual number of B lineage cells undergoing apoptosis per unit time in successive differentiation compartments. The findings indicate that, although influenced by local microenvironmental factors, apoptotic cell death occurs most markedly at two developmental stages associated with Ig heavy chain gene rearrangement and Ag receptor expression, respectively.     

Connexin43 gap junctions in normal, regenerating, and cultured mouse bone marrow and in human leukemias: their possible involvement in blood formation

Communicating channels called gap junctions are thought to play a ubiquitous part in cell growth and development. Based on earlier work, we have recently found functional evidence of their presence in human and mouse bone marrow. In this study we studied the cell-type association of the gap junction channel-forming protein, connexin, in mouse and human bone marrow under different physiological and pathological conditions and tested the pathway of communication in bone marrow cultures. For high-resolution antigen demonstration we took advantage of semi-thin resin sections, antigen retrieval methods, immunofluorescence, and confocal laser scanning microscopy. Connexin43 (Cx43) and its mRNA were consistently expressed in human and rodent marrow. Cx37 was found only in the arteriolar endothelium, but neither Cx32 nor -26 were expressed. In tissue sections, the immunostained junctions appeared as dots, which were digitally measured and counted. Their average size was 0.40 mm in human and 0.49 mm in mice marrow. There were at least twice as many gap junctions in the femoral midshaft of 6-week-old mice (1.75 x 10(5)/mm3) as in those older than 12 weeks (0.89 x 10(5)/mm3). Most Cx43 was associated with collagen III+ endosteal and adventitial stromal cells and with megakaryocytes. Elsewhere, they were few and randomly distributed between all kinds of hematopoietic cells. In the femoral epiphysis of juvenile mice, stromal cell processes full of Cx43 enmeshed three to six layers of hematopoietic cells near the endosteum. The same pattern was seen in the midshaft of regenerating mouse marrow 3 to 5 days after cytotoxic treatment with 5-fluorouracil. Functional tests in cultures showed the transfer of small fluorescent dyes, Lucifer Yellow and 2',7'-bis-(2-carboxyethyl)-5, 6-carboxyfluorescein, between stromal cells and in rare cases between stromal and hematopoietic cells too. The stromal cells were densely packed with Cx43 and we found aggregates of connexon particles in their membrane replicas. In normocellular human bone marrow, gap junctions were as rare as in adult mouse and similarly distributed, except that they were also on adipocytic membranes. In a few leukemic samples, characterized by an increased stromal/hematopoietic cell ratio, there were two- to fourfold more Cx43 (2.8 x 10(5) to 3.9 x 10(5)/mm3) than in the normal (1.0 x 10(5) to 1.2 x 10(5)/mm3). The cases included a hypoplastic acute lymphoblastic leukemia, an acute myeloid leukemia (French-American-British classification M4-5), a case of myelodysplastic syndrome with elevated number of megakaryocytes, and a CD34+ acute hemoblastosis, probably acute myeloid leukemia (French-American-British classification M7). Taken together, our results indicate that direct cell-cell communication may be involved in hematopoiesis, ie, in developmentally active epiphyseal bone marrow and when there is a demand for progenitors in regeneration. However, gap junctions may not play as important a role in resting adult hematopoiesis and in leukemias.